U.S. patent application number 13/940092 was filed with the patent office on 2014-06-12 for isoprene production using the dxp and mva pathway.
This patent application is currently assigned to The Goodyear Tire & Rubber Company. The applicant listed for this patent is Danisco US Inc., The Goodyear Tire & Rubber Company. Invention is credited to Gopal K. CHOTANI, Joseph C. MCAULIFFE, Michael C. MILLER, Rachel E. MUIR, Dmitrii V. VAVILINE, Walter WEYLER.
Application Number | 20140162337 13/940092 |
Document ID | / |
Family ID | 43356749 |
Filed Date | 2014-06-12 |
United States Patent
Application |
20140162337 |
Kind Code |
A1 |
CHOTANI; Gopal K. ; et
al. |
June 12, 2014 |
ISOPRENE PRODUCTION USING THE DXP AND MVA PATHWAY
Abstract
The invention provides for methods for producing isoprene from
cultured cells using various components of the DXP pathway and MVA
pathway, or components associated with the DXP pathway and MVA
pathway, iron-sulfur cluster-interacting redox polypeptides, and
isoprene synthase. The invention also provides compositions that
include these cultured cells.
Inventors: |
CHOTANI; Gopal K.;
(Cupertino, CA) ; MCAULIFFE; Joseph C.;
(Sunnyvale, CA) ; MILLER; Michael C.; (San
Francisco, CA) ; MUIR; Rachel E.; (Redwood City,
CA) ; VAVILINE; Dmitrii V.; (Palo Alto, CA) ;
WEYLER; Walter; (San Francisco, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Goodyear Tire & Rubber Company
Danisco US Inc. |
Akron
Palo Alto |
OH
CA |
US
US |
|
|
Assignee: |
The Goodyear Tire & Rubber
Company
Akron
OH
Danisco US Inc.
Palo Alto
CA
|
Family ID: |
43356749 |
Appl. No.: |
13/940092 |
Filed: |
July 11, 2013 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
12817134 |
Jun 16, 2010 |
8507235 |
|
|
13940092 |
|
|
|
|
61187941 |
Jun 17, 2009 |
|
|
|
61187930 |
Jun 17, 2009 |
|
|
|
61314985 |
Mar 17, 2010 |
|
|
|
61314979 |
Mar 17, 2010 |
|
|
|
Current U.S.
Class: |
435/167 ;
435/252.3; 435/252.31; 435/252.33; 435/252.34; 435/254.11;
435/254.2; 435/256.1; 435/256.7 |
Current CPC
Class: |
C12N 15/70 20130101;
C12P 5/007 20130101 |
Class at
Publication: |
435/167 ;
435/252.3; 435/252.31; 435/252.34; 435/252.33; 435/254.11;
435/256.7; 435/256.1; 435/254.2 |
International
Class: |
C12P 5/00 20060101
C12P005/00; C12N 15/70 20060101 C12N015/70 |
Claims
1. Cells comprising (i) a heterologous nucleic acid encoding an
iron-sulfur cluster-interacting redox polypeptide, a DXP pathway
polypeptide, a MVA pathway polypeptide, and an isoprene synthase
polypeptide or (ii) a duplicate copy of an endogenous nucleic acid
encoding an iron-sulfur cluster-interacting redox polypeptide, a
DXP pathway polypeptide, a MVA pathway polypeptide, and an isoprene
synthase polypeptide.
2. The cells of claim 1, wherein the cells further comprise a
heterologous nucleic acid or a duplicate copy of an endogenous
nucleic acid encoding an IDI (isopentenyl-diphosphate
delta-isomerase) polypeptide.
3. The cells of claim 1, wherein the cells in culture produce
greater than about 400 nmole/g.sub.wcm/hr of isoprene.
4. The cells of claim 1, wherein more than about 0.02 molar percent
of the carbon that the cells consume from a cell culture medium is
converted into isoprene.
5. A method of producing isoprene, the method comprising (a)
culturing cells comprising (i) a heterologous nucleic acid encoding
a heterologous nucleic acid encoding an iron-sulfur
cluster-interacting redox polypeptide, a DXP pathway polypeptide, a
MVA pathway polypeptide, and an isoprene synthase polypeptide or
(ii) a duplicate copy of an endogenous nucleic acid encoding an
iron-sulfur cluster-interacting redox polypeptide, a DXP pathway
polypeptide, a MVA pathway polypeptide, and an isoprene synthase
polypeptide under suitable culture conditions for the production of
isoprene, and (b) producing isoprene.
6. The method of claim 5, wherein the cells further comprise a
heterologous nucleic acid or a duplicate copy of an endogenous
nucleic acid encoding an IDI (isopentenyl-diphosphate
delta-isomerase) polypeptide.
7. The method of claim 5, wherein the cells in culture produce
greater than about 400 nmole/g.sub.wcm/hr of isoprene.
8. The method of claim 5, wherein more than about 0.02 molar
percent of the carbon that the cells consume from a cell culture
medium is converted into isoprene.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 12/817,134, filed Jun. 16, 2010, which claims
priority to U.S. Provisional Application No. 61/187,941, filed Jun.
17, 2009; U.S. Provisional Application No. 61/187,930, filed Jun.
17, 2009; U.S. Provisional Application No. 61/314,985, filed Mar.
17, 2010; U.S. Provisional Application No. 61/314,979, filed Mar.
17, 2010; the disclosures of which are herein incorporated by
reference in their entirety.
INCORPORATION BY REFERENCE
[0002] The content of the following submission on ASCII text file
is incorporated herein by reference in its entirety: a computer
readable form (CRF) of the Sequence Listing (file name:
643842001101_Sequence_Listing.txt, date created: Jul. 8, 2013,
size: 395,413 bytes).
FIELD OF THE INVENTION
[0003] The present invention relates generally to compositions and
methods for improving the production of isoprene from cultured
cells using the DXP pathway and MVA pathway.
BACKGROUND OF THE INVENTION
[0004] Isoprene (2-methyl-1,3-butadiene) is the critical starting
material for a variety of synthetic polymers, most notably
synthetic rubbers. Isoprene is naturally produced by a variety of
microbial, plant, and animal species. In particular, two pathways
have been identified for the biosynthesis of isoprene: the
mevalonate (MVA) pathway and the non-mevalonate (DXP) pathway (FIG.
19A). However, the yield of isoprene from naturally-occurring
organisms is commercially unattractive. About 800,000 tons per year
of cis-polyisoprene are produced from the polymerization of
isoprene; most of this polyisoprene is used in the tire and rubber
industry. Isoprene is also copolymerized for use as a synthetic
elastomer in other products such as footwear, mechanical products,
medical products, sporting goods, and latex.
[0005] Currently, the tire and rubber industry is based on the use
of natural and synthetic rubber. Natural rubber is obtained from
the milky juice of rubber trees or plants found in the rainforests
of Africa. Synthetic rubber is based primarily on butadiene
polymers. For these polymers, butadiene is obtained as a co-product
from ethylene and propylene manufacture.
[0006] While isoprene can be obtained by fractionating petroleum,
the purification of this material is expensive and time-consuming.
Petroleum cracking of the C5 stream of hydrocarbons produces only
about 15% isoprene. Thus, more economical methods for producing
isoprene are needed. In particular, methods that produce isoprene
at rates, titers, and purity that are sufficient to meet the
demands of a robust commercial process are desirable. Also desired
are systems for producing isoprene from inexpensive starting
materials.
BRIEF SUMMARY OF THE INVENTION
[0007] The invention provides, inter alia, compositions and methods
for the production of isoprene in increased amounts using various
DXP pathway genes and polypeptides and various MVA pathway genes
and polypeptides, iron-sulfur cluster-interacting redox genes and
polypeptides, isoprene synthase, and optionally, various genes and
polypeptides associated with the DXP pathway, various genes and
polypeptides associated with the MVA pathway, and IDI genes and
polypeptides. In one aspect, the invention features cells or cells
in culture which have been engineered for producing isoprene in
increased amounts by using a combination of various DXP pathway
genes and polypeptides, various MVA pathway genes and polypeptides,
iron-sulfur cluster-interacting redox genes and polypeptides,
isoprene synthase genes and polypeptides, and optionally, DXP
pathway associated genes and polypeptides, MVA pathway associated
genes and polypeptides, and IDI genes and polypeptides.
[0008] In some embodiments, the cells or cells in culture comprise
(i) a heterologous nucleic acid encoding an iron-sulfur
cluster-interacting redox polypeptide, a DXP pathway polypeptide, a
MVA pathway polypeptide, and an isoprene synthase polypeptide
and/or (ii) a duplicate copy of an endogenous nucleic acid encoding
an iron-sulfur cluster-interacting redox polypeptide, a DXP pathway
polypeptide, a MVA pathway polypeptide, and an isoprene synthase
polypeptide. In some embodiments, the cells or cells in culture
comprise (i) one or more copies of heterologous or endogenous
nucleic acid encoding an iron-sulfur cluster-interacting redox
polypeptide, (ii) one or more copies of heterologous or endogenous
nucleic acid encoding a DXP pathway polypeptide and/or a MVA
pathway polypeptide, and (iii) one or more copies of heterologous
or endogenous nucleic acid encoding an isoprene synthase
polypeptide. In some embodiments, the iron-sulfur
cluster-interacting redox polypeptide, the DXP pathway polypeptide,
a MVA pathway polypeptide, and isoprene synthase polypeptide are
operably linked to a promoter.
[0009] In some embodiments, the DXP pathway polypeptide is selected
from the group consisting of DXS (1-deoxy-D-xylulose-5-phosphate
synthase), DXR (1-deoxy-D-xylulose-5-phosphate reductoisomerase),
MCT (4-diphosphocytidyl-2C-methyl-D-erythritol synthase), CMK
(4-diphosphocytidyl-2-C-methyl-D-erythritol kinase), MCS
(2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase), HDS
(1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase), and HDR
(1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase). In some
embodiments, the DXP pathway polypeptide is DXS, HDS, or HDR. In
some embodiments, the cells further comprise a heterologous nucleic
acid or a duplicate copy of an endogenous nucleic acid encoding an
IDI (isopentenyl-diphosphate delta-isomerase) polypeptide.
[0010] In some embodiments, the MVA pathway polypeptide is selected
from the group consisting acetyl-CoA acetyltransferase (AA-CoA
thiolase), 3-hydroxy-3-methylglutaryl-CoA synthase (HMG-CoA
synthase), 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA
reductase), mevalonate kinase (MVK), phosphomevalonate kinase
(PMK), diphosphomevalonte decarboxylase (MVD), phosphomevalonate
decarboxylase (PMDC) and isopentenyl phosphate kinase (IPK). In
some embodiments, the cells further comprise a heterologous nucleic
acid or a duplicate copy of an endogenous nucleic acid encoding an
IDI (isopentenyl-diphosphate delta-isomerase) polypeptide.
[0011] In one embodiment, both the DXP and MVA pathways can be
present in any ratio to produce isoprene from each pathway in any
proportion in cells or cells in culture. In another embodiment,
about 10% to 50% of the isoprene is produced utilizing the DXP
pathway and the remainder is produced utilizing the MVA pathway. In
another embodiment, at least about 50% of the isoprene is produced
utilizing the DXP pathway and the remainder is produced utilizing
the MVA pathway.
[0012] In some embodiments, the invention provides cells or cells
in culture that produce greater than about 400 nmole of
isoprene/gram of cells for the wet weight of the cells/hour
(nmole/g.sub.wcm/hr) of isoprene. In some embodiments, the cells or
cells in culture convert more than about 0.002% of the carbon in a
cell culture medium into isoprene.
[0013] In some embodiments, the invention provides cells or cells
in culture where the level of HMBPP and DMAPP are maintained below
1 mM for the duration of the fermentation run. In other
embodiments, the invention provides cells in culture where the
level of HMBPP and DMAPP are maintained below 1 mM during the
exponential phase of the fermentation. In other embodiments, the
invention provides cells or cells in culture in which late DXP
pathway enzymes, particularly IspG and IspH are maintained at
levels consistent with minimizing phosphorylation level of Dxr.
[0014] In some embodiments of any of the aspects of the invention,
the iron-sulfur cluster-interacting redox polypeptide comprises
flavodoxin (e.g., flavodoxin I), flavodoxin reductase, ferredoxin
(e.g., ferredoxin I), ferredoxin-NADP+ oxidoreductase, and genes or
polypeptides encoding thereof (e.g., fpr and fldA).
[0015] In some embodiments, the cells or cells in culture comprise
(i) a heterologous nucleic acid encoding a ferredoxin polypeptide,
a ferredoxin-NADP+ oxidoreductase polypeptide, a DXP pathway
polypeptide, and an isoprene synthase polypeptide and/or (ii) a
duplicate copy of an endogenous nucleic acid encoding a ferredoxin
polypeptide, a ferredoxin-NADP+ oxidoreductase polypeptide, a DXP
pathway polypeptide, and an isoprene synthase polypeptide. In some
embodiments, the cells or cells in culture comprise IspG and fldA.
In another embodiment, the cells or cells in culture comprise IspG,
fldA, and IspH. In some embodiments, the ferredoxin polypeptide,
the ferredoxin-NADP+ oxidoreductase, the DXP pathway polypeptide,
and isoprene synthase polypeptide are operably linked to a
promoter. In some embodiments, the cells further comprise a
heterologous nucleic acid or a duplicate copy of an endogenous
nucleic acid encoding an IDI polypeptide.
[0016] In some embodiments, the cells in culture comprise (i) a
heterologous nucleic acid encoding a flavodoxin polypeptide, a DXP
pathway polypeptide, a MVA pathway polypeptide, and an isoprene
synthase polypeptide and/or (ii) a duplicate copy of an endogenous
nucleic acid encoding a flavodoxin polypeptide, a DXP pathway
polypeptide, a MVA pathway polypeptide, and an isoprene synthase
polypeptide. In some embodiments, the flavodoxin polypeptide, the
DXP pathway polypeptide, MVA pathway polypeptide, and isoprene
synthase polypeptide are operably linked to a promoter. In some
embodiments, the cells further comprise a heterologous nucleic acid
or a duplicate copy of an endogenous nucleic acid encoding an IDI
polypeptide.
[0017] In some embodiments, the cells are cultured in a culture
medium that includes a carbon source, such as, but not limited to,
a carbohydrate, glycerol, glycerine, dihydroxyacetone, one-carbon
source, oil, animal fat, animal oil, fatty acid, lipid,
phospholipid, glycerolipid, monoglyceride, diglyceride,
triglyceride, renewable carbon source, polypeptide (e.g., a
microbial or plant protein or peptide), yeast extract, component
from a yeast extract, or any combination of two or more of the
foregoing. In some embodiments, the cells are cultured under
limited glucose conditions.
[0018] In other aspects, the invention provides for methods of
producing isoprene, the method comprising (a) culturing cells
comprising (i) a heterologous nucleic acid encoding a heterologous
nucleic acid encoding an iron-sulfur cluster-interacting redox
polypeptide, a DXP pathway polypeptide, a MVA pathway polypeptide,
and an isoprene synthase polypeptide or (ii) a duplicate copy of an
endogenous nucleic acid encoding an iron-sulfur cluster-interacting
redox polypeptide, a DXP pathway polypeptide, a MVA pathway
polypeptide, and an isoprene synthase polypeptide under suitable
culture conditions for the production of isoprene, and (b)
producing isoprene. In one embodiment, the cells further comprise a
heterologous nucleic acid or a duplicate copy of an endogenous
nucleic acid encoding an IDI (isopentenyl-diphosphate
delta-isomerase) polypeptide. In other embodiments, the cells in
culture produce greater than about 400 nmole/g.sub.wcm/hr of
isoprene. In other embodiments, more than about 0.02 molar percent
of the carbon that the cells consume from a cell culture medium is
converted into isoprene.
[0019] In one aspect, the invention features methods of producing
isoprene, such as methods of using any of the cells described
herein to produce isoprene. In some embodiments, the method
involves culturing cells comprising (i) a heterologous nucleic acid
encoding a heterologous nucleic acid encoding an iron-sulfur
cluster-interacting redox polypeptide, a DXP pathway polypeptide,
and an isoprene synthase polypeptide, and/or (ii) a duplicate copy
of an endogenous nucleic acid encoding an iron-sulfur
cluster-interacting redox polypeptide, a DXP pathway polypeptide,
and an isoprene synthase polypeptide. In some embodiments, the
cells are cultured under suitable culture conditions for the
production of isoprene, and isoprene is produced. In some
embodiments, the iron-sulfur cluster-interacting redox polypeptide,
isoprene synthase polypeptide, and DXP pathway polypeptide are
operably linked to a promoter. In some embodiments, the DXP pathway
polypeptide is selected from the group consisting of DXS
(1-deoxy-D-xylulose-5-phosphate synthase), DXR
(1-deoxy-D-xylulose-5-phosphate reductoisomerase), MCT
(4-diphosphocytidyl-2C-methyl-D-erythritol synthase), CMK
(4-diphosphocytidyl-2-C-methyl-D-erythritol kinase), MCS
(2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase), HDS
(1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase), and HDR
(1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase). In some
embodiments, the DXP pathway polypeptide is DXS, HDS, or HDR. In
some embodiments, the cells further comprise a heterologous nucleic
acid or a duplicate copy of an endogenous nucleic acid encoding an
IDI (isopentenyl-diphosphate delta-isomerase) polypeptide. In some
embodiments, the method involves culturing cells under conditions
sufficient to produce greater than about 400 nmole/g.sub.wcm/hr of
isoprene. In some embodiments, the method involves culturing cells
under conditions sufficient to convert more than about 0.002%
(mol/mol) of the carbon in a cell culture medium into isoprene.
[0020] In various embodiments, the amount of isoprene produced
(such as the total amount of isoprene produced or the amount of
isoprene produced per liter of broth per hour per OD.sub.600)
during stationary phase is greater than or about 2 or more times
the amount of isoprene produced during the growth phase for the
same length of time. In some embodiments, the gas phase comprises
greater than or about 9.5% (volume) oxygen, and the concentration
of isoprene in the gas phase is less than the lower flammability
limit or greater than the upper flammability limit. In particular
embodiments, (i) the concentration of isoprene in the gas phase is
less than the lower flammability limit or greater than the upper
flammability limit, and (ii) the cells produce greater than about
400 nmole/g.sub.wcm/hr of isoprene.
[0021] In some embodiments, isoprene is only produced in stationary
phase. In some embodiments, isoprene is produced in both the growth
phase and stationary phase. In various embodiments, the amount of
isoprene produced (such as the total amount of isoprene produced or
the amount of isoprene produced per liter of broth per hour per
OD.sub.600) during stationary phase is greater than or about 2, 3,
4, 5, 10, 20, 30, 40, 50, or more times the amount of isoprene
produced during the growth phase for the same length of time.
[0022] In one aspect, the invention features compositions and
systems that comprise isoprene. In some embodiments, the
composition comprises greater than or about 2, 5, 10, 20, 30, 40,
50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or
1000 mg of isoprene. In some embodiments, the composition comprises
greater than or about 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100
g of isoprene (w/w) of the volatile organic fraction of the
composition is isoprene.
[0023] In some embodiments, the composition comprises greater than
or about 99.90, 99.92, 99.94, 99.96, 99.98, or 100% isoprene by
weight compared to the total weight of all C5 hydrocarbons in the
composition. In some embodiments, the composition comprises less
than or about 0.12, 0.10, 0.08, 0.06, 0.04, 0.02, 0.01, 0.005,
0.001, 0.0005, 0.0001, 0.00005, or 0.00001% C5 hydrocarbons other
than isoprene (such as 1,3-cyclopentadiene, cis-1,3-pentadiene,
trans-1,3-pentadiene, 1,4-pentadiene, 1-pentyne, 2-pentyne,
3-methyl-1-butyne, pent-4-ene-1-yne, trans-pent-3-ene-1-yne, or
cis-pent-3-ene-1-yne) by weight compared to the total weight of all
C5 hydrocarbons in the composition. In some embodiments, the
composition has less than or about 0.12, 0.10, 0.08, 0.06, 0.04,
0.02, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005, or 0.00001% for
1,3-cyclopentadiene, trans-1,3-pentadiene, cis-1,3-pentadiene,
1,4-pentadiene, 1-pentyne, 2-pentyne, 3-methyl-1-butyne,
pent-4-ene-1-yne, trans-pent-3-ene-1-yne, or cis-pent-3-ene-1-yne
by weight compared to the total weight of all C5 hydrocarbons in
the composition. In particular embodiments, the composition has
greater than about 2 mg of isoprene and has greater than or about
99.90, 99.92, 99.94, 99.96, 99.98, or 100% isoprene by weight
compared to the total weight of all C5 hydrocarbons in the
composition.
[0024] In some embodiments, the composition has less than or about
50, 40, 30, 20, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, or 0.005 ug/L of a
compound that inhibits the polymerization of isoprene for any
compound in the composition that inhibits the polymerization of
isoprene. In particular embodiments, the composition also has
greater than about 2 mg of isoprene.
[0025] In some embodiments, the composition comprises (i) a gas
phase that comprises isoprene and (ii) cells in culture that
produce greater than about 400 nmole/g.sub.wcm/hr of isoprene. In
some embodiments, the composition comprises a closed system, and
the gas phase comprises greater than or about 5, 10, 20, 30, 40,
50, 60, 70, 80, 90, 100 ug/L of isoprene when normalized to 1 mL of
1 OD.sub.600 cultured for 1 hour. In some embodiments, the
composition comprises an open system, and the gas phase comprises
greater than or about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100
ug/L of isoprene when sparged at a rate of 1 vvm. In some
embodiments, the volatile organic fraction of the gas phase
comprises greater than or about 99.90, 99.92, 99.94, 99.96, 99.98,
or 100% isoprene by weight compared to the total weight of all C5
hydrocarbons in the volatile organic fraction. In some embodiments,
the volatile organic fraction of the gas phase comprises less than
or about 0.12, 0.10, 0.08, 0.06, 0.04, 0.02, 0.01, 0.005, 0.001,
0.0005, 0.0001, 0.00005, or 0.00001% C5 hydrocarbons other than
isoprene (such as 1,3-cyclopentadiene, cis-1,3-pentadiene,
trans-1,3-pentadiene, 1,4-pentadiene, 1-pentyne, 2-pentyne,
3-methyl-1-butyne, pent-4-ene-1-yne, trans-pent-3-ene-1-yne, or
cis-pent-3-ene-1-yne) by weight compared to the total weight of all
C5 hydrocarbons in the volatile organic fraction. In some
embodiments, the volatile organic fraction of the gas phase has
less than or about 0.12, 0.10, 0.08, 0.06, 0.04, 0.02, 0.01, 0.005,
0.001, 0.0005, 0.0001, 0.00005, or 0.00001% for
1,3-cyclopentadiene, cis-1,3-pentadiene, trans-1,3-pentadiene,
1,4-pentadiene, 1-pentyne, 2-pentyne, 3-methyl-1-butyne,
pent-4-ene-1-yne, trans-pent-3-ene-1-yne, or cis-pent-3-ene-1-yne
by weight compared to the total weight of all C5 hydrocarbons in
the volatile organic fraction. In particular embodiments, the
volatile organic fraction of the gas phase has greater than about 2
mg of isoprene and has greater than or about 99.90, 99.92, 99.94,
99.96, 99.98, or 100% isoprene by weight compared to the total
weight of all C5 hydrocarbons in the volatile organic fraction.
[0026] In some embodiments, the volatile organic fraction of the
gas phase has less than or about 50, 40, 30, 20, 10, 5, 1, 0.5,
0.1, 0.05, 0.01, or 0.005 ug/L of a compound that inhibits the
polymerization of isoprene for any compound in the volatile organic
fraction of the gas phase that inhibits the polymerization of
isoprene. In particular embodiments, the volatile organic fraction
of the gas phase also has greater than about 2 mg of isoprene.
[0027] In some embodiments of any of the compositions of the
invention, at least a portion of the isoprene is in a gas phase. In
some embodiments, at least a portion of the isoprene is in a liquid
phase (such as a condensate). In some embodiments, at least a
portion of the isoprene is in a solid phase. In some embodiments,
at least a portion of the isoprene is adsorbed to a solid support,
such as a support that includes silica and/or activated carbon. In
some embodiments, the composition includes ethanol. In some
embodiments, the composition includes between about 75 to about 90%
by weight of ethanol, such as between about 75 to about 80%, about
80 to about 85%, or about 85 to about 90% by weight of ethanol. In
some embodiments, the composition includes between about 4 to about
15% by weight of isoprene, such as between about 4 to about 8%,
about 8 to about 12%, or about 12 to about 15% by weight of
isoprene.
[0028] In some embodiments, the invention also features systems
that include any of the cells and/or compositions described herein.
In some embodiments, the system includes a reactor that chamber
comprises cells in culture that produce greater than about 400,
500, 600, 700, 800, 900, 1,000, 1,250, 1,500, 1,750, 2,000, 2,500,
3,000, 4,000, 5,000, or more nmole/g.sub.wcm/hr isoprene. In some
embodiments, the system is not a closed system. In some
embodiments, at least a portion of the isoprene is removed from the
system. In some embodiments, the system includes a gas phase
comprising isoprene. In various embodiments, the gas phase
comprises any of the compositions described herein.
[0029] In one aspect, the invention provides a tire comprising
polyisoprene. In some embodiments, the polyisoprene is produced by
(i) polymerizing isoprene in any of the compositions described
herein or (ii) polymerizing isoprene recovered from any of the
compositions described herein. In some embodiments, the
polyisoprene comprises cis-1,4-polyisoprene.
[0030] In some embodiments of any of the compositions, systems, and
methods of the invention, a nonflammable concentration of isoprene
in the gas phase is produced. In some embodiments, the gas phase
comprises less than about 9.5% (volume) oxygen. In some
embodiments, the gas phase comprises greater than or about 9.5%
(volume) oxygen, and the concentration of isoprene in the gas phase
is less than the lower flammability limit or greater than the upper
flammability limit. In some embodiments, the portion of the gas
phase other than isoprene comprises between about 0% to about 100%
(volume) oxygen, such as between about 10% to about 100% (volume)
oxygen. In some embodiments, the portion of the gas phase other
than isoprene comprises between about 0% to about 99% (volume)
nitrogen. In some embodiments, the portion of the gas phase other
than isoprene comprises between about 1% to about 50% (volume)
CO.sub.2.
[0031] In some embodiments of any of the aspects of the invention,
the cells further comprise a heterologous nucleic acid or a
duplicate copy of an endogenous nucleic acid encoding a DXP pathway
associated polypeptide.
[0032] In some embodiments of any of the aspects of the invention,
the cells in culture produce isoprene at greater than or about 400,
500, 600, 700, 800, 900, 1,000, 1,250, 1,500, 1,750, 2,000, 2,500,
3,000, 4,000, 5,000, or more nmole/g.sub.wcm/hr isoprene. In some
embodiments of any of the aspects of the invention, the cells in
culture convert greater than or about 0.002, 0.005, 0.01, 0.02,
0.05, 0.1, 0.12, 0.14, 0.16, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8,
0.9, 1.0, 1.2, 1.4, 1.6%, or more of the carbon in the cell culture
medium into isoprene. In some embodiments of any of the aspects of
the invention, the cells in culture produce isoprene at greater
than or about 1, 10, 25, 50, 100, 150, 200, 250, 300, 400, 500,
600, 700, 800, 900, 1,000, 1,250, 1,500, 1,750, 2,000, 2,500,
3,000, 4,000, 5,000, 10,000, 100,000, or more ng of isoprene/gram
of cells for the wet weight of the cells/hr (ng/g.sub.wcm/h). In
some embodiments of any of the aspects of the invention, the cells
in culture produce a cumulative titer (total amount) of isoprene at
greater than or about 1, 10, 25, 50, 100, 150, 200, 250, 300, 400,
500, 600, 700, 800, 900, 1,000, 1,250, 1,500, 1,750, 2,000, 2,500,
3,000, 4,000, 5,000, 10,000, 50,000, 100,000, or more mg of
isoprene/L of broth (mg/L.sub.broth, wherein the volume of broth
includes the volume of the cells and the cell medium). Other
exemplary rates of isoprene production and total amounts of
isoprene production are disclosed herein.
[0033] In some embodiments of any of the aspects of the invention,
isoprene production can be further increased by using a mutant DXP
pathway polypeptide and nucleic acid derived from thereof. In some
embodiments, the mutant DXP pathway polypeptide is a HDR
polypeptide with the iron-sulfur cluster regulator (iscR) removed.
In some embodiments, the mutant DXP pathway polypeptide is a mutant
HDR polypeptide that produces solely DMAPP or a majority of DMAPP
relative to IPP.
[0034] In some embodiments of any of the aspects of the invention,
isoprene production can be further increased by increasing the
carbon flux through the DXP pathway and/or MVA pathway. In some
embodiments, the carbon flux can be increased by avoiding any
feedback inhibition of DXS activity by metabolites downstream the
DXP pathway or/and intermediates of other pathways that use a DXP
pathway polypeptide as a substrate. In some embodiments, the other
pathway that uses DXP pathway polypeptide as a substrate (e.g.,
DXP) is the thiamine (Vitamin B1) or pyridoxal (Vitamin B6)
pathway. In some embodiments, the carbon flux can be increased by
expressing a DXP pathway polypeptide from a different organism that
is not subject to inhibition by downstream products of the DXP
pathway. In some embodiments, the carbon flux can be increased by
deregulating glucose uptake. In other embodiments, the carbon flux
can be increased by maximizing the balance between the precursors
required for the DXP pathway and/or MVA pathway. In some
embodiments, the balance of the DXP pathway precursors, pyruvate
and glyceraldehydes-3-phosphate (G-3-P), can be achieved by
redirecting the carbon flux with the effect of elevating or
lowering pyruvate or G-3-P separately. In some embodiments, the
carbon flux can be increased by using a CRP (cAMP Receptor
Protein)-deleted mutant.
[0035] In some embodiments, the carbon flux can be increased by
using a strain (containing one or more DXP pathway genes or one or
more both DXP pathway and MVA pathway genes) containing a pyruvate
dehydrogenase E1 subunit variant. In some embodiments, the pyruvate
dehydrogenase (PDH) E1 subunit variant has an E636Q point
mutation.
[0036] In some embodiments of any of the aspects of the invention,
isoprene production can be further increased by utilizing the
downstream genes or polypeptides of the DXP pathway by introducing
a heterologous terpene synthase nucleic acid or a duplicate copy of
an endogenous terpene synthase nucleic acid into the cells, which
includes, but is not limited to ocimene synthase, farnesene
synthase, and artemesinin synthase.
[0037] In some embodiments of any of the aspects of the invention,
in some embodiments, the vector comprises a selective marker, such
as an antibiotic resistance nucleic acid.
[0038] In some embodiments of any of the aspects of the invention,
the heterologous isoprene synthase nucleic acid is operably linked
to a T7 promoter, such as a T7 promoter contained in a medium or
high copy plasmid. In some embodiments of any of the aspects of the
invention, the heterologous isoprene synthase nucleic acid is
operably linked to a Trc promoter, such as a Trc promoter contained
in a medium or high copy plasmid. In some embodiments of any of the
aspects of the invention, the heterologous isoprene synthase
nucleic acid is operably linked to a Lac promoter, such as a Lac
promoter contained in a low copy plasmid. In some embodiments of
any of the aspects of the invention, the heterologous isoprene
synthase nucleic acid is operably linked to an endogenous promoter,
such as an endogenous alkaline serine protease promoter. In some
embodiments, the heterologous isoprene synthase nucleic acid
integrates into a chromosome of the cells without a selective
marker.
[0039] In some embodiments, iron-sulfur cluster-interacting redox
nucleic acid, any one or more of the nucleic acids in the DXP
pathway, MVA pathway, and isoprene synthase nucleic acid are placed
under the control of a promoter or factor that is more active in
stationary phase than in the growth phase. In one embodiment, IDI
nucleic acid is also included for IDI expression to produce a
higher amount of isoprene than when IDI is not used. For example,
one or more iron-sulfur cluster-interacting redox nucleic acid, DXP
pathway nucleic acid, IDI nucleic acid, or isoprene synthase
nucleic acid may be placed under control of a stationary phase
sigma factor, such as RpoS. In some embodiments, one or more
iron-sulfur cluster-interacting redox nucleic acid, DXP pathway
nucleic acid, MVA pathway nucleic acid, IDI nucleic acid, or
isoprene synthase nucleic acid are placed under control of a
promoter inducible in stationary phase, such as a promoter
inducible by a response regulator active in stationary phase.
[0040] In some embodiments of any of the aspects of the invention,
cells expressing iron-sulfur cluster-interacting redox polypeptide,
isoprene synthase polypeptide, and DXP pathway polypeptide are
grown under non-inducing conditions. In some embodiments of any of
the aspects of the invention, cells expressing iron-sulfur
cluster-interacting redox polypeptide, DXP pathway polypeptide, IDI
polypeptide, and isoprene synthase polypeptide are grown under
non-inducing conditions. For example, the non-inducing condition is
that IPTG-induced expression from the Trc promoter regulated gene
constructs is not performed.
[0041] In some embodiments of any of the aspects of the invention,
the cells express a second DXP pathway polypeptide, in addition to
the first DXP pathway polypeptide, including DXS
(1-deoxy-D-xylulose-5-phosphate synthase), DXR
(1-deoxy-D-xylulose-5-phosphate reductoisomerase), MCT
(4-diphosphocytidyl-2C-methyl-D-erythritol synthase), CMK
(4-diphosphocytidyl-2-C-methyl-D-erythritol kinase), MCS
(2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase), HDS
(1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase), and HDR
(1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase). In some
embodiments of any of the aspects of the invention, the cells
express two or more DXP pathway polypeptides, in addition to the
first DXP pathway polypeptide as described above. In some
embodiments of any of the aspects of the invention, the cells
express 2, 3, 4, 5, 6, or 7 DXP pathway polypeptides, in addition
to the first DXP pathway polypeptide as described above.
[0042] In some embodiments of any of the aspects of the invention,
the cells express a second MVA pathway polypeptide, in addition to
the first MVA pathway polypeptide, including acetyl-CoA
acetyltransferase (AA-CoA thiolase), 3-hydroxy-3-methylglutaryl-CoA
synthase (HMG-CoA synthase), 3-hydroxy-3-methylglutaryl-CoA
reductase (HMG-CoA reductase), mevalonate kinase (MVK),
phosphomevalonate kinase (PMK), diphosphomevalonte decarboxylase
(MVD), phosphomevalonate decarboxylase (PMDC) and isopentenyl
phosphate kinase (IPK). In some embodiments of any of the aspects
of the invention, the cells express two or more MVA pathway
polypeptides, in addition to the first MVA pathway polypeptide as
described above. In some embodiments of any of the aspects of the
invention, the cells express 2, 3, 4, 5, 6, or 7 MVA pathway
polypeptides, in addition to the first MVA pathway polypeptide as
described above.
[0043] In some embodiments of any of the aspects of the invention,
at least a portion of the cells maintain the heterologous
iron-sulfur cluster-interacting redox nucleic acid, DXP pathway
nucleic acid, and isoprene synthase nucleic acid for at least or
about 5, 10, 20, 40, 50, 60, 65, or more cell divisions in a
continuous culture (such as a continuous culture without dilution).
In some embodiments of any of the aspects of the invention, at
least a portion of the cells maintain the heterologous iron-sulfur
cluster-interacting redox nucleic acid, IDI nucleic acid, DXP
pathway nucleic acid, and isoprene synthase nucleic acid for at
least or about 5, 10, 20, 40, 50, 60, 65, or more cell divisions in
a continuous culture (such as a continuous culture without
dilution). In some embodiments of any of the aspects of the
invention, at least a portion of the cells maintain the
heterologous isoprene synthase nucleic acid, DXS nucleic acid, IDI
nucleic acid, and iron-sulfur cluster-interacting redox nucleic
acid for at least or about 5, 10, 20, 40, 50, 60, 65, or more cell
divisions in a continuous culture (such as a continuous culture
without dilution). In some embodiments of any of the aspects of the
invention, the nucleic acid comprising the iron-sulfur
cluster-interacting redox nucleic acid, isoprene synthase nucleic
acid, DXP pathway nucleic acid, and/or IDI nucleic acid also
comprises a selective marker, such as an antibiotic resistance
nucleic acid.
[0044] In some embodiments of any of the aspects of the invention,
the isoprene synthase polypeptide is a polypeptide from a plant
such as Pueraria (e.g., Pueraria montana or Pueraria lobata) or
Populus (e.g., Populus tremuloides, Populus alba, Populus nigra,
Populus trichocarpa, or the hybrid, Populus alba.times.Populus
tremula).
[0045] In some embodiments of any of the aspects of the invention,
the cells are bacterial cells, such as gram-positive bacterial
cells (e.g., Bacillus cells such as Bacillus subtilis cells or
Streptomyces cells such as Streptomyces lividans, Streptomyces
coelicolor, or Streptomyces griseus cells) or cyanobacterial cells
(e.g., Thermosynechococcus cells such as Thermosynechococcus
elongates cells). In some embodiments of any of the aspects of the
invention, the cells are gram-negative bacterial cells (e.g.,
Escherichia cells such as Escherichia coli cells or Pantoea cells
such as Pantoea citrea cells) or cyanobacterial cells (e.g.,
Thermosynechococcus cells such as Thermosynechococcus elongates
cells). In some embodiments of any of the aspects of the invention,
the cells are fungal, cells such as filamentous fungal cells (e.g.,
Trichoderma cells such as Trichoderma reesei cells or Aspergillus
cells such as Aspergillus oryzae and Aspergillus niger), or yeast
cells (e.g., Yarrowia cells such as Yarrowia lipolytica cells).
[0046] In some embodiments of any of the aspects of the invention,
the microbial polypeptide carbon source includes one or more
polypeptides from yeast or bacteria. In some embodiments of any of
the aspects of the invention, the plant polypeptide carbon source
includes one or more polypeptides from soy, corn, canola, jatropha,
palm, peanut, sunflower, coconut, mustard, rapeseed, cottonseed,
palm kernel, olive, safflower, sesame, or linseed.
[0047] In one aspect, the invention features a product produced by
any of the compositions or methods of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0048] FIG. 1 is the nucleotide sequence of a kudzu isoprene
synthase gene codon-optimized for expression in E. coli (SEQ ID
NO:1). The atg start codon is in italics, the stop codon is in bold
and the added PstI site is underlined.
[0049] FIG. 2 is a map of pTrcKudzu.
[0050] FIGS. 3A-3C is the nucleotide sequence of pTrcKudzu (SEQ ID
NO:2). The RBS is underlined, the kudzu isoprene synthase start
codon is in bold capitol letters and the stop codon is in bold,
capitol, italics letters. The vector backbone is pTrcHis2B.
[0051] FIG. 4 is a map of pETNHisKudzu.
[0052] FIGS. 5A-5C is the nucleotide sequence of pETNHisKudzu (SEQ
ID NO:5).
[0053] FIG. 6 is a map of pCL-lac-Kudzu.
[0054] FIGS. 7A-7C is the nucleotide sequence of pCL-lac-Kudzu (SEQ
ID NO:7).
[0055] FIG. 8A is a graph showing the production of isoprene in E.
coli BL21 cells with no vector.
[0056] FIG. 8B is a graph showing the production of isoprene in E.
coli BL21 cells with pCL-lac-Kudzu
[0057] FIG. 8C is a graph showing the production of isoprene in E.
coli BL21 cells with pTrcKudzu.
[0058] FIG. 8D is a graph showing the production of isoprene in E.
coli BL21 cells with pETN-HisKudzu.
[0059] FIG. 9A is a graph showing OD over time of fermentation of
E. coli BL21/pTrcKudzu in a 14 liter fed batch fermentation.
[0060] FIG. 9B is a graph showing isoprene production over time of
fermentation of E. coli BL21/pTrcKudzu in a 14 liter fed batch
fermentation.
[0061] FIG. 10A is a graph showing the production of isoprene in
Panteoa citrea. Control cells without recombinant kudzu isoprene
synthase. Grey diamonds represent isoprene synthesis, black squares
represent OD.sub.600.
[0062] FIG. 10B is a graph showing the production of isoprene in
Panteoa citrea expressing pCL-lac Kudzu. Grey diamonds represent
isoprene synthesis, black squares represent OD.sub.600.
[0063] FIG. 10C is a graph showing the production of isoprene in
Panteoa citrea expressing pTrcKudzu. Grey diamonds represent
isoprene synthesis, black squares represent OD.sub.600.
[0064] FIG. 11 is a graph showing the production of isoprene in
Bacillus subtilis expressing recombinant isoprene synthase.
BG3594comK is a B. subtilis strain without plasmid (native isoprene
production). CF443-BG3594comK is a B. subtilis strain with pBSKudzu
(recombinant isoprene production). IS on the y-axis indicates
isoprene.
[0065] FIGS. 12A-12C is the nucleotide sequence of pBS Kudzu #2
(SEQ ID NO:56).
[0066] FIG. 13 is the nucleotide sequence of kudzu isoprene
synthase codon-optimized for expression in Yarrowia (SEQ ID
NO:8).
[0067] FIG. 14 is a map of pTrex3g comprising a kudzu isoprene
synthase gene codon-optimized for expression in Yarrowia.
[0068] FIGS. 15A-15C is the nucleotide sequence of vector
pSPZ1(MAP29Spb) (SEQ ID NO:11).
[0069] FIG. 16 is the nucleotide sequence of the synthetic kudzu
(Pueraria montana) isoprene gene codon-optimized for expression in
Yarrowia (SEQ ID NO:12).
[0070] FIG. 17 is the nucleotide sequence of the synthetic hybrid
poplar (Populus alba.times.Populus tremula) isoprene synthase gene
(SEQ ID NO:13). The ATG start codon is in bold and the stop codon
is underlined.
[0071] FIGS. 18A1-18A2 shows a schematic outlining construction of
vectors pYLA 1, pYL1 and pYL2 (SEQ ID NOS: 44, 45, 46, 47, 48 and
50).
[0072] FIG. 18B shows a schematic outlining construction of the
vector pYLA(POP1) (SEQ ID NOS: 43 and 44).
[0073] FIG. 18C shows a schematic outlining construction of the
vector pYLA(KZ1)
[0074] FIG. 18D shows a schematic outlining construction of the
vector pYLI(KZ1) (SEQ ID NOS: 41, 42).
[0075] FIG. 18E shows a schematic outlining construction of the
vector pYLI(MAP29)
[0076] FIG. 18F shows a schematic outlining construction of the
vector pYLA(MAP29)
[0077] FIG. 19 shows the MVA and DXP metabolic pathways for
isoprene (based on F. Bouvier et al., Progress in Lipid Res. 44:
357-429, 2005). The following description includes alternative
names for each polypeptide in the pathways and a reference that
discloses an assay for measuring the activity of the indicated
polypeptide (each of these references are each hereby incorporated
by reference in their entireties, particularly with respect to
assays for polypeptide activity for polypeptides in the MVA and DXP
pathways). Mevalonate Pathway: AACT; Acetyl-CoA acetyltransferase,
MvaE, EC 2.3.1.9. Assay: J. Bacteriol., 184: 2116-2122, 2002; HMGS;
Hydroxymethylglutaryl-CoA synthase, MvaS, EC 2.3.3.10. Assay: J.
Bacteriol., 184: 4065-4070, 2002; HMGR;
3-Hydroxy-3-methylglutaryl-CoA reductase, MvaE, EC 1.1.1.34. Assay:
J. Bacteriol., 184: 2116-2122, 2002; MVK; Mevalonate kinase, ERG12,
EC 2.7.1.36. Assay: Curr Genet 19:9-14, 1991. PMK;
Phosphomevalonate kinase, ERGS, EC 2.7.4.2, Assay: Mol Cell Biol.,
11:620-631, 1991; DPMDC; Diphosphomevalonate decarboxylase, MVD1,
EC 4.1.1.33. Assay: Biochemistry, 33:13355-13362, 1994; IDI;
Isopentenyl-diphosphate delta-isomerase, IDI1, EC 5.3.3.2. Assay:
J. Biol. Chem. 264:19169-19175, 1989. DXP Pathway: DXS;
1-deoxy-D-xylulose-5-phosphate synthase, dxs, EC 2.2.1.7. Assay:
PNAS, 94:12857-62, 1997; DXR; 1-Deoxy-D-xylulose 5-phosphate
reductoisomerase, dxr, EC 2.2.1.7. Assay: Eur. J. Biochem.
269:4446-4457, 2002; MCT; 4-Diphosphocytidyl-2C-methyl-D-erythritol
synthase, IspD, EC 2.7.7.60. Assay: PNAS, 97: 6451-6456, 2000; CMK;
4-Diphosphocytidyl-2-C-methyl-D-erythritol kinase, IspE, EC
2.7.1.148. Assay: PNAS, 97:1062-1067, 2000; MCS;
2C-Methyl-D-erythritol 2,4-cyclodiphosphate synthase, IspF, EC
4.6.1.12. Assay: PNAS, 96:11758-11763, 1999; HDS;
1-Hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase, ispG,
GcpE, EC 1.17.4.3. Assay: J. Org. Chem., 70:9168-9174, 2005; HDR;
1-Hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase, IspH,
LytB, EC 1.17.1.2. Assay: JACS, 126:12847-12855, 2004.
[0078] FIG. 20 shows graphs representing results of the GC-MS
analysis of isoprene production by recombinant Y. lipolytica
strains without (left) or with (right) a kudzu isoprene synthase
gene. The arrows indicate the elution time of the authentic
isoprene standard.
[0079] FIG. 21 is a map of pTrcKudzu yIDI DXS Kan.
[0080] FIGS. 22A-22D is the nucleotide sequence of pTrcKudzu yIDI
DXS Kan (SEQ ID NO:20).
[0081] FIG. 23A is a graph showing production of isoprene from
glucose in BL21/pTrcKudzukan. Time 0 is the time of induction with
IPTG (400 .mu.mol). The x-axis is time after induction; the y-axis
is OD.sub.600 and the y2-axis is total productivity of isoprene
(.mu.g/L headspace or specific productivity (.mu.g/L headspace/OD).
Diamonds represent OD.sub.600, circles represent total isoprene
productivity (.mu.g/L) and squares represent specific productivity
of isoprene (.mu.g/L/OD).
[0082] FIG. 23B is a graph showing production of isoprene from
glucose in BL21/pTrcKudzu yIDI kan. Time 0 is the time of induction
with IPTG (400 .mu.mol). The x-axis is time after induction; the
y-axis is OD.sub.600 and the y2-axis is total productivity of
isoprene (.mu.g/L headspace or specific productivity (.mu.g/L
headspace/OD). Diamonds represent OD.sub.600, circles represent
total isoprene productivity (.mu.g/L) and squares represent
specific productivity of isoprene (.mu.g/L/OD).
[0083] FIG. 23C is a graph showing production of isoprene from
glucose in BL21/pTrcKudzu DXS kan. Time 0 is the time of induction
with IPTG (400 .mu.mol). The x-axis is time after induction; the
y-axis is OD.sub.600 and the y2-axis is total productivity of
isoprene (.mu.g/L headspace or specific productivity (.mu.g/L
headspace/OD). Diamonds represent OD.sub.600, circles represent
total isoprene productivity (.mu.g/L) and squares represent
specific productivity of isoprene (.mu.g/L/OD).
[0084] FIG. 23D is a graph showing production of isoprene from
glucose in BL21/pTrcKudzu yIDI DXS kan. Time 0 is the time of
induction with IPTG (400 .mu.mol). The x-axis is time after
induction; the y-axis is OD.sub.600 and the y2-axis is total
productivity of isoprene (.mu.g/L headspace or specific
productivity (.mu.g/L headspace/OD). Diamonds represent OD.sub.600,
circles represent total isoprene productivity (.mu.g/L) and squares
represent specific productivity of isoprene (.mu.g/L/OD).
[0085] FIG. 23E is a graph showing production of isoprene from
glucose in BL21/pCL PtrcKudzu. Time 0 is the time of induction with
IPTG (400 .mu.mol). The x-axis is time after induction; the y-axis
is OD.sub.600 and the y2-axis is total productivity of isoprene
(.mu.g/L headspace or specific productivity (.mu.g/L headspace/OD).
Diamonds represent OD.sub.600, circles represent total isoprene
productivity (.mu.g/L) and squares represent specific productivity
of isoprene (.mu.g/L/OD).
[0086] FIG. 23F is a graph showing production of isoprene from
glucose in BL21/pCL PtrcKudzu yIDI. Time 0 is the time of induction
with IPTG (400 .mu.mol). The x-axis is time after induction; the
y-axis is OD.sub.600 and the y2-axis is total productivity of
isoprene (.mu.g/L headspace or specific productivity (.mu.g/L
headspace/OD). Diamonds represent OD.sub.600, circles represent
total isoprene productivity (.mu.g/L) and squares represent
specific productivity of isoprene (.mu.g/L/OD).
[0087] FIG. 23G is a graph showing production of isoprene from
glucose in BL21/pCL PtrcKudzu DXS. Time 0 is the time of induction
with IPTG (400 .mu.mol). The x-axis is time after induction; the
y-axis is OD.sub.600 and the y2-axis is total productivity of
isoprene (.mu.g/L headspace or specific productivity (.mu.g/L
headspace/OD). Diamonds represent OD.sub.600, circles represent
total isoprene productivity (.mu.g/L) and squares represent
specific productivity of isoprene (.mu.g/L/OD).
[0088] FIG. 23H is a graph showing production of isoprene from
glucose in BL21/pTrcKudzuIDIDXSkan. The arrow indicates the time of
induction with IPTG (400 .mu.mol). The x-axis is time after
induction; the y-axis is OD.sub.600 and the y2-axis is total
productivity of isoprene (.mu.g/L headspace or specific
productivity (.mu.g/L headspace/OD). Black diamonds represent
OD.sub.600, black triangles represent isoprene productivity
(.mu.g/L) and white squares represent specific productivity of
isoprene (.mu.g/L/OD).
[0089] FIG. 24 is a map of p9796-poplar.
[0090] FIGS. 25A-25B is a nucleotide sequence of p9796-poplar (SEQ
ID NO:21).
[0091] FIG. 26 is a map of pTrcPoplar.
[0092] FIGS. 27A-27C is a nucleotide sequence of pTrcPoplar (SEQ ID
NO:22).
[0093] FIG. 28 is a map of pTrcKudzu yIDI Kan.
[0094] FIGS. 29A-29C is a nucleotide sequence of pTrcKudzu yIDI Kan
(SEQ ID NO:23).
[0095] FIG. 30 is a map of pTrcKudzuDXS Kan.
[0096] FIGS. 31A-31C is a nucleotide sequence of pTrcKudzuDXS Kan
(SEQ ID NO:24).
[0097] FIG. 32 is a map of pCL PtrcKudzu.
[0098] FIGS. 33A-33C is a nucleotide sequence of pCL PtrcKudzu (SEQ
ID NO:25).
[0099] FIG. 34 is a map of pCL PtrcKudzu A3.
[0100] FIGS. 35A-35C is a nucleotide sequence of pCL PtrcKudzu A3
(SEQ ID NO:26).
[0101] FIG. 36 is a map of pCL PtrcKudzu yIDI.
[0102] FIGS. 37A-37C is a nucleotide sequence of pCL PtrcKudzu yIDI
(SEQ ID NO:27).
[0103] FIG. 38 is a map of pCL PtrcKudzu DXS.
[0104] FIGS. 39A-39D is a nucleotide sequence of pCL PtrcKudzu DXS
(SEQ ID NO:28).
[0105] FIG. 40 shows graphs representing isoprene production from
biomass feedstocks. Panel A shows isoprene production from corn
stover, Panel B shows isoprene production from bagasse, Panel C
shows isoprene production from softwood pulp, Panel D shows
isoprene production from glucose, and Panel E shows isoprene
production from cells with no additional feedstock. Grey squares
represent OD.sub.600 measurements of the cultures at the indicated
times post-inoculation and black triangles represent isoprene
production at the indicated times post-inoculation.
[0106] FIG. 41A shows a graph representing isoprene production by
BL21 (.lamda.DE3) pTrcKudzu yIDI DXS (kan) in a culture with no
glucose added. Squares represent OD.sub.600, and triangles
represent isoprene produced (.mu.g/ml).
[0107] FIG. 41B shows a graph representing isoprene production from
1% glucose feedstock invert sugar by BL21 (.lamda.DE3) pTrcKudzu
yIDI DXS (kan). Squares represent OD.sub.600, and triangles
represent isoprene produced (.mu.g/ml).
[0108] FIG. 41C shows a graph representing isoprene production from
1% invert sugar feedstock by BL21 (.lamda.DE3) pTrcKudzu yIDI DXS
(kan). Squares represent OD.sub.600, and triangles represent
isoprene produced (.mu.g/ml).
[0109] FIG. 41D shows a graph representing isoprene production from
1% AFEX corn stover feedstock by BL21 (.lamda.DE3) pTrcKudzu yIDI
DXS (kan). Squares represent OD.sub.600, and triangles represent
isoprene produced (.mu.g/ml).
[0110] FIG. 42 shows graphs demonstrating the effect of yeast
extract of isoprene production. Panel A shows the time course of
optical density within fermentors fed with varying amounts of yeast
extract. Panel B shows the time course of isoprene titer within
fermentors fed with varying amounts of yeast extract. The titer is
defined as the amount of isoprene produced per liter of
fermentation broth. Panel C shows the effect of yeast extract on
isoprene production in E. coli grown in fed-batch culture.
[0111] FIG. 43 shows graphs demonstrating isoprene production from
a 500 L bioreactor with E. coli cells containing the
pTrcKudzu+yIDI+DXS plasmid. Panel A shows the time course of
optical density within the 500-L bioreactor fed with glucose and
yeast extract. Panel B shows the time course of isoprene titer
within the 500-L bioreactor fed with glucose and yeast extract. The
titer is defined as the amount of isoprene produced per liter of
fermentation broth. Panel C shows the time course of total isoprene
produced from the 500-L bioreactor fed with glucose and yeast
extract.
[0112] FIG. 44 is a map of pBS Kudzu #2.
[0113] FIG. 45A is a graph showing growth during fermentation time
of Bacillus expressing recombinant kudzu isoprene synthase in 14
liter fed batch fermentation. Black diamonds represent a control
strain (BG3594comK) without recombinant isoprene synthase (native
isoprene production) and grey triangles represent Bacillus with
pBSKudzu (recombinant isoprene production).
[0114] FIG. 45B is a graph showing isoprene production during
fermentation time of Bacillus expressing recombinant kudzu isoprene
synthase in 14 liter fed batch fermentation. Black diamonds
represent a control strain (BG3594comK) without recombinant
isoprene synthase (native isoprene production) and grey triangles
represent Bacillus with pBSKudzu (recombinant isoprene
production).
[0115] FIGS. 46A-46D depict the growth rate and specific
productivity of isoprene generation for the empty vector (control),
HgS, and HgS-FldA strains.
[0116] FIG. 46E is a map of pBAD33.
[0117] FIGS. 46F and 46G are the nucleotide sequence of pBAD33 (SEQ
ID NO:51).
[0118] FIG. 46H is a map of pTrcHgS-pBAD33.
[0119] FIGS. 461 and 46J are the nucleotide sequence of
pTrcHgS-pBAD33 (SEQ ID NO:52).
[0120] FIG. 46K is a map of pTrcHgSfldA-pBAD33.
[0121] FIGS. 46L and 46M are the nucleotide sequence of
pTrcHgSfldA-pBAD33 (SEQ ID NO:53).
[0122] FIG. 47 shows the growth and isoprene production of strains
REM19-22 compared to REM23-26. The expression of isoprene synthase
in both sets of strains and the expression of the T. elongatus
genes in the test set of strains was induced with 200 uM IPTG at
time 0 when the cultures were at an OD.sub..lamda.600nm of
approximately 0.2-0.25. The data shown in the figure is that
obtained 4 hours after the addition of IPTG to the cultures. Cells
were grown shaking in the TM3 at 30.degree. C. Comparison of the
parental to test set strains indicates that isoprene production
increases 10%, 20%, 30%, and 80% over the parental strains for the
GI1.0-dxs, GI1.2-dxs, GI1.5-dxs, and GI-1.6-dxs test strains,
respectively.
[0123] FIG. 48 shows the increased levels of the GcpE product,
HDMAPP, accumulate in strains REM23-2. The concentrations of DXP
metabolites and larger isoprenoid molecules were determined for
REM19-26 (strain indicated on the x-axis) at a 5 hour
IPTG-induction period. The DXP metabolites and isoprenoids measured
are indicated in the figure legend; DXP, 1-deoxy-D-xylulose
5-phosphate; MEP, 2-C-methyl-D-erythritol 4-phosphate; cMEPP,
2-C-methyl-D-erythritol-2,4-cyclodiphosphate; HDMAPP,
(E)-4-hydroxy-3-methylbut-2-enyl diphosphate; DMAPP, dimethylallyl
diphosphate; IPP, isopentenyl diphosphate; FPP, farnesyl
pyrophosphate.
[0124] FIG. 49 shows specific productivity of isoprene production
in strain REM29 compared to REMH86.
[0125] FIG. 50 depicts a cartoon representation of the strategy
used to insert the GI 1.X-promoter series in front of dxs using the
RED/ET system. REM29 (blue) and REMH86 (yellow) were assayed for
growth rate (strains grew comparably) and isoprene production every
30 minutes across a 3 hour shake flask fermentation. At time 0 both
cultures were induced with 400 uM IPTG. Over the course of the
fermentation beginning at the first time point after induction, the
test strain produced approximately 16% higher isoprene levels than
the parental strain.
[0126] FIG. 51 is a map of T7-MEARR alba/pBBR1MCS-5.
[0127] FIG. 52 is a map of the Ptac-gcpE-petF-petH/pK184 construct
that was used to generate strains REM23-26.
[0128] FIGS. 53A-53B shows a cartoon representation of the T7-(-3)
alba/pBBR1MCS-5 (top) and T7-MTE alba/pBBR1MCS-5 (bottom)
constructs that were used to generate strains REMH76 and
REMH86.
[0129] FIG. 54 is a map of the Ptac-gcpE-lytB-petF-petH/pK184
construct that was used to generate strains REM31 and REM29.
[0130] FIGS. 55A-55C are the nucleotide sequence of T7-MEARR
alba/pBBR1MCS-5 (SEQ ID NO:73).
[0131] FIGS. 56A-56B are the nucleotide sequence of
Ptac-gcpE-petF-petH/pK184 (SEQ ID NO:74).
[0132] FIGS. 57A-57C are the nucleotide sequence of
T7-(-3.sub.--alba/pBBR1MCS-5 (SEQ ID NO:75).
[0133] FIGS. 58A-58C are the nucleotide sequence of T7-MTE
alba/pBBR1MCS-5 (SEQ ID NO:76).
[0134] FIGS. 59A-59B are the nucleotide sequence of
Ptab-gcpE-LytB-petF-petH/pK184 (SEQ ID NO:77).
[0135] FIG. 60 shows that .DELTA.iscR BL21(DE3) supports increased
isoprene production. Panel 60A shows the specific productivity of
REM12 compared to the otherwise isogenic .DELTA.iscR strain REM13.
Isoprene levels were determined 4.5 hours and 8 hours after
induction of the IPTG-inducible isoprene synthase and DXP enzymes
harbored by the strains. Data from three groups (A-C) of three
biological replicates for each strain are shown. Error bars depict
the standard deviation occurring between the biological replicates
of each group. From this data it was determined that isoprene
levels generated from the .DELTA.iscR strain were an average of 40%
and 73% higher than that produced by the wild-type strain at the
4.5 hour and 8 hour time point, respectively. Panel 60B shows the
growth rate of REM12 and REM13 isoprene-producing strains. The
growth rate of the same strains depicted in panel A was monitored
over the course of the eight hour experiment by periodically
measuring the optical density of the cultures at 600 nm. Time 0
corresponds to the time that 50 uM IPTG was added to the cultures.
Cells were grown shaking in TM3 at 30.degree. C. The higher
isoprene-producing strain .DELTA.iscR (REM13) grows at a reduced
rate relative to the lower isoprene-producing wild-type (REM12)
strain.
[0136] FIG. 61 is a cartoon representation of the strategy used to
delete the iscR locus using the RED/ET system.
[0137] FIG. 62 is a cartoon representation of the T7-MEARR
alba/pBBR1MCS-5.
[0138] FIG. 63 is a cartoon representation of the DXP operon
pET24a.
[0139] FIGS. 64A-64C are the nucleotide sequence of T7-MEARR
alba/pBBR1MCS-5 (SEQ ID NO:78).
[0140] FIGS. 65A-65D are the nucleotide sequence of DXP operon
pETt24a (SEQ ID NO:79).
[0141] FIG. 66 is a cartoon representation of the strategy used to
delete ispG and ispH using the RED/ET system.
[0142] FIG. 67 is a cartoon representation of the GI
1.6-gcpE-lytB-yidi/pCR-Blunt II-TOPO construct that was used to
generate strain MD09-219/GI1.6-gcpE-lytB-yidi/pCRII-TOPO (Kan).
[0143] FIG. 68 depicts the Pentose Phosphate (PPP) and
Entner-Doudoroff (ED) pathways (Fraenkel, J. Bact. 95:1267-1271
(1965), which is hereby incorporated by reference in its
entirety).
[0144] FIG. 69 is a map of pDu-39.
[0145] FIG. 70 is a map of pMCM596 pET24(MEA)alba-dxs-yIDI.
[0146] FIGS. 71A-71B are the nucleotide sequence of pDu-39(SEQ ID
NO:108).
[0147] FIGS. 72A-72C are the nucleotide sequence of MCM596 (SEQ ID
NO:109).
[0148] FIGS. 73A-C are the nucleotide sequence of pMCM596 (SEQ ID
NO:110).
[0149] FIG. 74 shows comparison of DXS sequences in microorganisms
synthesizing isoprenoids via the DXP pathway (E. coli, Chlorobium
tepidum TLS, Synechocystis sp. PCC6803, Gloeobacter violaceus PCC
7421, Clostridium botulinum B1 str. Okra, Mycobacterium
tuberculosis CDC1551) and via the MVA pathway (Myxococcus xanthus
DK 1622, Gramella forsetii KT0803, Flavobacterium johnsoniae UW101,
Lactobacillus johnsonii NCC 533, Lactobacillus gasseri ATCC 33323,
and Lactococcus lactis subsp. lactis 111403). Note the difference
in amino acid sequence at positions 200-260 in the two groups of
microorganisms.
[0150] FIGS. 75A and 75B are the nucleotide sequence of pDU-9 (SEQ
ID NO: 192).
[0151] FIG. 76 is a map of pDu9-pET-16b rev-yIDI.
[0152] FIG. 77 depicts GB-CMP-GI1.X-yidi construct design. The
final construct consists of Fragment A (Frag A) fused to Fragment B
(Frag B) to create a GI1.X promoter library transcribing yIDI with
the chloramphenicol antibiotic resistance marker upstream, and
flanking 50 bp regions of homology to the desired integration site
on the chromosome.
[0153] FIG. 78 depicts a plasmid map of pDW33. pBR322--plasmid
origin of replication; lacIq--lac repressor; Ptrc--the trc
promoter; lac operator--lac repressor binding site; P. alba IspS
(MEA)--gene encoding the isoprene synthase; rrn
terminator--transcription terminator; bla--beta lactamase gene.
[0154] FIG. 79 (includes five panels: FIGS. 79A, 79B, 79C, 79D, and
79E) shows the results of 15-L scale fermentation comparison of
strains CMP272, REMG39, and REM H8.sub.--12 for growth, isoprene
production, and product yield on carbon. Panel (A) isoprene titer
(g/L broth); Panel (B) specific productivity of isoprene generating
cultures; Panel (C) cell growth depicted by optical density (550
nm); Panel (D) cell growth shown by respiration (carbon evolution
rate, CER); Panel (E) overall percent yield of product from carbon
(weight in grams of isoprene/weight in grams of carbon fed*100).
The fermentation conditions are described in Example 24 Section F
(CMP272), G (REMG39), and Example 29 Section E (REM
H8.sub.--12).
[0155] FIG. 80 (includes three panels, 80A, 80B, and 80C) shows the
results of large scale fermentation comparison of strains CMP272,
REMG39, and REM H8.sub.--12 for DXP metabolites. Panels (A-C) The
same cells described in FIG. 79 are presented here. A legend
describing the metabolite profiles is shown at the bottom of each
panel. DXP, 1-Deoxy-D-xylulose 5-phosphate; MEP,
2-C-Methyl-D-erythritol 4-phosphate; CDP-ME, 4-(Cytidine
5'-diphospho)-2-C-methyl-D-erythritol; CDP-MEP,
2-Phospho-4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol; cMEPP,
2-C-methyl-D-erythritol-2,4-cyclodiphosphate; HDMAPP,
1-Hydroxy-2methyl-2-buten-4-yl 4-diphosphate; DMAPP, Dimethylallyl
diphosphate; IPP, Isopentenyl diphosphate; FPP, faresyl
pyrophosphate.
[0156] FIG. 81 (includes two panels: FIGS. 81A and 81B) depicts one
strategy for inserting GI1.X fldA into the BL21(DE3) chromosome.
Panel (A) The endogenous 150 bp BL21(DE3) fldA locus is shown. The
regions of homology within the GI1.X fldA PCR fragment to the
desired 5' and 3' integration sites on the chromosome are depicted
as gray block arrows. The half-arrowhead lines show where the PCR
primers used to verify the construct anneal to the chromosome. The
ribosome binding site (RBS), start codon of the encoded fldA mRNA,
and the endogenous DNA upstream of the fldA to be replaced by the
GI1.X proter series is shown. Panel (B) The 313 bp BL21(DE3) GI1.X
fldA region generated via Gene Bridges methods (GI1.6fldA of strain
REM I6.sub.--4) is shown. The inserted GI1.X promoter sequence(s)
is illustrated as a black block arrow; the placement of the FTR
scar sequences generated from use of the Gene Bridges insertion
method is indicated.
[0157] FIG. 82 depicts a plasmid map of GI1.6fldA/pCL.
repA--plasmid replication protein; aad--aminoglycoside
adenyltransferase; M13 for and M13 rev--binding sites for the
respective primers; RBS--ribosome binding site; fldA--E. coli fldA
gene.
[0158] FIG. 83 depicts a plasmid map of GI1.6fldA-IspG/pCL. Same
plasmid base as in FIG. 82: FldA--E. coli fldA gene; IspG--E. coli
ispG gene.
[0159] FIG. 84 depicts a plasmid map of GI1.6IspG/pCL. Same plasmid
base as FIGS. 82 and 83: IspG--E. coli ispG gene.
[0160] FIG. 85 (includes two panels, FIGS. 85A and 85B) depicts
small scale comparison of strains, REMC9.sub.--12, REME7.sub.--12,
and REMD6.sub.--12. Panel (A) Specific productivity (SP) of
isoprene production relative to growth. The y1 axis, specific
productivity of isoprene production (ug/L/OD/hr); y2 axis, cell
density (OD.sub.600). Specific productivity (solid bars) and
OD.sub.600 (diamonds). Measurements were taken at 3 and 4.5 h
post-induction (600 uM IPTG) from at least 2 biological replicates.
Panel (B) Intracellular metabolite concentrations. cMEPP:
2-C-methyl-D-erythritol 2,4-cyclodiphosphate;
HDMAPP--hydroxydimethylallyl diphosphate;
DMAPP--dimethylallyldiphosphate; IPP--isopentenyl diphosphate.
Y-axis: metabolite concentration in mM. Measurements shown were
taken at 3.75 h post-induction (600 uM IPTG); separate experimental
samples from (A); replicates produced similar results.
[0161] FIG. 86 (includes two panels: FIGS. 86A and 86B) shows the
results of small scale comparisons of strains REMG2.sub.--11,
REMG4.sub.--11 and REMG39. Panel (A) Specific productivity of
isoprene production relative to growth of. The y1 axis, specific
productivity of isoprene production (ug/L/OD/hr); y2 axis, cell
density (OD.sub.600). Specific productivity (solid bars) and OD600
(diamonds). Measurements are shown at 1 and 3.5 h post-induction
(400 uM IPTG) from at least 2 biological replicates. Panel (B)
Intracellular metabolite concentrations of strains. The y-axis is
metabolite concentration in mM. cMEPP: 2-C-methyl-D-erythritol
2,4-cyclodiphosphate; HDMAPP-hydroxydimethylallyl diphosphate.
Measurements are shown for the 3.5 h post-induction (400 uM IPTG)
samples from (A); replicates produced similar results (rows 1-3:
REM G2.sub.--11; rows 4-6: REM G4.sub.--11; rows 7-9: REMG39).
[0162] FIG. 87 depicts a plasmid map of pEWL454. The plasmid base
is pK184. p15A ori--plasmid origin of replication; RBS--ribosome
binding site; kan--kanamycin antibiotic resistance marker.
[0163] FIG. 88 depicts a plasmid map of PtacAnabaenaAspA
terminator/pEWL454. This is the same plasmid base as in FIG. 87.
Anabaena IspH--gene encoding the IspH enzyme from Anabaena.
[0164] FIG. 89 depicts the specific productivity of isoprene
production and intracellular metabolites of strains REMI7.sub.--11,
and REMH8.sub.--12. The two strains were compared at 3 and 3.75 h
following induction (500 uM IPTG). Isoprene measurements are shown
from at least 2 biological replicates; replicates are not shown for
the metabolite data, but produced similar results.
[0165] FIG. 90 (includes three panels: FIGS. 90A, 90B and 90C)
depicts the results from a 15-L scale fermentation of strain REM
H8.sub.--12 and REM G4.sub.--11 (A). Panel (A) isoprene titer (g/L
broth) for REMH 8.sub.--12 (open squares) and REM G4.sub.--11 (open
circles); Panel (B) cell growth depicted by optical density (550
nm); Panel (C) DXP metabolites. A legend describing the metabolite
profiles is shown at the bottom of (C); see FIG. 80 for metabolite
descriptions.
[0166] FIG. 91 depicts results from a preparative scale
inactivation of Dxr by DMAPP.
[0167] FIG. 92 (includes two panels: 92A and 92B) depicts isoprene
production by strains REM H8.sub.--12 and REM I7.sub.--11 harboring
an engineered DXP pathway and a lower MVA pathways. The top panel
shows isoprene production specifically due to MVA fed at indicated
concentrations to cultures grown on [U-.sup.13C]-glucose. The lower
panel shows isoprene production specifically arising from
[U-.sup.13C]-glucose]. Isoprene measurements were taken at
indicated times after induction of the cultures with IPTG. Isoprene
evolved was monitored by GC-MS with detection at m/z=67 as well as
m/z=73. While m/z=67 reports on isoprene from MVA (all .sup.12C),
m/z=73 reports on isoprene derived from [U-.sup.13C]-glucose.
[0168] FIG. 93 depicts a plasmid map of pDW15. mob--plasmid
mobilization region; AacC1 (Gent Resistance)--aminoglycoside
acetyltransferase, gentamicin resistance gene; M13 Reverse and M13
Forward--binding sites for the respective primers; Ptrc, Trc
promoter; mvaE and mvaS--E. faecalis genes encoding the
Acetoacetyl-Coenzyme A Thiolase/3-Hydroxy-3-Methylglutaryl-Coenzyme
A Reductase and 3-Hydroxy-3-Methylglutaryl-Coenzyme A Synthase,
respectively; RepA--plasmid replication protein.
[0169] FIG. 94 depicts a plasmid map of PTrp mMVK/pDW15. Same
plasmid base as in 1). Trp promoter; encoded M. mazei MVK--M. mazei
gene encoding Mevalonate Kinase; aspA terminator.
[0170] FIG. 95 depicts a plasmid map of pMCM900. FRT--Flip
recombinase target site; core Trc promoter--RNA polymerase binding
site; lac operator--Lad binding site; PMK orf--yeast
phosphomevalonate kinase coding sequence; MVD orf--yeast
diphosphomevalonate decarboxylase coding sequence; yIDI--yeast
isopentenyl diphosphate isomerase coding sequence; aspA
terminator--aspA transcriptional terminator; attTn7
downstream--glmS--downstream recombination targetting sequence;
KanR--kanamycin resistance gene; R6K on--plasmid origin of
replication; attTn7 upstream (pstS)--upstream recombination
targetting sequence.
[0171] FIG. 96 depicts the results for experiments for determining
the specific productivity relative to culture density in the
presence and absence of fosmidomycin for strain REM A2.sub.--17
grown on unlabeled glucose. The y1 axis, specific productivity of
isoprene production (ug/L OD hr); y2 axis, cell density
(OD.sub.600nm). Specific productivity (solid bars) and Cell density
(diamonds). Measurements were taken approx. 45 minutes
post-introduction of either 0 mM or 2 mM fosmidomycin; both
occurring approx. 3 hours after induction with 400 uM IPTG. The
data presented is the average of 3 biological; error bars are shown
for specific productivity and cell density values. The data
suggests a contribution of roughly 59% and 41% for isoprene
generated via the MVA pathway and DXP pathway, respectively; MVA
flux was determined by the fraction of isoprene produced during
exposure to fosmidomycin relative to the amount of isoprene
produced in the absence of the inhibitor.
[0172] FIG. 97 depicts the results for experiments for determining
the effect of fosmidomycin on accumulation of the DXP and MVA
pathway metabolites and isoprene emission rate in REM A2.sub.--17
strain. The metabolite concentrations in pelleted cells (same cells
depicted in FIG. 96) and isoprene emission rates were measured in
the cultures at the end of a 45 min. incubation in the presence and
in the absence of 2 mM fosmidomycin ("+FM" and "-FM",
respectively). The results are expressed as an average ratio of the
obtained concentrations and rates measured in three different
cultures.
[0173] FIG. 98 depicts the results for experiments for determining
the specific productivity relative to culture density in the
presence and absence of fosmidomycin for strain REM A2.sub.--17
grown on unlabeled and 1-.sup.13C labeled glucose. The y1 axis,
specific productivity of isoprene production (ug/L OD hr); y2 axis,
cell density (OD.sub.600nm). Specific productivity (solid bars) and
Cell density (diamonds). In lane 1: unlabeled culture without
tryptophan and without fosmidomycin; lane 2: 1-.sup.13C glucose
culture without tryptophan and without fosmidomycin; lane 3:
1-.sup.13C glucose culture with 50 uM tryptophan and without
fosmidomycin; lane 4: unlabeled culture without tryptophan and with
2 mM fosmidomycin; lane5: 1-.sup.13C glucose culture with 50 uM
tryptophan and with 2 mM fosmidomycin; lane 6: 1-.sup.13C glucose
culture with 50 uM tryptophan and with 2 mM fosmidomycin.
Measurements were taken approx. 45 minutes post-introduction of
either 0 mM or 2 mM fosmidomycin; both occurring approx. 3 hours
after induction with 400 uM IPTG. The data presented is the average
of 2 technical replicates; error bars are shown for specific
productivity values. The data suggests a contribution of roughly
52% and 48% for isoprene generated via the MVA pathway and DXP
pathway, respectively for the unlabeled culture. Similarly, the
data shows a 57% MVA-flux to 43% DXP-flux and 49% MVA-flux to 51%
DXP-flux contribution to the isoprene generated by the
1-.sup.13Cglucose culture without and with 50 uM tryptophan,
respectively. The repressed expression of the MVK enzyme mediated
by the presence of tryptophan in the growth media for cultures
represented by lanes 3 and 6 was reflected in the data as a 24% to
34% decrease in overall-flux compared to the cultures grown without
the addition of tryptophan to the growth media. MVA flux was
determined by the fraction of isoprene produced during exposure to
fosmidomycin relative to the amount of isoprene produced in the
absence of the inhibitor for each particular culture type.
[0174] FIG. 99 depicts the results for experiments for determining
the specific productivity relative to culture density in the
presence and absence of fosmidomycin for strain REM A2.sub.--17
grown on 3-.sup.13C glucose. The y1 axis, specific productivity of
isoprene production (ug/L OD hr); y2 axis, cell density
(OD.sub.600.). Specific productivity (solid bars) and Cell density
(diamonds). Measurements were taken approx. 1 hour
post-introduction of either 0 mM or 2 mM fosmidomycin; both
occurring approx. 3 hours after induction with 400 uM IPTG. The
data presented is the average of 2 technical replicates; error bars
are shown for specific productivity values. The data suggests a
contribution of roughly 58% and 42% for isoprene generated via the
MVA pathway and DXP pathway, respectively; MVA flux was determined
by the fraction of isoprene produced during exposure to
fosmidomycin relative to the amount of isoprene produced in the
absence of the inhibitor.
[0175] FIG. 100 (panels A and B) depicts the DXP and MVA
pathway-specific labeling pattern of isoprene resulting from: A)
1-.sup.13C glucose and B) 3-.sup.13C glucose catabolism via
glycolysis. Black circles indicate 100% abundance of .sup.13C atoms
at specified positions. Half-black circles indicate .sup.13C
abundance of 50% with the rest 50% being .sup.12C atoms coming from
the positions in glucose shown by open circles.
[0176] FIG. 101 (panels A and B) depicts the calculated
distributions of isoprene and cMEPP cumomers in REM A2.sub.--17
strain grown on: A) 1-.sup.13C glucose or B) 3-.sup.13C glucose in
the presence or in the absence of fosmidomycin (+FM and -FM,
respectively).
[0177] FIG. 102 (panels A and B) depicts the GC-MS spectra of: A)
unlabeled (synthetic) isoprene standard having natural abundance of
.sup.13C and B) isoprene produced by the REM A2.sub.--17 strain
grown on 3-.sup.13C glucose. Note that intensities of m/z 68, 69
and 70 peaks relative to the m/z 67 peak are higher in the REM
A2.sub.--17 strain compared to the isoprene standard because of
.sup.13C enrichment.
[0178] FIG. 103 depicts results of isoprene .sup.13C isotope
enrichment as a function of MVA/DXP pathway ratio.
[0179] FIG. 104 depicts an exemplary apparatus for generation,
collection and analysis of Bioisoprene.TM. product.
[0180] FIG. 105 depicts results showing the .sup.13C NMR spectrum
of natural .sup.13C-abundance isoprene.
[0181] FIG. 106 depicts results showing the .sup.13C NMR spectrum
of isoprene derived from a MVA/DXP dual pathway strain. Both C-4
and C-1/C-3 are .sup.13C-enriched relative to C-2, with a signal
intensity equal or less than the noise level demonstrates the
contribution of the both the MVA and DXP pathways to isoprene
synthesis in this strain.
[0182] FIG. 107 depicts a diagram for a portion of the PL.6 fkpB
locus. The nucleotide sequence of the region depicted in the figure
is indicated by the 323 bases listed below the diagram. The 5' and
3' regions of homology used to integrate the PL.6 promoter upstream
of fkpB are shown in gray. The sequence highlighted in black bold
text represents the exogenous sequence left in the region after
loopout of the Gene Bridges chloramphenicol resistance cassette,
referred to in the figure as the Gene Bridges scar, with the
remaining FRT (Flipase recognition target) site underlined. The
PL.6 promoter sequence is shown in regular black text. The -35,
-10, and RBS (ribosome binding site) positions are indicated in the
figure.
[0183] FIG. 108 (panels A, B, C, D) depicts a comparison of the
isoprene productivity of 4 strains. Panel A, typical isoprene
productivity of strain WW119 (parent to strains in panel B and C)
at two time points at 200 uM IPTG. This experiment was performed as
is described in the text for strains in panels B and C, except that
isoprene monitoring was limited to 2 and 4 hours. OD.sub.600 was
monitored throughout culture period for all strains at hourly
intervals. Panel B shows isoprene specific productivity for strains
REM 6.sub.--15 (PL.6 fkpB-ispH .DELTA.iscR) at several IPTG
concentrations. Panel C shows isoprene specific productivity for
strain REM D8.sub.--15 (PL.6 fkpB-ispH) at several IPTG
concentrations. The data is consistent with that .DELTA.iscR
rescues isoprene productivity lost upon introduction of
PL.6fkpB-ispH. Panel D shows isoprene specific productivity for
strain REM D7.sub.--15 at several IPTG concentrations.
[0184] FIG. 109 shows an image of E. coli ispH western blot. Lane
description is as follows: Lane 1, SeeBlue.RTM. Plus2 Pre-Stained
Standard, Invitrogen, Lane 2, E. coli ispH purified standard (0.4
.mu.g), Lane 3, REM A7.sub.--15 soluble fraction, Lane 4, REM
A7.sub.--15 insoluble fraction, Lane 5, REM A8.sub.--15 soluble
fraction, Lane 6, REM A8.sub.--15 insoluble fraction, Lane 6, REM
D1.sub.--14 soluble fraction, Lane 7, REM D1.sub.--14 insoluble
fraction, Lane 8, WW103 soluble fraction and, Lane 10, WW103
insoluble fraction. Development method: 1.degree. Ab Anti-Rabbit E.
coli ispH at 1:10,000 dilution, 2.degree. Ab Alexa Fluor.RTM. 488
goat anti-rabbit IgG (H+L), Invitrogen, 1:1,000 dilution; see text
for additional details. Gel was a Novagen 4 to 12% BT gel. Loading
was normalized to equal OD.sub.600. Pel, pellet; sup,
supernatant.
[0185] FIG. 110 shows E. coli ispH western blot quantitation.
Quantitation of the western data was by ImageQuant 5.2 (Molecular
Dynamics). Light shaded bars represent amount of ispH found in the
soluble fraction. Dark shaded bars represent amount of ispH found
in the insoluble fraction (SEQ ID NO: 193).
DETAILED DESCRIPTION OF THE INVENTION
[0186] The invention provides, inter alia, compositions and methods
for the production of isoprene in increased amounts using various
DXP pathway genes and polypeptides, various MVA pathway genes and
polypeptides, iron-sulfur cluster-interacting redox genes and
polypeptides, isoprene synthase genes and polypeptides, and
optionally, IDI genes and polypeptides and various genes and
polypeptides associated with the DXP pathway and/or MVA
pathway.
[0187] Unless defined otherwise, the meanings of all technical and
scientific terms used herein are those commonly understood by one
of skill in the art to which this invention belongs. Singleton, et
al., Dictionary of Microbiology and Molecular Biology, 2nd ed.,
John Wiley and Sons, New York (1994), and Hale & Marham, The
Harper Collins Dictionary of Biology, Harper Perennial, N.Y. (1991)
provide one of skill with a general dictionary of many of the terms
used in this invention. It is to be understood that this invention
is not limited to the particular methodology, protocols, and
reagents described, as these may vary. One of skill in the art will
also appreciate that any methods and materials similar or
equivalent to those described herein can also be used to practice
or test the invention. The headings provided herein are not
limitations of the various aspects or embodiments of the invention
which can be had by reference to the specification as a whole.
[0188] As used herein, the term "isoprene" or
"2-methyl-1,3-butadiene" (CAS#78-79-5) refers to the direct and
final volatile C5 hydrocarbon product from the elimination of
pyrophosphate from 3,3-dimethylallyl pyrophosphate (DMAPP), and
does not involve the linking or polymerization of one or more
isopentenyl diphosphate (IPP) molecules to one or more DMAPP
molecules. The term "isoprene" is not generally intended to be
limited to its method of production.
[0189] As used herein, the phrase, "various genes and polypeptides
associated with the DXP pathway," or "DXP pathway associated
nucleic acid(s) or polypeptide(s)" refers to any nucleic acid or
polypeptide that interacts with DXP pathway polypeptides or nucleic
acids, including, but not limited to, a terpene synthase (e.g.,
ocimene synthase, farnesene synthase, and artemesinin synthase),
either directly or indirectly.
[0190] For use herein, unless clearly indicated otherwise, use of
the terms "a", "an," and the like refers to one or more.
[0191] Reference to "about" a value or parameter herein includes
(and describes) embodiments that are directed to that value or
parameter per se. For example, description referring to "about X"
includes description of "X." Numeric ranges are inclusive of the
numbers defining the range.
[0192] It is understood that aspects and embodiments of the
invention described herein include "comprising," "consisting," and
"consisting essentially of" aspects and embodiments.
[0193] The present invention is based in part on the surprising
discovery that an increased amount of an iron-sulfur
cluster-interacting redox polypeptide increases the activity
demonstrated by the DXP pathway polypeptides (such as HDS (GcpE or
IspG) or HDR polypeptide (IspH or LytB). While not intending to be
bound to a particular theory, it is believed that the increased
expression of one or more endogenous or heterologous iron-sulfur
interacting redox nucleic acids or polypeptides improve the rate of
formation and the amount of DXP pathway polypeptides containing an
iron sulfur cluster (such as HDS or HDR), and/or stabilize DXP
pathway polypeptides containing an iron sulfur cluster (such as HDS
or HDR). This in turn increases the carbon flux to isoprene
synthesis in cells by increasing the synthesis of HMBPP and/or
DMAPP and decreasing the cMEPP and HMBPP pools in the DXP pathway.
For example, overexpression of an iron-sulfur cluster-interacting
redox polypeptide (flavodoxin I) in cells overexpressing a DXP
pathway polypeptide (DXS), isoprene synthase polypeptide, and IDI
polypeptide resulted in increased production of isoprene by about
1- to 2-fold in comparison to cells overexpressing DXP pathway
polypeptide, isoprene synthase polypeptide, and IDI polypeptide
only. See Example 8. Overexpression of one or more iron-sulfur
cluster-interacting redox polypeptide (ferredoxin and
ferredoxin-NADP+ oxidoreductase), one or more DXP pathway
polypeptide, isoprene synthase polypeptide, and IDI polypeptide
resulted in increased production of isoprene. See Example 9.
[0194] Accordingly, in one aspect of the invention, cells in
culture comprise (i) a heterologous nucleic acid encoding an
iron-sulfur cluster-interacting redox polypeptide, a heterologous
nucleic acid encoding DXP pathway polypeptide, and an heterologous
nucleic acid encoding isoprene synthase and/or (ii) a duplicate
copies of endogenous nucleic acids encoding an iron-sulfur
cluster-interacting redox polypeptide, a DXP pathway polypeptide,
and an isoprene synthase polypeptide. In some embodiments, the
cells in culture comprise (i) one or more copies of heterologous or
endogenous nucleic acid encoding an iron-sulfur cluster-interacting
redox polypeptide, (ii) one or more copies of heterologous or
endogenous nucleic acid encoding a DXP pathway polypeptide, and
(iii) one or more copies of heterologous or endogenous nucleic acid
encoding an isoprene synthase polypeptide.
[0195] In another aspect of the invention, provided are methods of
producing isoprene. In one embodiments, the method comprises (a)
culturing cells comprising (i) a heterologous nucleic acid encoding
a heterologous nucleic acid encoding an iron-sulfur
cluster-interacting redox polypeptide, a DXP pathway polypeptide,
and an isoprene synthase polypeptide and/or (ii) a duplicate copy
of an endogenous nucleic acid encoding an iron-sulfur
cluster-interacting redox polypeptide, a DXP pathway polypeptide,
and an isoprene synthase polypeptide under suitable culture
conditions for the production of isoprene, and (b) producing
isoprene.
[0196] As used herein, iron-sulfur cluster-interacting redox
polypeptide is a polypeptide that is capable of transferring
electrons to a polypeptide containing an iron-sulfur cluster. An
iron-sulfur cluster-interacting redox polypeptide includes, but is
not limited to, flavodoxin (e.g., flavodoxin I), flavodoxin
reductase, ferredoxin (e.g., ferredoxin I), ferredoxin-NADP+
oxidoreductase, and genes or polypeptides encoding thereof (e.g.,
fpr or fldA). For example, DXP pathway polypeptide HDS (GcpE) is a
metallo-enzyme possessing a [4Fe-4S].sup.2+ center and catalyzes
the reduction of cMEPP into HMBPP via two successive one-electron
transfers mediated by the reduction of [4Fe-4S].sup.2+ center in
the presence of flavodoxin/flavodoxin reductase (see, Wolff et al.,
FEBS Letters, 541:115-120 (2003)), which is hereby incorporated by
reference in its entirety). Similarly, DXP pathway polypeptide HDR
(LytB) is also a Fe/S protein catalyzing the reduction of HMBPP
into IPP or DMAPP via two successive one-electron transfers in the
presence of flavodoxin/flavodoxin reductase/NADPH system. See, for
example, Seemann, M. et al. Agnew. Chem. Int. Ed., 41: 4337-4339
(2002); Wolff, M. et al., FEBS Letters, 541: 115-120 (2003), which
are each hereby incorporated by reference in their entirety,
particularly with respect to the description of GcpE, LytB, and
flavodoxin/flavodoxin reductase/NADPH system).
[0197] As used herein, flavodoxin is a protein that is capable of
transferring electrons and contains the prosthetic group flavin
mononucleotide. In Escherichia coli (E. coli), flavodoxin is
encoded by the fldA gene and reduced by the FAD-containing protein
NADPH:ferredoxin oxidoreductase, and plays an essential role in the
DXP pathway for isoprenoid biosynthesis (see, example, Kia-Joo, P.
et al. FEBS Letters, 579: 3802-3806, 2005, which is hereby
incorporated by reference in its entirety).
[0198] As used herein, ferredoxin is a protein that is capable of
transferring electron and contains iron and labile sulfur in equal
amounts and plays an essential role in the DXP pathway for
isoprenoid biosynthesis. For example, HDS from plants and
cyanobacteria have been shown to be ferredoxin, rather than
flavodoxin-dependent, enzymes (Seemann et al., FEBS Lett.,
580(6):1547-52 (2006), which is hereby incorporated by reference in
its entirety).
[0199] As used herein, Fpr encodes flavodoxin/ferredoxin
NADPH-oxidoreductase and provides the necessary electron derived
from NADPH via FldA for HDS and HDR to perform their catalytic
functions (reviewed in report by L. A. Furgerson, The
Mevalonate-Independent Pathway to Isoprenoid Compounds: Discovery,
Elucidation, and Reaction Mechanisms, published Feb. 13, 2006,
which is hereby incorporated by reference in its entirety).
[0200] As used herein, the encoded DXS, DXR, MCT, CMK, MCS, HDS,
and HDR polypeptides are part of the DXP pathway for the
biosynthesis of isoprene (FIG. 19A).
[0201] DXS polypeptides convert pyruvate and
D-glyceraldehyde-3-phosphate into 1-deoxy-D-xylulose-5-phosphate
(DXP). While not intending to be bound by any particular theory, it
is believed that increasing the amount of DXS polypeptide increases
the flow of carbon through the DXP pathway, leading to greater
isoprene production.
[0202] DXR polypeptides convert 1-deoxy-D-xylulose 5-phosphate
(DXP) into 2-C-methyl-D-erythritol 4-phosphate (MEP). While not
intending to be bound by any particular theory, it is believed that
increasing the amount of DXS polypeptide increases the flow of
carbon through the DXP pathway, leading to greater isoprene
production.
[0203] MCT polypeptides convert 2-C-methyl-D-erythritol 4-phosphate
(MEP) into 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol
(CDP-Me). While not intending to be bound by any particular theory,
it is believed that increasing the amount of MCT polypeptide
increases the flow of carbon through the DXP pathway, leading to
greater isoprene production.
[0204] CMK polypeptides convert 4-(cytidine
5'-diphospho)-2-C-methyl-D-erythritol (CDP-ME) into
2-phospho-4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol
(CDP-MEP). While not intending to be bound by any particular
theory, it is believed that increasing the amount of CMK
polypeptide increases the flow of carbon through the DXP pathway,
leading to greater isoprene production.
[0205] MCS polypeptides convert 2-phospho-4-(cytidine
5'-diphospho)-2-C-methyl-D-erythritol (CDP-MEP) into
2-C-methyl-D-erythritol 2,4-cyclodiphoshphate (ME-CPP or cMEPP).
While not intending to be bound by any particular theory, it is
believed that increasing the amount of MCS polypeptide increases
the flow of carbon through the DXP pathway, leading to greater
isoprene production.
[0206] HDS polypeptides convert 2-C-methyl-D-erythritol
2,4-cyclodiphoshphate (ME-CPP or cMEPP) into
(E)-4-hydroxy-3-methylbut-2-en-1-yl-diphosphate (HMBPP or HDMAPP).
While not intending to be bound by any particular theory, it is
believed that increasing the amount of HDS polypeptide increases
the flow of carbon through the DXP pathway, leading to greater
isoprene production.
[0207] HDR polypeptides convert
(E)-4-hydroxy-3-methylbut-2-en-1-yl-diphosphate (HMBPP) into
isopentenyl diphosphate (IPP) and dimethylallyl diphosphate
(DMAPP). While not intending to be bound by any particular theory,
it is believed that increasing the amount of HDR polypeptide
increases the flow of carbon through the DXP pathway, leading to
greater isoprene production.
[0208] Isoprene synthase polypeptides convert dimethylallyl
diphosphate (DMAPP) into isoprene.
[0209] Heterologous iron-sulfur cluster-interacting redox
polypeptide, DXP pathway polypeptide, and isoprene synthase
polypeptide can be expressed in a variety of host cells, such as
Escherichia coli (E. coli), Panteoa citrea, Bacillus subtilis,
Yarrowia lipolytica, and Trichoderma reesei. All of these cells
produced more isoprene than the naturally occurring DXP pathway
alone.
[0210] As discussed further below, isoprene production by cells can
be enhanced by increasing the amount of expression of an
iron-sulfur cluster-interacting redox polypeptide, a DXP pathway
polypeptide, and an isoprene synthase polypeptide. The DXP pathway
polypeptides include DXS, DXR, MCT, CMK, MCS, HDS, and HDR. For
example, one or more DXP pathway nucleic acids can be introduced
into the cells, which includes DXS, DXR, MCT, CMK, MCS, HDS, and
HDR. The DXS, DXR, MCT, CMK, MCS, HDS, or HDR nucleic acid may be a
heterologous nucleic acid or a duplicate copy of an endogenous
nucleic acid. Similarly, the iron-sulfur cluster-interacting redox
nucleic acid may be a heterologous nucleic acid or duplicate copy
of an endogenous nucleic acid. Similarly, the isoprene synthase
nucleic acid may be a heterologous nucleic acid or a duplicate copy
of an endogenous nucleic acid. In some embodiments, the amount of
one or more iron-sulfur cluster-interacting redox polypeptide, one
or more of DXS, DXR, MCT, CMK, MCS, HDS, or HDR polypeptide, and
isoprene synthase polypeptide are increased by replacing one or
more endogenous iron-sulfur cluster-interacting redox promoters or
regulatory regions, one or more of the endogenous DXS, DXR, MCT,
CMK, MCS, HDS, or HDR promoters or regulatory regions, and isoprene
synthase promoter or regulatory region with other promoters and/or
regulatory regions that result in greater transcription of
iron-sulfur cluster-interacting redox nucleic acids, one or more of
DXS, DXR, MCT, CMK, MCS, HDS, or HDR nucleic acids, and isoprene
synthase nucleic acid.
[0211] In some embodiments, the presence of heterologous or extra
endogenous iron-sulfur cluster-interacting redox nucleic acid, DXP
pathway nucleic acid, and isoprene synthase nucleic acid cause
cells to grow more reproducibly and/or remain viable for longer
compared to the corresponding cell with only one or two of these
heterologous or extra endogenous nucleic acids. While not intending
to be bound to a particular theory, it is believed that the
overexpressing an iron sulfur cluster-interacting redox polypeptide
can increase the rate of formation or the amount of one or more DXP
pathway polypeptides (e.g., GcpE and/or LytB) or stabilizes one or
more DXP pathway polypeptides (e.g., GcpE and/or LytB), so that one
or more DXP pathway polypeptides are active for a longer period of
time, which in turn cause cells containing heterologous or extra
endogenous iron-sulfur cluster-interacting redox nucleic acid, DXP
pathway nucleic acid, and isoprene synthase nucleic acid to grow
more reproducibly and/or remain viable for longer compared to the
corresponding cell with only one or two of these heterologous or
extra endogenous nucleic acids. For example, cells containing
heterologous iron-sulfur cluster-interacting redox nucleic acid,
DXP pathway nucleic acid, and isoprene synthase nucleic acid grow
better than cells with only a DXP pathway nucleic acid, with only a
heterologous iron-sulfur cluster-interacting redox nucleic acid,
with a heterologous iron-sulfur cluster-interacting redox nucleic
acid and DXP pathway nucleic acid, iron-sulfur cluster-interacting
redox nucleic acid and isoprene synthase nucleic acid, or DXP
pathway nucleic acid and isoprene synthase nucleic acid. Also,
large amounts of iron-sulfur cluster-interacting redox polypeptide,
DXP pathway polypeptide, and isoprene synthase polypeptide can be
expressed in the cells without causing an excessive amount of
toxicity to the cells.
[0212] In some embodiments of any of the aspects of the invention,
the cells express a second DXP pathway polypeptide, in addition to
the first DXP pathway polypeptide, including DXS
(1-deoxy-D-xylulose-5-phosphate synthase), DXR
(1-deoxy-D-xylulose-5-phosphate reductoisomerase), MCT
(4-diphosphocytidyl-2C-methyl-D-erythritol synthase), CMK
(4-diphosphocytidyl-2-C-methyl-D-erythritol kinase), MCS
(2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase), HDS
(1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase), and HDR
(1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase). In some
embodiments of any of the aspects of the invention, the cells
express two or more DXP pathway polypeptides, in addition to the
first DXP pathway polypeptide as described above. In some
embodiments of any of the aspects of the invention, the cells
express 2, 3, 4, 5, 6, or 7 DXP pathway polypeptides, in addition
to the first DXP pathway polypeptide as described above.
[0213] Additionally, isoprene production by cells that contain a
heterologous iron-sulfur cluster-interacting redox nucleic acid,
DXP pathway nucleic acid (e.g., DXS, DXR, MCT, CMK, MCS, HDS, or
HDR), and isoprene synthase nucleic acid can be enhanced by
increasing the amount of an IDI polypeptide expressed by the
cells.
[0214] In some embodiments, isoprene production by cells that
contain a heterologous iron-sulfur cluster-interacting redox
nucleic acid, DXS nucleic acid, and isoprene synthase nucleic acid
can be enhanced by increasing the amount of an IDI polypeptide
expressed by the cells. In other embodiments, isoprene production
by cells that contain a heterologous iron-sulfur
cluster-interacting redox nucleic acid, HDS (IspG or GcpE), and
isoprene synthase nucleic acids can be enhanced by increasing the
amount of an IDI polypeptide expressed by the cells. In some
embodiments, the cells comprise IspG and fldA. In another
embodiment, the cells comprise IspG, fldA, and IspH.
[0215] In some embodiments, isoprene production by cells that
contain a heterologous flavodoxin nucleic acid, DXS nucleic acid,
and isoprene synthase nucleic acid can be enhanced by increasing
the amount of an IDI polypeptide expressed by the cells. In other
embodiments, isoprene production by cells that contain a
heterologous flavodoxin nucleic acid, HDS (IspG or GcpE) nucleic
acid, and isoprene synthase nucleic acid can be enhanced by
increasing the amount of an IDI polypeptide expressed by the
cells.
[0216] In some embodiments, isoprene production by cells that
contain a heterologous ferredoxin nucleic acid, ferredoxin-NADP+
oxidoreductase nucleic acid, DXS nucleic acid, and isoprene
synthase nucleic acid can be enhanced by increasing the amount of
an IDI polypeptide expressed by the cells. In other embodiments,
isoprene production by cells that contain a heterologous ferredoxin
nucleic acid, ferredoxin-NADP+ oxidoreductase nucleic acid, HDS
(IspG or GcpE) nucleic acid, and isoprene synthase nucleic acid can
be enhanced by increasing the amount of an IDI polypeptide
expressed by the cells.
[0217] In some embodiments, isoprene production by cells that
contain a heterologous iron-sulfur cluster-interacting redox
nucleic acid, HDR (IspH or LytB) nucleic acid, and isoprene
synthase nucleic acid can be enhanced by increasing the amount of
an IDI (isopentenyl-diphosphate delta-isomerase) polypeptide
expressed by the cells. In some embodiments, the cells comprise
IspG and fldA. In another embodiment, the cells comprise IspG,
fldA, and IspH.
[0218] In some embodiments, isoprene production by cells that
contain a heterologous flavodoxin, HDR (IspH or LytB), and isoprene
synthase nucleic acids can be enhanced by increasing the amount of
an IDI (isopentenyl-diphosphate delta-isomerase) polypeptide
expressed by the cells.
[0219] In some embodiments, isoprene production by cells that
contain a heterologous ferredoxin nucleic acid, ferredoxin-NADP+
oxidoreductase nucleic acid, HDR (IspH or LytB) nucleic acid, and
isoprene synthase nucleic acids can be enhanced by increasing the
amount of an IDI (isopentenyl-diphosphate delta-isomerase)
polypeptide expressed by the cells.
[0220] In some embodiments, isoprene production by cells that
contain a heterologous ferredoxin nucleic acid, ferredoxin-NADP+
oxidoreductase nucleic acid, HDS and HDR nucleic acids, and
isoprene synthase nucleic acid can be enhanced by increasing the
amount of an IDI (isopentenyl-diphosphate delta-isomerase)
polypeptide expressed by the cells.
[0221] IDI polypeptides catalyze the interconversion of isopentenyl
diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). While not
intending to be bound by any particular theory, it is believed that
increasing the amount of IDI polypeptide in cells increases the
amount (and conversion rate) of IPP that is converted into DMAPP,
which in turn is converted into isoprene.
[0222] The IDI nucleic acid may be a heterologous nucleic acid or a
duplicate copy of an endogenous nucleic acid. In some embodiments,
the amount of iron-sulfur cluster-interacting redox polypeptide,
one or more of DXP pathway polypeptide (e.g., DXS, DXR, MCT, CMK,
MCS, HDS, or HDR), isoprene synthase polypeptide, and IDI
polypeptide are increased by replacing endogenous iron-sulfur
cluster-interacting redox promoter or regulatory region, one or
more of the endogenous DXP pathway promoter or regulatory region,
and IDI promoters or regulatory region with other promoters and/or
regulatory regions that result in greater transcription of
iron-sulfur cluster-interacting redox nucleic acid, DXP pathway
nucleic acid, isoprene synthase nucleic acid, and IDI nucleic
acid.
[0223] Heterologous IDI polypeptides can also be expressed in a
variety of host cells in the presence of isoprene synthase, such as
Escherichia coli (E. coli), Panteoa citrea, Bacillus subtilis,
Yarrowia lipolytica, and Trichoderma reesei. All of these cells
produced more isoprene than when IDI is not used.
[0224] Additionally, isoprene production by cells that contain a
heterologous iron-sulfur cluster-interacting redox nucleic acid,
DXP pathway nucleic acid (e.g., DXS, DXR, MCT, CMK, MCS, HDS, or
HDR), isoprene synthase nucleic acid, and optionally IDI nucleic
acid, can be enhanced by increasing the amount of a DXP pathway
associated polypeptide expressed by the cells
[0225] In some embodiments of any of the aspects of the invention,
isoprene production can be further increased by using a mutant DXP
pathway polypeptide or nucleic acid derived from thereof. In some
embodiments, the mutant DXP pathway polypeptide is a HDR
polypeptide with the iron-sulfur cluster regulator (iscR) removed.
In some embodiments, the mutant DXP pathway polypeptide is a mutant
HDR polypeptide that produces solely DMAPP or a majority of DMAPP
relative to IPP. For example, the use of the LytBG120D in a DXP
pathway-mediated isoprene production strain allows the unique
generation of an isoprenoid product that is derived almost entirely
from DMAPP. See Example 18.
[0226] As used herein, iscR is encoded by an ORF located
immediately upstream of genes coding for the E. coli Fe--S cluster
assembly proteins. In the DXP pathway, the implementation of a gene
cassette directing the overexpression of the isc operon involved in
the assembly of iron-sulfur clusters into an E. coli strain
engineered for HDR protein anaerobically purified from this strain
by a factor of at least 200. (Grawert et al., J Am Chem Soc.
126(40):12847-55 (2004); Schwartz et al., PNAS, 98(26):14751-3
(2001); Akhtar and Jones, Appl. Microbiol. Biotechnol. 78(5):853-62
(2008), which are each hereby incorporated by reference in their
entireties).
[0227] In some embodiments of any of the aspects of the invention,
isoprene production can be further increased by increasing the
carbon flux through the DXP pathway. In some embodiments, the
carbon flux can be increased by avoiding any feedback inhibition of
DXS activity by metabolites downstream the DXP pathway or/and
intermediates of other pathways that use a DXP pathway polypeptide
as a substrate (e.g., DXR). In some embodiments, the feedback
inhibition by some DXP pathway polypeptides (e.g., DXR) can be
alleviated by rebalancing pathyway enzymes and maintaining levels
of HMBPP and DMAPP at concentrations below 1 to 2 mM DMAPP and 1 to
2 mM HMBPP. In some embodiments, the level of HMBPP and DMAPP are
maintained below 1 mM for the duration of the fermentation run. In
other embodiments, the level of HMBPP and DMAPP are maintained
below 1 mM during the exponential phase of the fermentation. In
other embodiments, late DXP pathway enzymes, particularly IspG and
IspH, are maintained at levels consistent with minimizing
phosphorylation level of Dxr.
[0228] In some embodiments, the other pathway that uses DXP pathway
polypeptide as a substrate (e.g., DXP) is the thiamine (Vitamin B1)
or pyridoxal (Vitamin B6) pathway. In some embodiments, the carbon
flux can be increased by expressing a DXP pathway polypeptide from
a different organism that is not subject to inhibition by
downstream products of the DXP pathway. In some embodiments, the
carbon flux can be increased by deregulating glucose uptake. In
other embodiments, the carbon flux can be increased by maximizing
the balance between the precursors required for the DXP pathway. In
some embodiments, the balance of the DXP pathway precursors,
pyruvate and glyceraldehydes-3-phosphate (G-3-P) can be achieved by
redirecting the carbon flux with the effect of elevating or
lowering pyruvate or G-3-P separately. In some embodiments, the
carbon flux can be increased by using a strain (containing one or
more DXP pathway genes or one or more both DXP pathway and MVA
pathway genes) containing a pyruvate dehydrogenase E1 subunit
variant. In some embodiments, the pyruvate dehydrogenase (PDH) E1
subunit variant has an E636Q point mutation. In some embodiments,
the carbon flux can be increased by using a CRP-deleted mutant. As
used herein, CRP (cAMP Receptor Protein) is a positive regulator
protein activated by cyclic AMP. It is required for RNA polymerase
to initiate transcription of certain (catabolite-sensitive) operons
of E. coli.
[0229] In some embodiments of any of the aspects of the invention,
isoprene production can be further increased by utilizing the
downstream genes or polypeptides of the DXP pathway by introducing
a heterologous terpene synthase nucleic acid or a duplicate copy of
an endogenous terpene synthase nucleic acid into the cells, which
includes, but is not limited to ocimene synthase, farnesene
synthase, and artemesinin synthase.
[0230] In some embodiments, a renewable carbon source is used for
the production of isoprene. In some embodiments, the concentrations
of isoprene and any oxidants are within the nonflammable ranges to
reduce or eliminate the risk that a fire may occur during
production or recovery of isoprene. See for example, U.S. Appl. No.
61/133,947, which is hereby incorporated by reference in its
entirety, particularly with respect to flammability modeling and
testing of isoprene in Example 13 and WO2010/003007. The
compositions and methods of the present invention are desirable
because they allow high isoprene yield per cell, high carbon yield,
high isoprene purity, high productivity, low energy usage, low
production cost and investment, and minimal side reactions. This
efficient, large scale, biosynthetic process for isoprene
production provides an isoprene source for synthetic isoprene-based
rubber and provides a desirable, low-cost alternative to using
natural rubber.
[0231] In some embodiments, at least a portion of the cells
maintain the heterologous iron-sulfur cluster-interacting redox
nucleic acid, DXP pathway nucleic acid, DXP pathway associated
nucleic acid, and isoprene synthase nucleic acid for at least about
5, 10, 20, 50, 75, 100, 200, 300, or more cell divisions in a
continuous culture (such as a continuous culture without dilution).
In some embodiments, at least a portion of the cells maintain the
heterologous iron-sulfur cluster-interacting redox nucleic acid,
DXP pathway nucleic acid, DXP pathway associated nucleic acid,
isoprene synthase nucleic acid, and IDI nucleic acid for at least
about 5, 10, 20, 50, 75, 100, 200, 300, or more cell divisions in a
continuous culture (such as a continuous culture without dilution).
In some embodiments of any of the aspects of the invention, the
nucleic acid comprising the heterologous or duplicate copy of an
endogenous iron-sulfur cluster-interacting redox nucleic acid, DXP
pathway nucleic acid, isoprene synthase nucleic acid, and/or IDI
nucleic acid and DXP pathway associated nucleic acid also comprises
a selective marker, such as a kanamycin, ampicillin, carbenicillin,
gentamicin, hygromycin, phleomycin, bleomycin, neomycin, or
chloramphenicol antibiotic resistance nucleic acid.
[0232] The amount of isoprene produced can be further increased by
adding yeast extract to the cell culture medium. For example, the
amount of isoprene produced that are linearly proportional to the
amount of yeast extract in the cell medium for the concentrations
are tested. Increasing the amount of yeast extract in the presence
of glucose can result in more isoprene being produced than
increasing the amount of glucose in the presence of yeast extract.
Also, increasing the amount of yeast extract can allow the cells to
produce a high level of isoprene for a longer length of time and
improved the health of the cells.
[0233] Isoprene production can also be demonstrated using three
types of hydrolyzed biomass (bagasse, corn stover, and soft wood
pulp) as the carbon source. If desired, any other biomass carbon
source can be used in the compositions and methods of the
invention. Biomass carbon sources are desirable because they are
cheaper than many conventional cell mediums, thereby facilitating
the economical production of isoprene.
[0234] In some embodiments, an oil is included in the cell medium.
See, for example, U.S. 61/134,094, which is hereby incorporated by
reference in its entirety, particularly with respect to oils
included in the cell medium In some embodiments, more than one oil
(such as 2, 3, 4, 5, or more oils) is included in the cell medium.
While not intending to be bound to any particular theory, it is
believed that (i) the oil may increase the amount of carbon in the
cells that is available for conversion to isoprene, (ii) the oil
may increase the amount of glyceraldehyde 3-phosphate and/or
pyruvate in the cells, thereby increasing the carbon flow through
the DXP pathway, and/or (ii) the oil may provide extra nutrients to
the cells, which is desirable since a lot of the carbon in the
cells is converted to isoprene rather than other products. In some
embodiments, cells that are cultured in a cell medium containing
oil naturally use the DXP pathway to produce isoprene or are
genetically modified to contain nucleic acids for the entire DXP
pathway. In some embodiments, the oil is partially or completely
hydrolyzed before being added to the cell culture medium to
facilitate the use of the oil by the host cells.
Exemplary Polypeptides and Nucleic Acids
[0235] Various iron-sulfur cluster-interacting redox polypeptides
and nucleic acids, DXP pathway polypeptides and nucleic acids, DXP
pathway associated polypeptides and nucleic acids, isoprene
synthase polypeptides and nucleic acids, and IDI polypeptides and
nucleic acids can be used in the compositions and methods of the
invention.
[0236] As used herein, "polypeptides" includes polypeptides,
proteins, peptides, fragments of polypeptides, and fusion
polypeptides. In some embodiments, the fusion polypeptide includes
part or all of a first polypeptide (e.g., an iron-sulfur
cluster-interacting redox polypeptide, DXP pathway polypeptide, DXP
pathway associated polypeptide, isoprene synthase polypeptide, and
IDI polypeptide, or catalytically active fragment thereof) and may
optionally include part or all of a second polypeptide (e.g., a
peptide that facilitates purification or detection of the fusion
polypeptide, such as a His-tag). In some embodiments, the fusion
polypeptide has an activity of two or more DXP pathway
polypeptides.
[0237] In various embodiments, a polypeptide has at least or about
50, 100, 150, 175, 200, 250, 300, 350, 400, or more amino acids. In
some embodiments, the polypeptide fragment contains at least or
about 25, 50, 75, 100, 150, 200, 300, or more contiguous amino
acids from a full-length polypeptide and has at least or about 5%,
10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% of an
activity of a corresponding full-length polypeptide. In particular
embodiments, the polypeptide includes a segment of or the entire
amino acid sequence of any naturally-occurring iron-sulfur
cluster-interacting redox polypeptide, DXP pathway polypeptide, DXP
pathway associated polypeptide, isoprene synthase polypeptide, or
IDI polypeptide. In some embodiments, the polypeptide has one or
more mutations compared to the sequence of a wild-type (i.e., a
sequence occurring in nature) iron-sulfur cluster-interacting redox
polypeptide, DXP pathway polypeptide, DXP pathway associated
polypeptide, isoprene synthase polypeptide, or IDI polypeptide.
[0238] In some embodiments, the polypeptide is an isolated
polypeptide. As used herein, an "isolated polypeptide" is not part
of a library of polypeptides, such as a library of 2, 5, 10, 20, 50
or more different polypeptides and is separated from at least one
component with which it occurs in nature. An isolated polypeptide
can be obtained, for example, by expression of a recombinant
nucleic acid encoding the polypeptide.
[0239] In some embodiments, the polypeptide is a heterologous
polypeptide. By "heterologous polypeptide" is meant a polypeptide
whose amino acid sequence is not identical to that of another
polypeptide naturally expressed in the same host cell. In
particular, a heterologous polypeptide is not identical to a
wild-type nucleic acid that is found in the same host cell in
nature.
[0240] As used herein, a "nucleic acid" refers to two or more
deoxyribonucleotides and/or ribonucleotides in either single or
double-stranded form. In some embodiments, the nucleic acid is a
recombinant nucleic acid. By "recombinant nucleic acid" means a
nucleic acid of interest that is free of one or more nucleic acids
(e.g., genes) which, in the genome occurring in nature of the
organism from which the nucleic acid of interest is derived, flank
the nucleic acid of interest. The term therefore includes, for
example, a recombinant DNA which is hereby incorporated into a
vector, into an autonomously replicating plasmid or virus, or into
the genomic DNA of a prokaryote or eukaryote, or which exists as a
separate molecule (e.g., a cDNA, a genomic DNA fragment, or a cDNA
fragment produced by PCR or restriction endonuclease digestion)
independent of other sequences. In various embodiments, a nucleic
acid is a recombinant nucleic acid. In some embodiments, an
iron-sulfur cluster-interacting redox nucleic acid, DXP pathway
nucleic acid, DXP pathway associated nucleic acid, isoprene
synthase nucleic acid, or IDI nucleic acid is operably linked to
another nucleic acid encoding all or a portion of another
polypeptide such that the recombinant nucleic acid encodes a fusion
polypeptide that includes an iron-sulfur cluster-interacting redox
polypeptide, DXP pathway polypeptide, DXP pathway associated
polypeptide, isoprene synthase polypeptide, and/or IDI and all or
part of another polypeptide (e.g., a peptide that facilitates
purification or detection of the fusion polypeptide, such as a
His-tag). In some embodiments, part or all of a recombinant nucleic
acid is chemically synthesized. It is to be understood that
mutations, including single nucleotide mutations, can occur within
a nucleic acid as defined herein.
[0241] In some embodiments, the nucleic acid is a heterologous
nucleic acid. By "heterologous nucleic acid" is meant a nucleic
acid whose nucleic acid sequence is not identical to that of
another nucleic acid naturally found in the same host cell.
[0242] In particular embodiments, the nucleic acid includes a
segment of or the entire nucleic acid sequence of any
naturally-occurring iron-sulfur cluster-interacting redox nucleic
acid, DXP pathway nucleic acid, DXP pathway associated nucleic
acid, isoprene synthase nucleic, or IDI nucleic acid. In some
embodiments, the nucleic acid includes at least or about 50, 100,
150, 200, 300, 400, 500, 600, 700, 800, or more contiguous
nucleotides from a naturally-occurring iron-sulfur
cluster-interacting redox nucleic acid, DXP pathway nucleic acid,
DXP pathway associated nucleic acid, isoprene synthase nucleic, or
IDI nucleic acid. In some embodiments, the nucleic acid has one or
more mutations compared to the sequence of a wild-type (i.e., a
sequence occurring in nature) iron-sulfur cluster-interacting redox
nucleic acid, DXP pathway nucleic acid, DXP pathway associated
nucleic acid, isoprene synthase nucleic acid, or IDI nucleic acid.
In some embodiments, the nucleic acid has one or more mutations
(e.g., a silent mutation) that increase the transcription or
translation of iron-sulfur cluster-interacting redox nucleic acid,
DXP pathway nucleic acid, DXP pathway associated nucleic acid,
isoprene synthase nucleic acid, or IDI nucleic acid. In some
embodiments, the nucleic acid is a degenerate variant of any
nucleic acid encoding an iron-sulfur cluster-interacting redox
polypeptide, DXP pathway polypeptide, DXP pathway associated
polypeptide, isoprene synthase polypeptide, or IDI polypeptide.
[0243] "Codon degeneracy" refers to divergence in the genetic code
permitting variation of the nucleotide sequence without affecting
the amino acid sequence of an encoded polypeptide. The skilled
artisan is well aware of the "codon-bias" exhibited by a specific
host cell in usage of nucleotide codons to specify a given amino
acid. Therefore, when synthesizing a nucleic acid for improved
expression in a host cell, it is desirable in some embodiments to
design the nucleic acid such that its frequency of codon usage
approaches the frequency of preferred codon usage of the host
cell.
[0244] The accession numbers of exemplary isoprene synthase and DXP
pathway polypeptides and nucleic acids are listed in Appendix 1
(the accession numbers of Appendix 1 and their corresponding
sequences are herein incorporated by reference in their entireties,
particularly with respect to the amino acid and nucleic acid
sequences of isoprene synthase and/or DXP pathway polypeptides and
nucleic acids). The Kegg database also contains the amino acid and
nucleic acid sequences of numerous exemplary isoprene synthase
and/or DXP pathway polypeptides and nucleic acids (see, for
example, the world-wide web at
"genome.jp/kegg/pathway/map/map00100.html" and the sequences
therein, which are each hereby incorporated by reference in their
entireties, particularly with respect to the amino acid and nucleic
acid sequences of isoprene synthase and/or DXP pathway polypeptides
and nucleic acids). In some embodiments, one or more of the
isoprene synthase and/or DXP pathway polypeptides and/or nucleic
acids have a sequence identical to a sequence publicly available on
Dec. 12, 2007 or Sep. 14, 2008 such as any of the sequences that
correspond to any of the accession numbers in Appendix 1 or any of
the sequences present in the Kegg database. Additional exemplary
isoprene synthase and/or DXP pathway polypeptides and nucleic acids
are described further below.
Exemplary Isoprene Synthase Polypeptides and Nucleic Acids
[0245] As noted above, isoprene synthase polypeptides convert
dimethylallyl diphosphate (DMAPP) into isoprene. Exemplary isoprene
synthase polypeptides include polypeptides, fragments of
polypeptides, peptides, and fusions polypeptides that have at least
one activity of an isoprene synthase polypeptide. Standard methods
can be used to determine whether a polypeptide has isoprene
synthase polypeptide activity by measuring the ability of the
polypeptide to convert DMAPP into isoprene in vitro, in a cell
extract, or in vivo. In an exemplary assay, cell extracts are
prepared by growing a strain (e.g., the E. coli/pTrcKudzu strain
described herein) in the shake flask method as described in Example
1. After induction is complete, approximately 10 mL of cells are
pelleted by centrifugation at 7000.times.g for 10 minutes and
resuspended in 5 ml of PEB without glycerol. The cells are lysed
using a French Pressure cell using standard procedures.
Alternatively the cells are treated with lysozyme (Ready-Lyse
lysozyme solution; EpiCentre) after a freeze/thaw at -80 C.
[0246] Isoprene synthase polypeptide activity in the cell extract
can be measured, for example, as described in Silver et al., J.
Biol. Chem. 270:13010-13016, 1995 and references therein, which are
each hereby incorporated by reference in their entireties,
particularly with respect to assays for isoprene synthase
polypeptide activity. DMAPP (Sigma) is evaporated to dryness under
a stream of nitrogen and rehydrated to a concentration of 100 mM in
100 mM potassium phosphate buffer pH 8.2 and stored at -20.degree.
C. To perform the assay, a solution of 5 .mu.L of 1M MgCl.sub.2, 1
mM (250 .mu.g/ml) DMAPP, 65 .mu.L of Plant Extract Buffer (PEB) (50
mM Tris-HCl, pH 8.0, 20 mM MgCl.sub.2, 5% glycerol, and 2 mM DTT)
is added to 25 .mu.L of cell extract in a 20 ml Headspace vial with
a metal screw cap and teflon coated silicon septum (Agilent
Technologies) and cultured at 37.degree. C. for 15 minutes with
shaking. The reaction is quenched by adding 200 .mu.L of 250 mM
EDTA and quantified by GC/MS as described in Example 1, part
II.
[0247] Exemplary isoprene synthase nucleic acids include nucleic
acids that encode a polypeptide, fragment of a polypeptide,
peptide, or fusion polypeptide that has at least one activity of an
isoprene synthase polypeptide. Exemplary isoprene synthase
polypeptides and nucleic acids include naturally-occurring
polypeptides and nucleic acids from any of the source organisms
described herein as well as mutant polypeptides and nucleic acids
derived from any of the source organisms described herein.
[0248] In some embodiments, the isoprene synthase polypeptide or
nucleic acid is from the family Fabaceae, such as the Faboideae
subfamily. In some embodiments, the isoprene synthase polypeptide
or nucleic acid is a polypeptide or nucleic acid from Pueraria
montana (kudzu) (Sharkey et al., Plant Physiology 137: 700-712,
2005), Pueraria lobata, poplar (such as Populus alba, Populus
nigra, Populus trichocarpa, or Populus alba.times.tremula
(CAC35696) Miller et al., Planta 213: 483-487, 2001) aspen (such as
Populus tremuloides) Silver et al., JBC 270(22): 13010-1316, 1995),
or English Oak (Quercus robur) (Zimmer et al., WO 98/02550), which
are each hereby incorporated by reference in their entireties,
particularly with respect to isoprene synthase nucleic acids and
the expression of isoprene synthase polypeptides. Suitable isoprene
synthases include, but are not limited to, those identified by
Genbank Accession Nos. AY341431, AY316691, AY279379, AJ457070, and
AY182241, which are each hereby incorporated by reference in their
entireties, particularly with respect to sequences of isoprene
synthase nucleic acids and polypeptides. In some embodiments, the
isoprene synthase polypeptide or nucleic acid is not a
naturally-occurring polypeptide or nucleic acid from Quercus robur
(i.e., the isoprene synthase polypeptide or nucleic acid is an
isoprene synthase polypeptide or nucleic acid other than a
naturally-occurring polypeptide or nucleic acid from Quercus
robur). In some embodiments, the isoprene synthase nucleic acid or
polypeptide is a naturally-occurring polypeptide or nucleic acid
from poplar. In some embodiments, the isoprene synthase nucleic
acid or polypeptide is not a naturally-occurring polypeptide or
nucleic acid from poplar.
Exemplary DXP Pathway Polypeptides and Nucleic Acids
[0249] Exemplary DXP pathways polypeptides include, but are not
limited to any of the following polypeptides: DXS polypeptides, DXR
polypeptides, MCT polypeptides, CMK polypeptides, MCS polypeptides,
HDS polypeptides, HDR polypeptides, IDI polypeptides, and
polypeptides (e.g., fusion polypeptides) having an activity of one,
two, or more of the DXP pathway polypeptides. In particular, DXP
pathway polypeptides include polypeptides, fragments of
polypeptides, peptides, and fusions polypeptides that have at least
one activity of a DXP pathway polypeptide. Exemplary DXP pathway
nucleic acids include nucleic acids that encode a polypeptide,
fragment of a polypeptide, peptide, or fusion polypeptide that has
at least one activity of a DXP pathway polypeptide. Exemplary DXP
pathway polypeptides and nucleic acids include naturally-occurring
polypeptides and nucleic acids from any of the source organisms
described herein as well as mutant polypeptides and nucleic acids
derived from any of the source organisms described herein.
[0250] In particular, DXS polypeptides convert pyruvate and
D-glyceraldehyde 3-phosphate into 1-deoxy-d-xylulose 5-phosphate
(DXP). Standard methods can be used to determine whether a
polypeptide has DXS polypeptide activity by measuring the ability
of the polypeptide to convert pyruvate and D-glyceraldehyde
3-phosphate in vitro, in a cell extract, or in vivo.
[0251] DXR polypeptides convert 1-deoxy-d-xylulose 5-phosphate
(DXP) into 2-C-methyl-D-erythritol 4-phosphate (MEP). Standard
methods can be used to determine whether a polypeptide has DXR
polypeptides activity by measuring the ability of the polypeptide
to convert DXP in vitro, in a cell extract, or in vivo.
[0252] MCT polypeptides convert 2-C-methyl-D-erythritol 4-phosphate
(MEP) into 4-(cytidine 5'-diphospho)-2-methyl-D-erythritol
(CDP-ME). Standard methods can be used to determine whether a
polypeptide has MCT polypeptides activity by measuring the ability
of the polypeptide to convert MEP in vitro, in a cell extract, or
in vivo.
[0253] CMK polypeptides convert 4-(cytidine
5'-diphospho)-2-C-methyl-D-erythritol (CDP-ME) into
2-phospho-4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol
(CDP-MEP). Standard methods can be used to determine whether a
polypeptide has CMK polypeptides activity by measuring the ability
of the polypeptide to convert CDP-ME in vitro, in a cell extract,
or in vivo.
[0254] MCS polypeptides convert 2-phospho-4-(cytidine
5'-diphospho)-2-C-methyl-D-erythritol (CDP-MEP) into
2-C-methyl-D-erythritol 2,4-cyclodiphosphate (ME-CPP or cMEPP).
Standard methods can be used to determine whether a polypeptide has
MCS polypeptides activity by measuring the ability of the
polypeptide to convert CDP-MEP in vitro, in a cell extract, or in
vivo.
[0255] HDS polypeptides convert 2-C-methyl-D-erythritol
2,4-cyclodiphosphate into (E)-4-hydroxy-3-methylbut-2-en-1-yl
diphosphate (HMBPP or HDMAPP). Standard methods can be used to
determine whether a polypeptide has HDS polypeptides activity by
measuring the ability of the polypeptide to convert ME-CPP in
vitro, in a cell extract, or in vivo.
[0256] HDR polypeptides convert (E)-4-hydroxy-3-methylbut-2-en-1-yl
diphosphate into isopentenyl diphosphate (IPP) and dimethylallyl
diphosphate (DMAPP). Standard methods can be used to determine
whether a polypeptide has HDR polypeptides activity by measuring
the ability of the polypeptide to convert HMBPP in vitro, in a cell
extract, or in vivo.
[0257] IDI polypeptides convert isopentenyl diphosphate into
dimethylallyl diphosphate. Standard methods can be used to
determine whether a polypeptide has IDI polypeptides activity by
measuring the ability of the polypeptide to convert isopentenyl
diphosphate in vitro, in a cell extract, or in vivo.
Exemplary MVA Pathway Polypeptides and Nucleic Acids
[0258] In some aspects of the invention, the cells described in any
of the compositions or methods described herein comprise a nucleic
acid encoding an MVA pathway polypeptide. In some embodiments, the
MVA pathway polypeptide is an endogenous polypeptide. In some
embodiments, the cells comprise one or more additional copies of an
endogenous nucleic acid encoding an MVA pathway polypeptide. In
some embodiments, the endogenous nucleic acid encoding an MVA
pathway polypeptide operably linked to a constitutive promoter. In
some embodiments, the endogenous nucleic acid encoding an MVA
pathway polypeptide operably linked to a constitutive promoter. In
some embodiments, the endogenous nucleic acid encoding an MVA
pathway polypeptide is operably linked to a strong promoter. In a
particular embodiment, the cells are engineered to over-express the
endogenous MVA pathway polypeptide relative to wild-type cells.
[0259] In some embodiments, the MVA pathway polypeptide is a
heterologous polypeptide. In some embodiments, the cells comprise
more than one copy of a heterologous nucleic acid encoding an MVA
pathway polypeptide. In some embodiments, the heterologous nucleic
acid encoding an MVA pathway polypeptide is operably linked to a
constitutive promoter. In some embodiments, the heterologous
nucleic acid encoding an MVA pathway polypeptide is operably linked
to a strong promoter.
[0260] Exemplary MVA pathway polypeptides include acetyl-CoA
acetyltransferase (AA-CoA thiolase) polypeptides,
3-hydroxy-3-methylglutaryl-CoA synthase (HMG-CoA synthase)
polypeptides, 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA
reductase) polypeptides, mevalonate kinase (MVK) polypeptides,
phosphomevalonate kinase (PMK) polypeptides, diphosphomevalonte
decarboxylase (MVD) polypeptides, phosphomevalonate decarboxylase
(PMDC) polypeptides, isopentenyl phosphate kinase (IPK)
polypeptides, IDI polypeptides, and polypeptides (e.g., fusion
polypeptides) having an activity of two or more MVA pathway
polypeptides. In particular, MVA pathway polypeptides include
polypeptides, fragments of polypeptides, peptides, and fusions
polypeptides that have at least one activity of an MVA pathway
polypeptide. Exemplary MVA pathway nucleic acids include nucleic
acids that encode a polypeptide, fragment of a polypeptide,
peptide, or fusion polypeptide that has at least one activity of an
MVA pathway polypeptide. Exemplary MVA pathway polypeptides and
nucleic acids include naturally-occurring polypeptides and nucleic
acids from any of the source organisms described herein. In
addition, variants of MVA pathway polypeptide that confer the
result of better isoprene production can also be used as well.
[0261] Types of MVA pathway polypeptides and/or DXP pathway
polypeptides which can be used and methods of making microorganisms
(e.g., facultative anaerobes such as E. coli) encoding MVA pathway
polypeptides and/or DXP pathway polypeptides are also described in
International Patent Application Publication No. WO2009/076676;
U.S. patent application Ser. Nos. 12/496,573, 12/560,390,
12/560,317, 12/560,370, 12/560,305, and 12/560,366; and U.S.
Provisional Patent Application Nos. 61/187,930, 61/187,934, and
61/187,959.
[0262] One of skill in the art can readily select and/or use
suitable promoters to optimize the expression of isoprene synthase
or and one or more MVA pathway polypeptides and/or one or more DXP
pathway polypeptides. Similarly, one of skill in the art can
readily select and/or use suitable vectors (or transfer vehicle) to
optimize the expression of isoprene synthase or and one or more MVA
pathway polypeptides and/or one or more DXP pathway polypeptides.
In some embodiments, the vector contains a selective marker.
Examples of selectable markers include, but are not limited to,
antibiotic resistance nucleic acids (e.g., kanamycin, ampicillin,
carbenicillin, gentamicin, hygromycin, phleomycin, bleomycin,
neomycin, or chloramphenicol) and/or nucleic acids that confer a
metabolic advantage, such as a nutritional advantage on the host
cell. In some embodiments, an isoprene synthase or MVA pathway
nucleic acid integrates into a chromosome of the cells without a
selective marker.
Exemplary Iron-Sulfur Cluster-Interacting Redox Polypeptides and
Nucleic Acids
[0263] As noted above, the iron-sulfur cluster-interacting redox
polypeptide plays an essential role in the DXP pathway for
isoprenoid biosynthesis. Exemplary iron-sulfur cluster-interacting
redox polypeptides include polypeptides, fragments of polypeptides,
peptides, and fusions polypeptides that have at least one activity
of a iron-sulfur cluster-interacting redox polypeptide. Standard
methods can be used to determine whether a polypeptide has
iron-sulfur cluster-interacting redox polypeptide activity by using
a hydrogenase-linked assay measuring the rate of
metronidazole[1-(2-hydroxyethyl)-2-methyl-5-nitroimidazole]
reduction (Chen and Blanchard, Analytical Biochem, 93:216-222
(1979)), which is hereby incorporated by reference in its entirety,
especially with respect to the hydrogenase-linked assay for
ferredoxin and flavodoxin).
[0264] Exemplary iron-sulfur cluster-interacting redox polypeptide
nucleic acids include nucleic acids that encode a polypeptide,
fragment of a polypeptide, peptide, or fusion polypeptide that has
at least one activity of an iron-sulfur cluster-interacting redox
polypeptide. Exemplary iron-sulfur cluster-interacting redox
polypeptides and nucleic acids include naturally-occurring
polypeptides and nucleic acids from any of the source organisms
described herein as well as mutant polypeptides and nucleic acids
derived from any of the source organisms described herein.
Exemplary Methods for Isolating Nucleic Acids
[0265] Iron-sulfur cluster-interacting redox nucleic acid, DXP
pathway nucleic acid, DXP pathway associated nucleic acid, isoprene
synthase nucleic, or IDI nucleic acid can be isolated using
standard methods. Methods of obtaining desired nucleic acids from a
source organism of interest (such as a bacterial genome) are common
and well known in the art of molecular biology (see, for example,
WO 2004/033646 and references cited therein, which are each hereby
incorporated by reference in their entireties, particularly with
respect to the isolation of nucleic acids of interest). For
example, if the sequence of the nucleic acid is known (such as any
of the known nucleic acids described herein), suitable genomic
libraries may be created by restriction endonuclease digestion and
may be screened with probes complementary to the desired nucleic
acid sequence. Once the sequence is isolated, the DNA may be
amplified using standard primer directed amplification methods such
as polymerase chain reaction (PCR) (U.S. Pat. No. 4,683,202, which
is hereby incorporated by reference in its entirety, particularly
with respect to PCR methods) to obtain amounts of DNA suitable for
transformation using appropriate vectors.
[0266] Alternatively, iron-sulfur cluster-interacting redox nucleic
acid, DXP pathway nucleic acid, DXP pathway associated nucleic
acid, isoprene synthase nucleic, and/or IDI nucleic acid (such as
any isoprene synthase nucleic acid, iron-sulfur cluster-interacting
redox nucleic acid, DXP pathway nucleic acid, and/or IDI nucleic
acid with a known nucleic acid sequence) can be chemically
synthesized using standard methods.
[0267] Additional iron-sulfur cluster-interacting redox nucleic
acid, DXP pathway nucleic acid, DXP pathway associated nucleic
acid, isoprene synthase nucleic, and/or IDI nucleic acid which may
be suitable for use in the compositions and methods described
herein can be identified using standard methods. For example,
cosmid libraries of the chromosomal DNA of organisms known to
produce isoprene naturally can be constructed in organisms such as
E. coli, and then screened for isoprene production. In particular,
cosmid libraries may be created where large segments of genomic DNA
(35-45 kb) are packaged into vectors and used to transform
appropriate hosts. Cosmid vectors are unique in being able to
accommodate large quantities of DNA. Generally cosmid vectors have
at least one copy of the cos DNA sequence which is needed for
packaging and subsequent circularization of the heterologous DNA.
In addition to the cos sequence, these vectors also contain an
origin of replication such as ColEI and drug resistance markers
such as a nucleic acid resistant to ampicillin or neomycin. Methods
of using cosmid vectors for the transformation of suitable
bacterial hosts are well described in Sambrook et al., Molecular
Cloning: A Laboratory Manual, 2.sup.nd ed., Cold Spring Harbor,
1989, which is hereby incorporated by reference in its entirety,
particularly with respect to transformation methods.
[0268] Typically to clone cosmids, heterologous DNA is isolated
using the appropriate restriction endonucleases and ligated
adjacent to the cos region of the cosmid vector using the
appropriate ligases. Cosmid vectors containing the linearized
heterologous DNA are then reacted with a DNA packaging vehicle such
as bacteriophage. During the packaging process, the cos sites are
cleaved and the heterologous DNA is packaged into the head portion
of the bacterial viral particle. These particles are then used to
transfect suitable host cells such as E. coli. Once injected into
the cell, the heterologous DNA circularizes under the influence of
the cos sticky ends. In this manner, large segments of heterologous
DNA can be introduced and expressed in host cells.
[0269] Additional methods for obtaining iron-sulfur
cluster-interacting redox nucleic acid, DXP pathway nucleic acid,
DXP pathway associated nucleic acid, isoprene synthase nucleic
acid, and/or IDI nucleic acid include screening a metagenomic
library by assay (such as the headspace assay (see for example, in
U.S. application Ser. No. 12/335,071 and PCT/US2008/086809, which
are hereby incorporated by reference in their entireties,
particularly with respect to headspace assay for isoprene
production in Example 1 and 7) or by PCR using primers directed
against nucleotides encoding for a length of conserved amino acids
(for example, at least 3 conserved amino acids). Conserved amino
acids can be identified by aligning amino acid sequences of known
iron-sulfur cluster-interacting redox polypeptide, DXP pathway
polypeptide, DXP pathway associated polypeptide, isoprene synthase
polypeptide, and/or IDI polypeptide. Conserved amino acids for
isoprene synthase polypeptides can be identified based on aligned
sequences of known isoprene synthase polypeptides. An organism
found to produce isoprene naturally can be subjected to standard
protein purification methods (which are well known in the art) and
the resulting purified polypeptide can be sequenced using standard
methods. Other methods are found in the literature (see, for
example, Julsing et al., Applied. Microbiol. Biotechnol. 75:
1377-84, 2007; Withers et al., Appl Environ Microbiol.
73(19):6277-83, 2007, which are each hereby incorporated by
reference in their entireties, particularly with respect to
identification of nucleic acids involved in the synthesis of
isoprene).
[0270] Additionally, standard sequence alignment and/or structure
prediction programs can be used to identify additional DXP pathway
polypeptides and nucleic acids based on the similarity of their
primary and/or predicted polypeptide secondary structure with that
of known DXP pathway polypeptides and nucleic acids. Standard
databases such as the swissprot-trembl database (world-wide web at
"expasy.org", Swiss Institute of Bioinformatics Swiss-Prot group
CMU-1 rue Michel Servet CH-1211 Geneva 4, Switzerland) can also be
used to identify isoprene synthase, flavodoxin I, DXP pathway,
and/or IDI polypeptides and nucleic acids. The secondary and/or
tertiary structure of an iron-sulfur cluster-interacting redox
polypeptide, DXP pathway polypeptide, DXP pathway associated
polypeptide, isoprene synthase polypeptide, and/or IDI polypeptide
can be predicted using the default settings of standard structure
prediction programs, such as PredictProtein (630 West, 168 Street,
BB217, New York, N.Y. 10032, USA). Alternatively, the actual
secondary and/or tertiary structure of an iron-sulfur
cluster-interacting redox polypeptide, DXP pathway polypeptide, DXP
pathway associated polypeptide, isoprene synthase polypeptide,
and/or IDI polypeptide can be determined using standard methods.
Additional iron-sulfur cluster-interacting redox nucleic acid, DXP
pathway nucleic acid, DXP pathway associated nucleic acid, isoprene
synthase nucleic acid, and/or IDI nucleic acid can also be
identified by hybridization to probes generated from known
iron-sulfur cluster-interacting redox nucleic acid, DXP pathway
nucleic acid, DXP pathway associated nucleic acid, isoprene
synthase nucleic acid, and/or IDI nucleic acid.
Exemplary Promoters and Vectors
[0271] Any of the iron-sulfur cluster-interacting redox nucleic
acid, DXP pathway nucleic acid, DXP pathway associated nucleic
acid, isoprene synthase nucleic acid, or IDI nucleic acid described
herein can be included in one or more vectors. Accordingly, the
invention also features vectors with one more nucleic acids
encoding any of the iron-sulfur cluster-interacting redox
polypeptide, DXP pathway polypeptide, DXP pathway associated
polypeptide, isoprene synthase polypeptide, and/or IDI polypeptide
that are described herein. As used herein, a "vector" means a
construct that is capable of delivering, and desirably expressing
one or more nucleic acids of interest in a host cell. Examples of
vectors include, but are not limited to, plasmids, viral vectors,
DNA or RNA expression vectors, cosmids, and phage vectors. In some
embodiments, the vector contains a nucleic acid under the control
of an expression control sequence.
[0272] As used herein, an "expression control sequence" means a
nucleic acid sequence that directs transcription of a nucleic acid
of interest. An expression control sequence can be a promoter, such
as a constitutive or an inducible promoter, or an enhancer. An
"inducible promoter" is a promoter that is active under
environmental or developmental regulation. The expression control
sequence is operably linked to the nucleic acid segment to be
transcribed.
[0273] In some embodiments, the vector contains a selective marker.
The term "selective marker" refers to a nucleic acid capable of
expression in a host cell that allows for ease of selection of
those host cells containing an introduced nucleic acid or vector.
Examples of selectable markers include, but are not limited to,
antibiotic resistance nucleic acids (e.g., kanamycin, ampicillin,
carbenicillin, gentamicin, hygromycin, phleomycin, bleomycin,
neomycin, or chloramphenicol) and/or nucleic acids that confer a
metabolic advantage, such as a nutritional advantage on the host
cell. Exemplary nutritional selective markers include those markers
known in the art as amdS, argB, and pyr4. Markers useful in vector
systems for transformation of Trichoderma are known in the art
(see, e.g., Finkelstein, Chapter 6 in Biotechnology of Filamentous
Fungi, Finkelstein et al., Eds. Butterworth-Heinemann, Boston,
Mass., Chap. 6., 1992; and Kinghorn et al., Applied Molecular
Genetics of Filamentous Fungi, Blackie Academic and Professional,
Chapman and Hall, London, 1992, which are each hereby incorporated
by reference in their entireties, particularly with respect to
selective markers). In some embodiments, the selective marker is
the amdS nucleic acid, which encodes the enzyme acetamidase,
allowing transformed cells to grow on acetamide as a nitrogen
source. The use of an A. nidulans amdS nucleic acid as a selective
marker is described in Kelley et al., EMBO J. 4:475-479, 1985 and
Penttila et al., Gene 61:155-164, 1987 (which are each hereby
incorporated by reference in their entireties, particularly with
respect to selective markers). In some embodiments, an isoprene
synthase, flavodoxin I, DXP pathway, or IDI nucleic acid integrates
into a chromosome of the cells without a selective marker.
[0274] Suitable vectors are those which are compatible with the
host cell employed. Suitable vectors can be derived, for example,
from a bacterium, a virus (such as bacteriophage T7 or a M-13
derived phage), a cosmid, a yeast, or a plant. Protocols for
obtaining and using such vectors are known to those in the art
(see, for example, Sambrook et al., Molecular Cloning: A Laboratory
Manual, 2.sup.nd ed., Cold Spring Harbor, 1989, which is hereby
incorporated by reference in its entirety, particularly with
respect to the use of vectors).
[0275] Promoters are well known in the art. Any promoter that
functions in the host cell can be used for expression of an
iron-sulfur cluster-interacting redox nucleic acid, DXP pathway
nucleic acid, DXP pathway associated nucleic acid, isoprene
synthase nucleic acid, or IDI nucleic acid in the host cell.
Initiation control regions or promoters, which are useful to drive
expression of iron-sulfur cluster-interacting redox nucleic acid,
DXP pathway nucleic acid, DXP pathway associated nucleic acid,
isoprene synthase nucleic acid, and/or IDI nucleic acid in various
host cells are numerous and familiar to those skilled in the art
(see, for example, WO 2004/033646 and references cited therein,
which are each hereby incorporated by reference in their
entireties, particularly with respect to vectors for the expression
of nucleic acids of interest). Virtually any promoter capable of
driving these nucleic acids is suitable for the present invention
including, but not limited to, CYC1, HIS3, GAL1, GAL10, ADH1, PGK,
PHO5, GAPDH, ADCI, TRP1, URA3, LEU2, ENO, and TPI (useful for
expression in Saccharomyces); AOX1 (useful for expression in
Pichia); and lac, trp, P.sub.L, P.sub.R, T7, tac, and trc (useful
for expression in E. coli).
[0276] In some embodiments, a glucose isomerase promoter is used
(see, for example, U.S. Pat. No. 7,132,527 and references cited
therein, which are each hereby incorporated by reference in their
entireties, particularly with respect promoters and plasmid systems
for expressing polypeptides of interest). Reported glucose
isomerase promoter mutants can be used to vary the level of
expression of the polypeptide encoded by a nucleic acid operably
linked to the glucose isomerase promoter (U.S. Pat. No. 7,132,527).
In various embodiments, the glucose isomerase promoter is contained
in a low, medium, or high copy plasmid (U.S. Pat. No.
7,132,527).
[0277] In various embodiments, an iron-sulfur cluster-interacting
redox nucleic acid, DXP pathway nucleic acid, DXP pathway
associated nucleic acid, isoprene synthase nucleic acid, or IDI
nucleic acid is contained in a low copy plasmid (e.g., a plasmid
that is maintained at about 1 to about 4 copies per cell), medium
copy plasmid (e.g., a plasmid that is maintained at about 10 to
about 15 copies per cell), or high copy plasmid (e.g., a plasmid
that is maintained at about 50 or more copies per cell). In some
embodiments, the heterologous or extra endogenous iron-sulfur
cluster-interacting redox nucleic acid, DXP pathway nucleic acid,
DXP pathway associated nucleic acid, isoprene synthase nucleic
acid, or IDI nucleic acid is operably linked to a T7 promoter. In
some embodiments, the heterologous or extra endogenous iron-sulfur
cluster-interacting redox nucleic acid, DXP pathway nucleic acid,
DXP pathway associated nucleic acid, isoprene synthase nucleic
acid, or IDI nucleic acid operably linked to a T7 promoter is
contained in a medium or high copy plasmid. In some embodiments,
the heterologous or extra endogenous iron-sulfur
cluster-interacting redox nucleic acid, DXP pathway nucleic acid,
DXP pathway associated nucleic acid, isoprene synthase nucleic
acid, or IDI nucleic acid is operably linked to a Trc promoter. In
some embodiments, the heterologous or extra endogenous is
iron-sulfur cluster-interacting redox nucleic acid, DXP pathway
nucleic acid, DXP pathway associated nucleic acid, isoprene
synthase nucleic acid, or IDI nucleic acid operably linked to a Trc
promoter is contained in a medium or high copy plasmid. In some
embodiments, the heterologous or extra endogenous iron-sulfur
cluster-interacting redox nucleic acid, DXP pathway nucleic acid,
DXP pathway associated nucleic acid, isoprene synthase nucleic
acid, or IDI nucleic acid is operably linked to a Lac promoter. In
some embodiments, the heterologous or extra endogenous iron-sulfur
cluster-interacting redox nucleic acid, DXP pathway nucleic acid,
DXP pathway associated nucleic acid, isoprene synthase nucleic
acid, or IDI nucleic acid operably linked to a Lac promoter is
contained in a low copy plasmid. In some embodiments, the
heterologous or extra endogenous iron-sulfur cluster-interacting
redox nucleic acid, DXP pathway nucleic acid, DXP pathway
associated nucleic acid, isoprene synthase nucleic acid, or IDI
nucleic acid is operably linked to an endogenous promoter, such as
an endogenous Escherichia, Panteoa, Bacillus, Yarrowia,
Streptomyces, Trichoderma or Thermosynechococcus promoter or an
endogenous alkaline serine protease iron-sulfur cluster-interacting
redox promoter, DXP pathway promoter, DXP pathway associated
promoter, isoprene synthase promoter, or IDI promoter. In some
embodiments, the heterologous or extra endogenous isoprene synthase
nucleic acid, iron-sulfur cluster-interacting redox nucleic acid,
DXP pathway nucleic acid, DXP pathway associated nucleic acid,
and/or IDI nucleic acid operably linked to an endogenous promoter
is contained in a high copy plasmid. In some embodiments, the
vector is a replicating plasmid that does not integrate into a
chromosome in the cells. In some embodiments, part or all of the
vector integrates into a chromosome in the cells.
[0278] In some embodiments, the vector is any vector which when
introduced into a fungal host cell is integrated into the host cell
genome and is replicated. Reference is made to the Fungal Genetics
Stock Center Catalogue of Strains (FGSC, the world-wide web at
"fgsc.net" and the references cited therein, which are each hereby
incorporated by reference in their entireties, particularly with
respect to vectors) for a list of vectors. Additional examples of
suitable expression and/or integration vectors are provided in
Sambrook et al., Molecular Cloning: A Laboratory Manual, 2.sup.nd
ed., Cold Spring Harbor, 1989, Current Protocols in Molecular
Biology (F. M. Ausubel et al. (eds) 1987, Supplement 30, section
7.7.18); van den Hondel et al. in Bennett and Lasure (Eds.) More
Gene Manipulations in Fungi, Academic Press pp. 396-428, 1991; and
U.S. Pat. No. 5,874,276, which are each hereby incorporated by
reference in their entireties, particularly with respect to
vectors. Particularly useful vectors include pFB6, pBR322, PUC18,
pUC100, and pENTR/D.
[0279] In some embodiments, iron-sulfur cluster-interacting redox
nucleic acid, DXP pathway nucleic acid, DXP pathway associated
nucleic acid, isoprene synthase nucleic acid, or IDI nucleic acid
is operably linked to a suitable promoter that shows
transcriptional activity in a fungal host cell. The promoter may be
derived from one or more nucleic acids encoding a polypeptide that
is either endogenous or heterologous to the host cell. In some
embodiments, the promoter is useful in a Trichoderma host. Suitable
non-limiting examples of promoters include cbh1, cbh2, egl1, egl2,
pepA, hfb1, hfb2, xyn1, and amy. In some embodiments, the promoter
is one that is native to the host cell. For example, in some
embodiments when T. reesei is the host, the promoter is a native T.
reesei promoter. In some embodiments, the promoter is T. reesei
cbh1, which is an inducible promoter and has been deposited in
GenBank under Accession No. D86235, which is hereby incorporated by
reference in its entirety, particularly with respect to promoters.
In some embodiments, the promoter is one that is heterologous to
the fungal host cell. Other examples of useful promoters include
promoters from the genes of A. awamori and A. niger glucoamylase
(glaA) (Nunberg et al., Mol. Cell Biol. 4:2306-2315, 1984 and Boel
et al., EMBO J. 3:1581-1585, 1984, which are each hereby
incorporated by reference in their entireties, particularly with
respect to promoters); Aspergillus niger alpha amylases,
Aspergillus oryzae TAKA amylase, T. reesei xln1, and the T. reesei
cellobiohydrolase 1 (EP 137280, which is hereby incorporated by
reference in its entirety, particularly with respect to
promoters).
[0280] In some embodiments, the expression vector also includes a
termination sequence. Termination control regions may also be
derived from various genes native to the host cell. In some
embodiments, the termination sequence and the promoter sequence are
derived from the same source. In another embodiment, the
termination sequence is endogenous to the host cell. A particularly
suitable terminator sequence is cbh1 derived from a Trichoderma
strain (such as T. reesei). Other useful fungal terminators include
the terminator from an A. niger or A. awamori glucoamylase nucleic
acid (Nunberg et al., Mol. Cell Biol. 4:2306-2315, 1984 and Boel et
al., EMBO J. 3:1581-1585, 1984; which are each hereby incorporated
by reference in their entireties, particularly with respect to
fungal terminators). Optionally, a termination site may be
included. For effective expression of the polypeptides, DNA
encoding the polypeptide are linked operably through initiation
codons to selected expression control regions such that expression
results in the formation of the appropriate messenger RNA.
[0281] In some embodiments, the promoter, coding, region, and
terminator all originate from the iron-sulfur cluster-interacting
redox nucleic acid, DXP pathway nucleic acid, DXP pathway
associated nucleic acid, isoprene synthase nucleic acid, or IDI
nucleic acid to be expressed. In some embodiments, the coding
region for iron-sulfur cluster-interacting redox nucleic acid, DXP
pathway nucleic acid, DXP pathway associated nucleic acid, isoprene
synthase nucleic acid, or IDI nucleic acid is inserted into a
general-purpose expression vector such that it is under the
transcriptional control of the expression construct promoter and
terminator sequences. In some embodiments, genes or part thereof
are inserted downstream of the strong cbh1 promoter.
[0282] An iron-sulfur cluster-interacting redox nucleic acid, DXP
pathway nucleic acid, DXP pathway associated nucleic acid, isoprene
synthase nucleic acid, or IDI nucleic acid can be incorporated into
a vector, such as an expression vector, using standard techniques
(Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold
Spring Harbor, 1982, which is hereby incorporated by reference in
its entirety, particularly with respect to the screening of
appropriate DNA sequences and the construction of vectors). Methods
used to ligate the DNA construct comprising a nucleic acid of
interest (such as an iron-sulfur cluster-interacting redox nucleic
acid, DXP pathway nucleic acid, DXP pathway associated nucleic
acid, isoprene synthase nucleic acid, or IDI nucleic acid), a
promoter, a terminator, and other sequences and to insert them into
a suitable vector are well known in the art. For example,
restriction enzymes can be used to cleave the iron-sulfur
cluster-interacting redox nucleic acid, DXP pathway nucleic acid,
DXP pathway associated nucleic acid, isoprene synthase nucleic
acid, or IDI nucleic acid and the vector. Then, the compatible ends
of the cleaved iron-sulfur cluster-interacting redox nucleic acid,
DXP pathway nucleic acid, DXP pathway associated nucleic acid,
isoprene synthase nucleic acid, or IDI nucleic acid and the cleaved
vector can be ligated. Linking is generally accomplished by
ligation at convenient restriction sites. If such sites do not
exist, the synthetic oligonucleotide linkers are used in accordance
with conventional practice (see, Sambrook et al., Molecular
Cloning: A Laboratory Manual, 2.sup.nd ed., Cold Spring Harbor,
1989, and Bennett and Lasure, More Gene Manipulations in Fungi,
Academic Press, San Diego, pp 70-76, 1991, which are each hereby
incorporated by reference in their entireties, particularly with
respect to oligonucleotide linkers). Additionally, vectors can be
constructed using known recombination techniques (e.g., Invitrogen
Life Technologies, Gateway Technology).
[0283] In some embodiments, it may be desirable to over-express
iron-sulfur cluster-interacting redox nucleic acid, DXP pathway
nucleic acid, DXP pathway associated nucleic acid, isoprene
synthase nucleic acid, or IDI nucleic acid at levels far higher
than currently found in naturally-occurring cells. This result may
be accomplished by the selective cloning of the nucleic acids
encoding those polypeptides into multicopy plasmids or placing
those nucleic acids under a strong inducible or constitutive
promoter. Methods for over-expressing desired polypeptides are
common and well known in the art of molecular biology and examples
may be found in Sambrook et al., Molecular Cloning: A Laboratory
Manual, 2.sup.nd ed., Cold Spring Harbor, 1989, which is hereby
incorporated by reference in its entirety, particularly with
respect to cloning techniques.
[0284] The following resources include descriptions of additional
general methodology useful in accordance with the invention:
Kreigler, Gene Transfer and Expression; A Laboratory Manual, 1990
and Ausubel et al., Eds. Current Protocols in Molecular Biology,
1994, which are each hereby incorporated by reference in their
entireties, particularly with respect to molecular biology and
cloning techniques.
Exemplary Source Organisms
[0285] Iron-sulfur cluster-interacting redox nucleic acid, DXP
pathway nucleic acid, DXP pathway associated nucleic acid, isoprene
synthase nucleic acid, or IDI nucleic acid (and their encoded
polypeptides) can be obtained from any organism that naturally
contains iron-sulfur cluster-interacting redox nucleic acid, DXP
pathway nucleic acid, DXP pathway associated nucleic acid, isoprene
synthase nucleic acid, and/or IDI nucleic acid. As noted above,
isoprene is formed naturally by a variety of organisms, such as
bacteria, yeast, plants, and animals. Organisms contain the MVA
pathway, DXP pathway, or both the MVA and DXP pathways for
producing isoprene (FIG. 19A). Thus, DXS, DXR, MCT, CMK, MCS, HDS,
or HDR nucleic acids can be obtained, e.g., from any organism that
contains the DXP pathway or contains both the MVA and DXP pathways.
IDI and isoprene synthase nucleic acids can be obtained, e.g., from
any organism that contains the MVA pathway, DXP pathway, or both
the MVA and DXP pathways.
[0286] In some embodiments, the nucleic acid sequence of the
iron-sulfur cluster-interacting redox nucleic acid, DXP pathway
nucleic acid, DXP pathway associated nucleic acid, isoprene
synthase nucleic acid, or IDI nucleic acid is identical to the
sequence of a nucleic acid that is produced by any of the following
organisms in nature. In some embodiments, the amino acid sequence
of iron-sulfur cluster-interacting redox polypeptide, DXP pathway
polypeptide, DXP pathway associated polypeptide, isoprene synthase
polypeptide, or IDI polypeptide is identical to the sequence of a
polypeptide that is produced by any of the following organisms in
nature. In some embodiments, the iron-sulfur cluster-interacting
redox nucleic acid, DXP pathway nucleic acid, DXP pathway
associated nucleic acid, isoprene synthase nucleic acid, or IDI
nucleic acid or its encoded polypeptide is a mutant nucleic acid or
polypeptide derived from any of the organisms described herein. As
used herein, "derived from" refers to the source of the nucleic
acid or polypeptide into which one or more mutations is introduced.
For example, a polypeptide that is "derived from a plant
polypeptide" refers to polypeptide of interest that results from
introducing one or more mutations into the sequence of a wild-type
(i.e., a sequence occurring in nature) plant polypeptide.
[0287] In some embodiments, the source organism is a fungus,
examples of which are species of Aspergillus such as A. oryzae and
A. niger, species of Saccharomyces such as S. cerevisiae, species
of Schizosaccharomyces such as S. pombe, and species of Trichoderma
such as T. reesei. In some embodiments, the source organism is a
filamentous fungal cell. The term "filamentous fungi" refers to all
filamentous forms of the subdivision Eumycotina (see, Alexopoulos,
C. J. (1962), Introductory Mycology, Wiley, New York). These fungi
are characterized by a vegetative mycelium with a cell wall
composed of chitin, cellulose, and other complex polysaccharides.
The filamentous fungi are morphologically, physiologically, and
genetically distinct from yeasts. Vegetative growth by filamentous
fungi is by hyphal elongation and carbon catabolism is obligatory
aerobic. The filamentous fungal parent cell may be a cell of a
species of, but not limited to, Trichoderma, (e.g., Trichoderma
reesei, the asexual morph of Hypocrea jecorina, previously
classified as T. longibrachiatum, Trichoderma viride, Trichoderma
koningii, Trichoderma harzianum) (Sheir-Neirs et al., Appl.
Microbiol. Biotechnol 20: 46-53, 1984; ATCC No. 56765 and ATCC No.
26921); Penicillium sp., Humicola sp. (e.g., H. insolens, H.
lanuginose, or H. grisea); Chrysosporium sp. (e.g., C.
lucknowense), Gliocladium sp., Aspergillus sp. (e.g., A. oryzae, A.
niger, A. sojae, A. japonicus, A. nidulans, or A. awamori) (Ward et
al., Appl. Microbiol. Biotechnol. 39: 7380743, 1993 and Goedegebuur
et al., Genet 41: 89-98, 2002), Fusarium sp., (e.g., F. roseum, F.
graminum F. cerealis, F. oxysporuim, or F. venenatum), Neurospora
sp., (e.g., N. crassa), Hypocrea sp., Mucor sp., (e.g., M. miehei),
Rhizopus sp. and Emericella sp. (see also, Innis et al., Sci. 228:
21-26, 1985). The term "Trichoderma" or "Trichoderma sp." or
"Trichoderma spp." refer to any fungal genus previously or
currently classified as Trichoderma.
[0288] In some embodiments, the fungus is A. nidulans, A. awamori,
A. oryzae, A. aculeatus, A. niger, A. japonicus, T. reesei, T.
viride, F. oxysporum, or F. solani. Aspergillus strains are
disclosed in Ward et al., Appl. Microbiol. Biotechnol. 39:738-743,
1993 and Goedegebuur et al., Curr Gene 41:89-98, 2002, which are
each hereby incorporated by reference in their entireties,
particularly with respect to fungi. In particular embodiments, the
fungus is a strain of Trichoderma, such as a strain of T. reesei.
Strains of T. reesei are known and non-limiting examples include
ATCC No. 13631, ATCC No. 26921, ATCC No. 56764, ATCC No. 56765,
ATCC No. 56767, and NRRL 15709, which are each hereby incorporated
by reference in their entireties, particularly with respect to
strains of T. reesei. In some embodiments, the host strain is a
derivative of RL-P37. RL-P37 is disclosed in Sheir-Neiss et al.,
Appl. Microbiol. Biotechnology 20:46-53, 1984, which is hereby
incorporated by reference in its entirety, particularly with
respect to strains of T. reesei.
[0289] In some embodiments, the source organism is a yeast, such as
Saccharomyces sp., Schizosaccharomyces sp., Pichia sp., or Candida
sp.
[0290] In some embodiments, the source organism is a bacterium,
such as strains of Bacillus such as B. lichenformis or B. subtilis,
strains of Pantoea such as P. citrea, strains of Pseudomonas such
as P. alcaligenes, strains of Streptomyces such as S. lividans or
S. rubiginosus, strains of Thermosynechococcus such as T.
elongatus, strains of Sinorhizobium such as S. meliloti, strains of
Helicobacter such as H. pylori, strains of Agrobacterium such as A.
tumefaciens, strains of Deinococcus such as D. radiodurans, strains
of Listeria such as L. monocytogenes, strains of Lactobacillus such
as L. spp, or strains of Escherichia such as E. coli.
[0291] As used herein, "the genus Bacillus" includes all species
within the genus "Bacillus," as known to those of skill in the art,
including but not limited to B. subtilis, B. licheniformis, B.
lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B.
amyloliquefaciens, B. clausii, B. halodurans, B. megaterium, B.
coagulans, B. circulans, B. lautus, and B. thuringiensis. It is
recognized that the genus Bacillus continues to undergo taxonomical
reorganization. Thus, it is intended that the genus include species
that have been reclassified, including but not limited to such
organisms as B. stearothermophilus, which is now named "Geobacillus
stearothermophilus." The production of resistant endospores in the
presence of oxygen is considered the defining feature of the genus
Bacillus, although this characteristic also applies to the recently
named Alicyclobacillus, Amphibacillus, Aneurinibacillus,
Anoxybacillus, Brevibacillus, Filobacillus, Gracilibacillus,
Halobacillus, Paenibacillus, Salibacillus, Thennobacillus,
Ureibacillus, and Virgibacillus.
[0292] In some embodiments, the source organism is a gram-positive
bacterium. Non-limiting examples include strains of Streptomyces
(e.g., S. lividans, S. coelicolor, or S. griseus), Bacillus,
Listeria (e.g., L. monocytogenes) or Lactobacillus (e.g., L. spp).
In some embodiments, the source organism is a gram-negative
bacterium, such as E. coli, Pseudomonas sp, or H. pylori.
[0293] In some embodiments, the source organism is a plant, such as
a plant from the family Fabaceae, such as the Faboideae subfamily.
In some embodiments, the source organism is kudzu, poplar (such as
Populus alba.times.tremula CAC35696), aspen (such as Populus
tremuloides), Quercus robur, Arabidopsis (such as A. thaliana), or
Zea (such as Z. mays).
[0294] In some embodiments, the source organism is an algae, such
as a green algae, red algae, glaucophytes, chlorarachniophytes,
euglenids, chromista, or dinoflagellates.
[0295] In some embodiments, the source organism is a
cyanobacterium, such as cyanobacteria classified into any of the
following groups based on morphology: Chroococcales,
Pleurocapsales, Oscillatoriales, Nostocales, or Stigonematales. In
some embodiments, the cyanobacterium is Thermosynechococcus
elongates.
Exemplary Host Cells
[0296] A variety of host cells can be used to express iron-sulfur
cluster-interacting redox polypeptide, DXP pathway polypeptide, DXP
pathway associated polypeptide, MVA pathway polypeptide, MVA
pathway associated polypeptide, isoprene synthase polypeptide, or
IDI polypeptide and to produce isoprene in the methods of the
claimed invention. Exemplary host cells include cells from any of
the organisms listed in the prior section under the heading
"Exemplary Source Organisms." The host cell may be a cell that
naturally produces isoprene or a cell that does not naturally
produce isoprene. In some embodiments, the host cell naturally
produces isoprene using the DXP pathway and an isoprene synthase,
and one or more DXP pathway polypeptide and iron-sulfur
cluster-interacting redox polypeptides are added to enhance
production of isoprene using this pathway. In some embodiments, the
host cell naturally produces isoprene using the DXP pathway and
isoprene synthase, and one or more DXP pathway nucleic acids, one
or more iron-sulfur cluster-interacting redox nucleic acids, and
IDI are added to enhance production of isoprene using this
pathway.
Exemplary Transformation Methods
[0297] iron-sulfur cluster-interacting redox nucleic acid, DXP
pathway nucleic acid, DXP pathway associated nucleic acid, isoprene
synthase nucleic acid, or IDI nucleic acid or its vectors
containing them can be inserted into a host cell (e.g., a plant
cell, a fungal cell, a yeast cell, or a bacterial cell described
herein) using standard techniques for expression of the encoded
iron-sulfur cluster-interacting redox polypeptide, DXP pathway
polypeptide, DXP pathway associated polypeptide, isoprene synthase
polypeptide, and/or IDI polypeptide. Introduction of a DNA
construct or vector into a host cell can be performed using
techniques such as transformation, electroporation, nuclear
microinjection, transduction, transfection (e.g., lipofection
mediated or DEAE-Dextrin mediated transfection or transfection
using a recombinant phage virus), incubation with calcium phosphate
DNA precipitate, high velocity bombardment with DNA-coated
microprojectiles, and protoplast fusion. General transformation
techniques are known in the art (see, e.g., Current Protocols in
Molecular Biology (F. M. Ausubel et al. (eds) Chapter 9, 1987;
Sambrook et al., Molecular Cloning: A Laboratory Manual, 2.sup.nd
ed., Cold Spring Harbor, 1989; and Campbell et al., Curr. Genet.
16:53-56, 1989, which are each hereby incorporated by reference in
their entireties, particularly with respect to transformation
methods). The expression of heterologous polypeptide in Trichoderma
is described in U.S. Pat. No. 6,022,725; U.S. Pat. No. 6,268,328;
U.S. Pat. No. 7,262,041; WO 2005/001036; Harkki et al.; Enzyme
Microb. Technol. 13:227-233, 1991; Harkki et al., Bio Technol.
7:596-603, 1989; EP 244,234; EP 215,594; and Nevalainen et al.,
"The Molecular Biology of Trichoderma and its Application to the
Expression of Both Homologous and Heterologous Genes," in Molecular
Industrial Mycology, Eds. Leong and Berka, Marcel Dekker Inc., NY
pp. 129-148, 1992, which are each hereby incorporated by reference
in their entireties, particularly with respect to transformation
and expression methods). Reference is also made to Cao et al.,
(Sci. 9:991-1001, 2000; EP 238023; and Yelton et al., Proceedings.
Natl. Acad. Sci. USA 81:1470-1474, 1984 (which are each hereby
incorporated by reference in their entireties, particularly with
respect to transformation methods) for transformation of
Aspergillus strains. The introduced nucleic acids may be integrated
into chromosomal DNA or maintained as extrachromosomal replicating
sequences.
[0298] Any method known in the art may be used to select
transformants. In one non-limiting example, stable transformants
including an amdS marker are distinguished from unstable
transformants by their faster growth rate and the formation of
circular colonies with a smooth, rather than ragged outline on
solid culture medium containing acetamide. Additionally, in some
cases a further test of stability is conducted by growing the
transformants on a solid non-selective medium (e.g., a medium that
lacks acetamide), harvesting spores from this culture medium, and
determining the percentage of these spores which subsequently
germinate and grow on selective medium containing acetamide.
[0299] In some embodiments, fungal cells are transformed by a
process involving protoplast formation and transformation of the
protoplasts followed by regeneration of the cell wall in a known
manner. In one specific embodiment, the preparation of Trichoderma
sp. for transformation involves the preparation of protoplasts from
fungal mycelia (see, Campbell et al., Curr. Genet. 16:53-56, 1989,
which is hereby incorporated by reference in its entirety,
particularly with respect to transformation methods). In some
embodiments, the mycelia are obtained from germinated vegetative
spores. The mycelia are treated with an enzyme that digests the
cell wall resulting in protoplasts. The protoplasts are then
protected by the presence of an osmotic stabilizer in the
suspending medium. These stabilizers include sorbitol, mannitol,
potassium chloride, magnesium sulfate, and the like. Usually the
concentration of these stabilizers varies between 0.8 M and 1.2 M.
It is desirable to use about a 1.2 M solution of sorbitol in the
suspension medium.
[0300] Uptake of DNA into the host Trichoderma sp. strain is
dependent upon the calcium ion concentration. Generally, between
about 10 mM CaCl.sub.2 and 50 mM CaCl.sub.2 is used in an uptake
solution. In addition to the calcium ion in the uptake solution,
other compounds generally included are a buffering system such as
TE buffer (10 Mm Tris, pH 7.4; 1 mM EDTA) or 10 mM MOPS, pH 6.0
buffer (morpholinepropanesulfonic acid) and polyethylene glycol
(PEG). While not intending to be bound to any particular theory, it
is believed that the polyethylene glycol acts to fuse the cell
membranes, thus permitting the contents of the medium to be
delivered into the cytoplasm of the Trichoderma sp. strain and the
plasmid DNA to be transferred to the nucleus. This fusion
frequently leaves multiple copies of the plasmid DNA integrated
into the host chromosome.
[0301] Usually a suspension containing the Trichoderma sp.
protoplasts or cells that have been subjected to a permeability
treatment at a density of 10.sup.5 to 10.sup.7/mL (such as
2.times.10.sup.6/mL) are used in the transformation. A volume of
100 .mu.L of these protoplasts or cells in an appropriate solution
(e.g., 1.2 M sorbitol and 50 mM CaCl.sub.2) are mixed with the
desired DNA. Generally, a high concentration of PEG is added to the
uptake solution. From 0.1 to 1 volume of 25% PEG 4000 can be added
to the protoplast suspension. In some embodiments, about 0.25
volumes are added to the protoplast suspension. Additives such as
dimethyl sulfoxide, heparin, spermidine, potassium chloride, and
the like may also be added to the uptake solution and aid in
transformation. Similar procedures are available for other fungal
host cells (see, e.g., U.S. Pat. Nos. 6,022,725 and 6,268,328,
which are each hereby incorporated by reference in their
entireties, particularly with respect to transformation
methods).
[0302] Generally, the mixture is then cultured at approximately
0.degree. C. for a period of between 10 to 30 minutes. Additional
PEG is then added to the mixture to further enhance the uptake of
the desired nucleic acid sequence. The 25% PEG 4000 is generally
added in volumes of 5 to 15 times the volume of the transformation
mixture; however, greater and lesser volumes may be suitable. The
25% PEG 4000 is desirably about 10 times the volume of the
transformation mixture. After the PEG is added, the transformation
mixture is then cultured either at room temperature or on ice
before the addition of a sorbitol and CaCl.sub.2 solution. The
protoplast suspension is then further added to molten aliquots of a
growth medium. When the growth medium includes a growth selection
(e.g., acetamide or an antibiotic) it permits the growth of
transformants only.
[0303] The transformation of bacterial cells may be performed
according to conventional methods, e.g., as described in Sambrook
et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor,
1982, which is hereby incorporated by reference in its entirety,
particularly with respect to transformation methods.
Exemplary Cell Culture Media
[0304] The invention also includes a cell or a population of cells
in culture that produce isoprene. By "cells in culture" is meant
two or more cells in a solution (e.g., a cell medium) that allows
the cells to undergo one or more cell divisions. "Cells in culture"
do not include plant cells that are part of a living, multicellular
plant containing cells that have differentiated into plant tissues.
In various embodiments, the cell culture includes at least or about
10, 20, 50, 100, 200, 500, 1,000, 5,000, 10,000 or more cells.
[0305] Any carbon source can be used to cultivate the host cells.
The term "carbon source" refers to one or more carbon-containing
compounds capable of being metabolized by a host cell or organism.
For example, the cell medium used to cultivate the host cells may
include any carbon source suitable for maintaining the viability or
growing the host cells.
[0306] In some embodiments, the carbon source is a carbohydrate
(such as monosaccharide, disaccharide, oligosaccharide, or
polysaccharids), invert sugar (e.g., enzymatically treated sucrose
syrup), glycerol, glycerine (e.g., a glycerine byproduct of a
biodiesel or soap-making process), dihydroxyacetone, one-carbon
source, oil (e.g., a plant or vegetable oil such as corn, palm, or
soybean oil), animal fat, animal oil, fatty acid (e.g., a saturated
fatty acid, unsaturated fatty acid, or polyunsaturated fatty acid),
lipid, phospholipid, glycerolipid, monoglyceride, diglyceride,
triglyceride, polypeptide (e.g., a microbial or plant protein or
peptide), renewable carbon source (e.g., a biomass carbon source
such as a hydrolyzed biomass carbon source), yeast extract,
component from a yeast extract, polymer, acid, alcohol, aldehyde,
ketone, amino acid, succinate, lactate, acetate, ethanol, or any
combination of two or more of the foregoing. In some embodiments,
the carbon source is a product of photosynthesis, including, but
not limited to, glucose.
[0307] Exemplary monosaccharides include glucose and fructose;
exemplary oligosaccharides include lactose and sucrose, and
exemplary polysaccharides include starch and cellulose. Exemplary
carbohydrates include C6 sugars (e.g., fructose, mannose,
galactose, or glucose) and C5 sugars (e.g., xylose or arabinose).
In some embodiments, the cell medium includes a carbohydrate as
well as a carbon source other than a carbohydrate (e.g., glycerol,
glycerine, dihydroxyacetone, one-carbon source, oil, animal fat,
animal oil, fatty acid, lipid, phospholipid, glycerolipid,
monoglyceride, diglyceride, triglyceride, renewable carbon source,
or a component from a yeast extract). In some embodiments, the cell
medium includes a carbohydrate as well as a polypeptide (e.g., a
microbial or plant protein or peptide). In some embodiments, the
microbial polypeptide is a polypeptide from yeast or bacteria. In
some embodiments, the plant polypeptide is a polypeptide from soy,
corn, canola, jatropha, palm, peanut, sunflower, coconut, mustard,
rapeseed, cottonseed, palm kernel, olive, safflower, sesame, or
linseed.
[0308] In some embodiments, the concentration of the carbohydrate
is at least or about 5 grams per liter of broth (g/L, wherein the
volume of broth includes both the volume of the cell medium and the
volume of the cells), such as at least or about 10, 15, 20, 30, 40,
50, 60, 80, 100, 150, 200, 300, 400, or more g/L. In some
embodiments, the concentration of the carbohydrate is between about
50 and about 400 g/L, such as between about 100 and about 360 g/L,
between about 120 and about 360 g/L, or between about 200 and about
300 g/L. In some embodiments, this concentration of carbohydrate
includes the total amount of carbohydrate that is added before
and/or during the culturing of the host cells.
[0309] In some embodiments, the cells are cultured under limited
glucose conditions. By "limited glucose conditions" is meant that
the amount of glucose that is added is less than or about 105%
(such as about 100%) of the amount of glucose that is consumed by
the cells. In particular embodiments, the amount of glucose that is
added to the culture medium is approximately the same as the amount
of glucose that is consumed by the cells during a specific period
of time. In some embodiments, the rate of cell growth is controlled
by limiting the amount of added glucose such that the cells grow at
the rate that can be supported by the amount of glucose in the cell
medium. In some embodiments, glucose does not accumulate during the
time the cells are cultured. In various embodiments, the cells are
cultured under limited glucose conditions for greater than or about
1, 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, or 70 hours. In
various embodiments, the cells are cultured under limited glucose
conditions for greater than or about 5, 10, 15, 20, 25, 30, 35, 40,
50, 60, 70, 80, 90, 95, or 100% of the total length of time the
cells are cultured. While not intending to be bound by any
particular theory, it is believed that limited glucose conditions
may allow more favorable regulation of the cells.
[0310] In some embodiments, the cells are cultured in the presence
of an excess of glucose. In particular embodiments, the amount of
glucose that is added is greater than about 105% (such as about or
greater than 110, 120, 150, 175, 200, 250, 300, 400, or 500%) or
more of the amount of glucose that is consumed by the cells during
a specific period of time. In some embodiments, glucose accumulates
during the time the cells are cultured.
[0311] Exemplary lipids are any substance containing one or more
fatty acids that are C4 and above fatty acids that are saturated,
unsaturated, or branched.
[0312] Exemplary oils are lipids that are liquid at room
temperature. In some embodiments, the lipid contains one or more C4
or above fatty acids (e.g., contains one or more saturated,
unsaturated, or branched fatty acid with four or more carbons). In
some embodiments, the oil is obtained from soy, corn, canola,
jatropha, palm, peanut, sunflower, coconut, mustard, rapeseed,
cottonseed, palm kernel, olive, safflower, sesame, linseed,
oleagineous microbial cells, Chinese tallow, or any combination of
two or more of the foregoing.
[0313] Exemplary fatty acids include compounds of the formula
RCOOH, where "R" is a hydrocarbon. Exemplary unsaturated fatty
acids include compounds where "R" includes at least one
carbon-carbon double bond. Exemplary unsaturated fatty acids
include, but are not limited to, oleic acid, vaccenic acid,
linoleic acid, palmitelaidic acid, and arachidonic acid. Exemplary
polyunsaturated fatty acids include compounds where "R" includes a
plurality of carbon-carbon double bonds. Exemplary saturated fatty
acids include compounds where "R" is a saturated aliphatic group.
In some embodiments, the carbon source includes one or more
C.sub.12-C.sub.22 fatty acids, such as a C.sub.12 saturated fatty
acid, a C.sub.14 saturated fatty acid, a C.sub.16 saturated fatty
acid, a C.sub.18 saturated fatty acid, a C.sub.20 saturated fatty
acid, or a C.sub.22 saturated fatty acid. In an exemplary
embodiment, the fatty acid is palmitic acid. In some embodiments,
the carbon source is a salt of a fatty acid (e.g., an unsaturated
fatty acid), a derivative of a fatty acid (e.g., an unsaturated
fatty acid), or a salt of a derivative of fatty acid (e.g., an
unsaturated fatty acid). Suitable salts include, but are not
limited to, lithium salts, potassium salts, sodium salts, and the
like. Di- and triglycerols are fatty acid esters of glycerol.
[0314] In some embodiments, the concentration of the lipid, oil,
fat, fatty acid, monoglyceride, diglyceride, or triglyceride is at
least or about 1 gram per liter of broth (g/L, wherein the volume
of broth includes both the volume of the cell medium and the volume
of the cells), such as at least or about 5, 10, 15, 20, 30, 40, 50,
60, 80, 100, 150, 200, 300, 400, or more g/L. In some embodiments,
the concentration of the lipid, oil, fat, fatty acid,
monoglyceride, diglyceride, or triglyceride is between about 10 and
about 400 g/L, such as between about 25 and about 300 g/L, between
about 60 and about 180 g/L, or between about 75 and about 150 g/L.
In some embodiments, the concentration includes the total amount of
the lipid, oil, fat, fatty acid, monoglyceride, diglyceride, or
triglyceride that is added before and/or during the culturing of
the host cells. In some embodiments, the carbon source includes
both (i) a lipid, oil, fat, fatty acid, monoglyceride, diglyceride,
or triglyceride and (ii) a carbohydrate, such as glucose. In some
embodiments, the ratio of the lipid, oil, fat, fatty acid,
monoglyceride, diglyceride, or triglyceride to the carbohydrate is
about 1:1 on a carbon basis (i.e., one carbon in the lipid, oil,
fat, fatty acid, monoglyceride, diglyceride, or triglyceride per
carbohydrate carbon). In particular embodiments, the amount of the
lipid, oil, fat, fatty acid, monoglyceride, diglyceride, or
triglyceride is between about 60 and 180 g/L, and the amount of the
carbohydrate is between about 120 and 360 g/L.
[0315] Exemplary microbial polypeptide carbon sources include one
or more polypeptides from yeast or bacteria. Exemplary plant
polypeptide carbon sources include one or more polypeptides from
soy, corn, canola, jatropha, palm, peanut, sunflower, coconut,
mustard, rapeseed, cottonseed, palm kernel, olive, safflower,
sesame, or linseed.
[0316] Exemplary renewable carbon sources include cheese whey
permeate, cornsteep liquor, sugar beet molasses, barley malt, and
components from any of the foregoing. Exemplary renewable carbon
sources also include glucose, hexose, pentose and xylose present in
biomass, such as corn, switchgrass, sugar cane, cell waste of
fermentation processes, and protein by-product from the milling of
soy, corn, or wheat. In some embodiments, the biomass carbon source
is a lignocellulosic, hemicellulosic, or cellulosic material such
as, but are not limited to, a grass, wheat, wheat straw, bagasse,
sugar cane bagasse, soft wood pulp, corn, corn cob or husk, corn
kernel, fiber from corn kernels, corn stover, switch grass, rice
hull product, or a by-product from wet or dry milling of grains
(e.g., corn, sorghum, rye, triticate, barley, wheat, and/or
distillers grains). Exemplary cellulosic materials include wood,
paper and pulp waste, herbaceous plants, and fruit pulp. In some
embodiments, the carbon source includes any plant part, such as
stems, grains, roots, or tubers. In some embodiments, all or part
of any of the following plants are used as a carbon source: corn,
wheat, rye, sorghum, triticate, rice, millet, barley, cassava,
legumes, such as beans and peas, potatoes, sweet potatoes, bananas,
sugarcane, and/or tapioca. In some embodiments, the carbon source
is a biomass hydrolysate, such as a biomass hydrolysate that
includes both xylose and glucose or that includes both sucrose and
glucose.
[0317] In some embodiments, the renewable carbon source (such as
biomass) is pretreated before it is added to the cell culture
medium. In some embodiments, the pretreatment includes enzymatic
pretreatment, chemical pretreatment, or a combination of both
enzymatic and chemical pretreatment (see, for example, Farzaneh et
al., Bioresource Technology 96 (18): 2014-2018, 2005; U.S. Pat. No.
6,176,176; U.S. Pat. No. 6,106,888; which are each hereby
incorporated by reference in their entireties, particularly with
respect to the pretreatment of renewable carbon sources). In some
embodiments, the renewable carbon source is partially or completely
hydrolyzed before it is added to the cell culture medium.
[0318] In some embodiments, the renewable carbon source (such as
corn stover) undergoes ammonia fiber expansion (AFEX) pretreatment
before it is added to the cell culture medium (see, for example,
Farzaneh et al., Bioresource Technology 96 (18): 2014-2018, 2005).
During AFEX pretreatment, a renewable carbon source is treated with
liquid anhydrous ammonia at moderate temperatures (such as about 60
to about 100.degree. C.) and high pressure (such as about 250 to
about 300 psi) for about 5 minutes. Then, the pressure is rapidly
released. In this process, the combined chemical and physical
effects of lignin solubilization, hemicellulose hydrolysis,
cellulose decrystallization, and increased surface area enables
near complete enzymatic conversion of cellulose and hemicellulose
to fermentable sugars. AFEX pretreatment has the advantage that
nearly all of the ammonia can be recovered and reused, while the
remaining serves as nitrogen source for microbes in downstream
processes. Also, a wash stream is not required for AFEX
pretreatment. Thus, dry matter recovery following the AFEX
treatment is essentially 100%. AFEX is basically a dry to dry
process. The treated renewable carbon source is stable for long
periods and can be fed at very high solid loadings in enzymatic
hydrolysis or fermentation processes. Cellulose and hemicellulose
are well preserved in the AFEX process, with little or no
degradation. There is no need for neutralization prior to the
enzymatic hydrolysis of a renewable carbon source that has
undergone AFEX pretreatment. Enzymatic hydrolysis of AFEX-treated
carbon sources produces clean sugar streams for subsequent
fermentation use.
[0319] In some embodiments, the concentration of the carbon source
(e.g., a renewable carbon source) is equivalent to at least or
about 0.1, 0.5, 1, 1.5 2, 3, 4, 5, 10, 15, 20, 30, 40, or 50%
glucose (w/v). The equivalent amount of glucose can be determined
by using standard HPLC methods with glucose as a reference to
measure the amount of glucose generated from the carbon source. In
some embodiments, the concentration of the carbon source (e.g., a
renewable carbon source) is equivalent to between about 0.1 and
about 20% glucose, such as between about 0.1 and about 10% glucose,
between about 0.5 and about 10% glucose, between about 1 and about
10% glucose, between about 1 and about 5% glucose, or between about
1 and about 2% glucose.
[0320] In some embodiments, the carbon source includes yeast
extract or one or more components of yeast extract. In some
embodiments, the concentration of yeast extract is at least 1 gram
of yeast extract per liter of broth (g/L, wherein the volume of
broth includes both the volume of the cell medium and the volume of
the cells), such at least or about 5, 10, 15, 20, 30, 40, 50, 60,
80, 100, 150, 200, 300, or more g/L. In some embodiments, the
concentration of yeast extract is between about 1 and about 300
g/L, such as between about 1 and about 200 g/L, between about 5 and
about 200 g/L, between about 5 and about 100 g/L, or between about
5 and about 60 g/L. In some embodiments, the concentration includes
the total amount of yeast extract that is added before and/or
during the culturing of the host cells. In some embodiments, the
carbon source includes both yeast extract (or one or more
components thereof) and another carbon source, such as glucose. In
some embodiments, the ratio of yeast extract to the other carbon
source is about 1:5, about 1:10, or about 1:20 (w/w).
[0321] Additionally the carbon source may also be one-carbon
substrates such as carbon dioxide, or methanol. Glycerol production
from single carbon sources (e.g., methanol, formaldehyde, or
formate) has been reported in methylotrophic yeasts (Yamada et al.,
Agric. Biol. Chem., 53(2) 541-543, 1989, which is hereby
incorporated by reference in its entirety, particularly with
respect to carbon sources) and in bacteria (Hunter et. al.,
Biochemistry, 24, 4148-4155, 1985, which is hereby incorporated by
reference in its entirety, particularly with respect to carbon
sources). These organisms can assimilate single carbon compounds,
ranging in oxidation state from methane to formate, and produce
glycerol. The pathway of carbon assimilation can be through
ribulose monophosphate, through serine, or through
xylulose-momophosphate (Gottschalk, Bacterial Metabolism, Second
Edition, Springer-Verlag: New York, 1986, which is hereby
incorporated by reference in its entirety, particularly with
respect to carbon sources). The ribulose monophosphate pathway
involves the condensation of formate with ribulose-5-phosphate to
form a six carbon sugar that becomes fructose and eventually the
three carbon product glyceraldehyde-3-phosphate. Likewise, the
serine pathway assimilates the one-carbon compound into the
glycolytic pathway via methylenetetrahydrofolate.
[0322] In addition to one and two carbon substrates, methylotrophic
organisms are also known to utilize a number of other carbon
containing compounds such as methylamine, glucosamine and a variety
of amino acids for metabolic activity. For example, methylotrophic
yeast are known to utilize the carbon from methylamine to form
trehalose or glycerol (Bellion et al., Microb. Growth Cl Compd.,
[Int. Symp.], 7.sup.th ed., 415-32. Editors: Murrell et al.,
Publisher: Intercept, Andover, UK, 1993, which is hereby
incorporated by reference in its entirety, particularly with
respect to carbon sources). Similarly, various species of Candida
metabolize alanine or oleic acid (Sulter et al., Arch. Microbiol.
153(5), 485-9, 1990, which is hereby incorporated by reference in
its entirety, particularly with respect to carbon sources).
[0323] In some embodiments, cells are cultured in a standard medium
containing physiological salts and nutrients (see, e.g., Pourquie,
J. et al., Biochemistry and Genetics of Cellulose Degradation, eds.
Aubert et al., Academic Press, pp. 71-86, 1988 and Ilmen et al.,
Appl. Environ. Microbiol. 63:1298-1306, 1997, which are each hereby
incorporated by reference in their entireties, particularly with
respect to cell medias). Exemplary growth media are common
commercially prepared media such as Luria Bertani (LB) broth,
Sabouraud Dextrose (SD) broth, or Yeast medium (YM) broth. Other
defined or synthetic growth media may also be used, and the
appropriate medium for growth of particular host cells are known by
someone skilled in the art of microbiology or fermentation
science.
[0324] In addition to an appropriate carbon source, the cell medium
desirably contains suitable minerals, salts, cofactors, buffers,
and other components known to those skilled in the art suitable for
the growth of the cultures or the enhancement of isoprene
production (see, for example, WO 2004/033646 and references cited
therein and WO 96/35796 and references cited therein, which are
each hereby incorporated by reference in their entireties,
particularly with respect cell medias and cell culture conditions).
In some embodiments where an isoprene synthase nucleic acid,
iron-sulfur cluster-interacting redox nucleic acid, DXP pathway
nucleic acid, DXP pathway associated nucleic acid, or IDI nucleic
acid is under the control of an inducible promoter, the inducing
agent (e.g., a sugar, metal salt or antimicrobial), is desirably
added to the medium at a concentration effective to induce
expression of an isoprene synthase polypeptide, iron-sulfur
cluster-interacting redox polypeptide, DXP pathway polypeptide, DXP
pathway associated polypeptide, or IDI polypeptide. In some
embodiments, cell medium has an antibiotic (such as kanamycin) that
corresponds to the antibiotic resistance nucleic acid (such as a
kanamycin resistance nucleic acid) on a vector that has one or more
isoprene synthase nucleic acid, iron-sulfur cluster-interacting
redox nucleic acid, DXP pathway nucleic acid, DXP pathway
associated nucleic acid, or IDI nucleic acid.
Exemplary Cell Culture Conditions
[0325] Materials and methods suitable for the maintenance and
growth of bacterial cultures are well known in the art. Exemplary
techniques may be found in Manual of Methods for General
Bacteriology Gerhardt et al., eds), American Society for
Microbiology, Washington, D.C. (1994) or Brock in Biotechnology: A
Textbook of Industrial Microbiology, Second Edition (1989) Sinauer
Associates, Inc., Sunderland, Mass., which are each hereby
incorporated by reference in their entireties, particularly with
respect to cell culture techniques. In some embodiments, the cells
are cultured in a culture medium under conditions permitting the
expression of one or more isoprene synthase polypeptide,
iron-sulfur cluster-interacting redox polypeptide, DXP pathway
polypeptide, DXP pathway associated polypeptide, or IDI polypeptide
encoded by a nucleic acid inserted into the host cells.
[0326] Standard cell culture conditions can be used to culture the
cells (see, for example, WO 2004/033646 and references cited
therein, which are each hereby incorporated by reference in their
entireties, particularly with respect to cell culture and
fermentation conditions). Cells are grown and maintained at an
appropriate temperature, gas mixture, and pH (such as at about 20
to about 37.degree. C., at about 6% to about 84% CO.sub.2, and at a
pH between about 5 to about 9). In some embodiments, cells are
grown at 35.degree. C. in an appropriate cell medium. In some
embodiments, e.g., cultures are cultured at approximately
28.degree. C. in appropriate medium in shake cultures or fermentors
until desired amount of isoprene production is achieved. In some
embodiments, the pH ranges for fermentation are between about pH
5.0 to about pH 9.0 (such as about pH 6.0 to about pH 8.0 or about
6.5 to about 7.0). Reactions may be performed under aerobic,
anoxic, or anaerobic conditions based on the requirements of the
host cells. Exemplary culture conditions for a given filamentous
fungus are known in the art and may be found in the scientific
literature and/or from the source of the fungi such as the American
Type Culture Collection and Fungal Genetics Stock Center.
[0327] In various embodiments, the cells are grown using any known
mode of fermentation, such as batch, fed-batch, or continuous
processes. In some embodiments, a batch method of fermentation is
used. Classical batch fermentation is a closed system where the
composition of the media is set at the beginning of the
fermentation and is not subject to artificial alterations during
the fermentation. Thus, at the beginning of the fermentation the
cell medium is inoculated with the desired host cells and
fermentation is permitted to occur adding nothing to the system.
Typically, however, "batch" fermentation is batch with respect to
the addition of carbon source and attempts are often made at
controlling factors such as pH and oxygen concentration. In batch
systems, the metabolite and biomass compositions of the system
change constantly until the time the fermentation is stopped.
Within batch cultures, cells moderate through a static lag phase to
a high growth log phase and finally to a stationary phase where
growth rate is diminished or halted. In some embodiments, cells in
log phase are responsible for the bulk of the isoprene production.
In some embodiments, cells in stationary phase produce
isoprene.
[0328] In some embodiments, a variation on the standard batch
system is used, such as the Fed-Batch system. Fed-Batch
fermentation processes comprise a typical batch system with the
exception that the carbon source is added in increments as the
fermentation progresses. Fed-Batch systems are useful when
catabolite repression is apt to inhibit the metabolism of the cells
and where it is desirable to have limited amounts of carbon source
in the cell medium. Fed-batch fermentations may be performed with
the carbon source (e.g., glucose) in a limited or excess amount.
Measurement of the actual carbon source concentration in Fed-Batch
systems is difficult and is therefore estimated on the basis of the
changes of measurable factors such as pH, dissolved oxygen, and the
partial pressure of waste gases such as CO.sub.2. Batch and
Fed-Batch fermentations are common and well known in the art and
examples may be found in Brock, Biotechnology: A Textbook of
Industrial Microbiology, Second Edition (1989) Sinauer Associates,
Inc., which is hereby incorporated by reference in its entirety,
particularly with respect to cell culture and fermentation
conditions.
[0329] In some embodiments, continuous fermentation methods are
used. Continuous fermentation is an open system where a defined
fermentation medium is added continuously to a bioreactor and an
equal amount of conditioned medium is removed simultaneously for
processing. Continuous fermentation generally maintains the
cultures at a constant high density where cells are primarily in
log phase growth.
[0330] Continuous fermentation allows for the modulation of one
factor or any number of factors that affect cell growth or isoprene
production. For example, one method maintains a limiting nutrient
such as the carbon source or nitrogen level at a fixed rate and
allows all other parameters to moderate. In other systems, a number
of factors affecting growth can be altered continuously while the
cell concentration (e.g., the concentration measured by media
turbidity) is kept constant. Continuous systems strive to maintain
steady state growth conditions. Thus, the cell loss due to media
being drawn off is balanced against the cell growth rate in the
fermentation. Methods of modulating nutrients and growth factors
for continuous fermentation processes as well as techniques for
maximizing the rate of product formation are well known in the art
of industrial microbiology and a variety of methods are detailed by
Brock, Biotechnology: A Textbook of Industrial Microbiology, Second
Edition (1989) Sinauer Associates, Inc., which is hereby
incorporated by reference in its entirety, particularly with
respect to cell culture and fermentation conditions.
[0331] In some embodiments, cells are immobilized on a substrate as
whole cell catalysts and subjected to fermentation conditions for
isoprene production.
[0332] In some embodiments, bottles of liquid culture are placed in
shakers in order to introduce oxygen to the liquid and maintain the
uniformity of the culture. In some embodiments, an incubator is
used to control the temperature, humidity, shake speed, and/or
other conditions in which a culture is grown. The simplest
incubators are insulated boxes with an adjustable heater, typically
going up to .about.65.degree. C. More elaborate incubators can also
include the ability to lower the temperature (via refrigeration),
or the ability to control humidity or CO.sub.2 levels. Most
incubators include a timer; some can also be programmed to cycle
through different temperatures, humidity levels, etc. Incubators
can vary in size from tabletop to units the size of small
rooms.
[0333] If desired, a portion or all of the cell medium can be
changed to replenish nutrients and/or avoid the build up of
potentially harmful metabolic byproducts and dead cells. In the
case of suspension cultures, cells can be separated from the media
by centrifuging or filtering the suspension culture and then
resuspending the cells in fresh media. In the case of adherent
cultures, the media can be removed directly by aspiration and
replaced. In some embodiments, the cell medium allows at least a
portion of the cells to divide for at least or about 5, 10, 20, 40,
50, 60, 65, or more cell divisions in a continuous culture (such as
a continuous culture without dilution).
[0334] In some embodiments, a constitutive or leaky promoter (such
as a Trc promoter) is used and a compound (such as IPTG) is not
added to induce expression of the isoprene synthase nucleic acid,
iron-sulfur cluster-interacting redox nucleic acid, DXP pathway
nucleic acid, DXP pathway associated nucleic acid, or IDI nucleic
acid, operably linked to the promoter. In some embodiments, a
compound (such as IPTG) is added to induce expression of the
isoprene synthase nucleic acid, iron-sulfur cluster-interacting
redox nucleic acid, DXP pathway nucleic acid, DXP pathway
associated nucleic acid, or IDI nucleic acid operably linked to the
promoter.
Exemplary Methods for Decoupling Isoprene Production from Cell
Growth.
[0335] The invention provides, inter alia, compositions and methods
for increasing the production of isoprene from cultured cells. When
feedstock is used, it is desirable for the carbon from the
feedstock to be converted to isoprene rather than to the growth and
maintenance of the cells. In some embodiments, the cells are grown
to a low to medium OD.sub.600, then production of isoprene is
started or increased. This strategy permits a large portion of the
carbon to be converted to isoprene.
[0336] In some embodiments, cells reach an optical density such
that they no longer divide or divide extremely slowly, but continue
to make isoprene for several hours (such as about 2, 4, 6, 8, 10,
15, 20, 25, 30, or more hours). In some cases, the optical density
at 550 nm decreases over time (such as a decrease in the optical
density after the cells are no longer in an exponential growth
phase due to cell lysis), and the cells continue to produce a
substantial amount of isoprene. In some embodiments, the optical
density at 550 nm of the cells increases by less than or about 50%
(such as by less than or about 40, 30, 20, 10, 5, or 0%) over a
certain time period (such as greater than or about 5, 10, 15, 20,
25, 30, 40, 50 or 60 hours), and the cells produce isoprene at
greater than or about 1, 10, 25, 50, 100, 150, 200, 250, 300, 400,
500, 600, 700, 800, 900, 1,000, 1,250, 1,500, 1,750, 2,000, 2,500,
3,000, 4,000, 5,000, or more nmole of isoprene/gram of cells for
the wet weight of the cells/hour (nmole/g.sub.wcm/hr) during this
time period. In some embodiments, the amount of isoprene is between
about 2 to about 5,000 nmole/g.sub.wcm/hr, such as between about 2
to about 100 nmole/g.sub.wcm/hr, about 100 to about 500
nmole/g.sub.wcm/hr, about 150 to about 500 nmole/g.sub.wcm/hr,
about 500 to about 1,000 nmole/g.sub.wcm/hr, about 1,000 to about
2,000 nmole/g.sub.wcm/hr, or about 2,000 to about 5,000
nmole/g.sub.wcm/hr. In some embodiments, the amount of isoprene is
between about 20 to about 5,000 nmole/g.sub.wcm/hr, about 100 to
about 5,000 nmole/g.sub.wcm/hr, about 200 to about 2,000
nmole/g.sub.wcm/hr, about 200 to about 1,000 nmole/g.sub.wcm/hr,
about 300 to about 1,000 nmole/g.sub.wcm/hr, or about 400 to about
1,000 nmole/g.sub.wcm/hr.
[0337] In some embodiments, the optical density at 550 nm of the
cells increases by less than or about 50% (such as by less than or
about 40, 30, 20, 10, 5, or 0%) over a certain time period (such as
greater than or about 5, 10, 15, 20, 25, 30, 40, 50 or 60 hours),
and the cells produce a cumulative titer (total amount) of isoprene
at greater than or about 1, 10, 25, 50, 100, 150, 200, 250, 300,
400, 500, 600, 700, 800, 900, 1,000, 1,250, 1,500, 1,750, 2,000,
2,500, 3,000, 4,000, 5,000, 10,000, 50,000, 100,000, or more mg of
isoprene/L of broth (mg/L.sub.broth, wherein the volume of broth
includes the volume of the cells and the cell medium) during this
time period. In some embodiments, the amount of isoprene is between
about 2 to about 5,000 mg/L.sub.broth, such as between about 2 to
about 100 mg/L.sub.broth, about 100 to about 500 mg/L.sub.broth,
about 500 to about 1,000 mg/L.sub.broth, about 1,000 to about 2,000
mg/L.sub.broth, or about 2,000 to about 5,000 mg/L.sub.broth. In
some embodiments, the amount of isoprene is between about 20 to
about 5,000 mg/L.sub.broth, about 100 to about 5,000
mg/L.sub.broth, about 200 to about 2,000 mg/L.sub.broth, about 200
to about 1,000 mg/L.sub.broth, about 300 to about 1,000
mg/L.sub.broth, or about 400 to about 1,000 mg/L.sub.broth.
[0338] In some embodiments, the optical density at 550 nm of the
cells increases by less than or about 50% (such as by less than or
about 40, 30, 20, 10, 5, or 0%) over a certain time period (such as
greater than or about 5, 10, 15, 20, 25, 30, 40, 50 or 60 hours),
and the cells convert greater than or about 0.0015, 0.002, 0.005,
0.01, 0.02, 0.05, 0.1, 0.12, 0.14, 0.16, 0.2, 0.3, 0.4, 0.5, 0.6,
0.7, 0.8, 0.9, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.5, 3.0, 3.5, 4.0,
5.0, 6.0, 7.0, or 8.0% of the carbon in the cell culture medium
into isoprene during this time period. In some embodiments, the
percent conversion of carbon into isoprene is between such as about
0.002 to about 4.0%, about 0.002 to about 3.0%, about 0.002 to
about 2.0%, about 0.002 to about 1.6%, about 0.002 to about 0.005%,
about 0.005 to about 0.01%, about 0.01 to about 0.05%, about 0.05
to about 0.15%, 0.15 to about 0.2%, about 0.2 to about 0.3%, about
0.3 to about 0.5%, about 0.5 to about 0.8%, about 0.8 to about
1.0%, or about 1.0 to about 1.6%. In some embodiments, the percent
conversion of carbon into isoprene is between about 0.002 to about
0.4%, 0.002 to about 0.16%, 0.04 to about 0.16%, about 0.005 to
about 0.3%, about 0.01 to about 0.3%, or about 0.05 to about
0.3%.
[0339] In some embodiments, isoprene is only produced in stationary
phase. In some embodiments, isoprene is produced in both the growth
phase and stationary phase. In various embodiments, the amount of
isoprene produced (such as the total amount of isoprene produced or
the amount of isoprene produced per liter of broth per hour per
OD.sub.600) during stationary phase is greater than or about 2, 3,
4, 5, 10, 20, 30, 40, 50, or more times the amount of isoprene
produced during the growth phase for the same length of time. In
various embodiments, greater than or about 5, 10, 20, 30, 40, 50,
60, 70, 80, 90, 95, 99% or more of the total amount of isoprene
that is produced (such as the production of isoprene during a
fermentation for a certain amount of time, such as 20 hours) is
produced while the cells are in stationary phase. In various
embodiments, greater than or about 5, 10, 20, 30, 40, 50, 60, 70,
80, 90, 95, 99% or more of the total amount of isoprene that is
produced (such as the production of isoprene during a fermentation
for a certain amount of time, such as 20 hours) is produced while
the cells divide slowly or not at all such that the optical density
at 550 nm of the cells increases by less than or about 50% (such as
by less than or about 40, 30, 20, 10, 5, or 0%). In some
embodiments, isoprene is only produced in the growth phase.
[0340] In some embodiments, one or more isoprene synthase nucleic
acid, iron-sulfur cluster-interacting redox nucleic acid, DXP
pathway nucleic acid, DXP pathway associated nucleic acid, and/or
IDI nucleic acid are placed under the control of a promoter or
factor that is more active in stationary phase than in the growth
phase. For example, one or more isoprene synthase nucleic acid,
iron-sulfur cluster-interacting redox nucleic acid, DXP pathway
nucleic acid, DXP pathway associated nucleic acid, and/or IDI
nucleic acid may be placed under control of a stationary phase
sigma factor, such as RpoS. In some embodiments, one or more
isoprene synthase nucleic acid, iron-sulfur cluster-interacting
redox nucleic acid, DXP pathway nucleic acid, DXP pathway
associated nucleic acid, and/or IDI nucleic acid are placed under
control of a promoter inducible in stationary phase, such as a
promoter inducible by a response regulator active in stationary
phase.
Production of Isoprene within Safe Operating Ranges
[0341] The invention provides, inter alia, compositions and methods
for increasing the production of isoprene from cultured cells. The
production of isoprene within safe operating levels according to
its flammability characteristics simplifies the design and
construction of commercial facilities, vastly improves the ability
to operate safely, and limits the potential for fires to occur. In
particular, the optimal ranges for the production of isoprene are
within the safe zone, i.e., the nonflammable range of isoprene
concentrations. In one such aspect, the invention features a method
for the production of isoprene within the nonflammable range of
isoprene concentrations (outside the flammability envelope of
isoprene).
[0342] Thus, computer modeling and experimental testing were used
to determine the flammability limits of isoprene (such as isoprene
in the presence of O.sub.2, N.sub.2, CO.sub.2, or any combination
of two or more of the foregoing gases) in order to ensure process
safety. The flammability envelope is characterized by the lower
flammability limit (LFL), the upper flammability limit (UFL), the
limiting oxygen concentration (LOC), and the limiting temperature.
For a system to be flammable, a minimum amount of fuel (such as
isoprene) must be in the presence of a minimum amount of oxidant,
typically oxygen. The LFL is the minimum amount of isoprene that
must be present to sustain burning, while the UFL is the maximum
amount of isoprene that can be present. Above this limit, the
mixture is fuel rich and the fraction of oxygen is too low to have
a flammable mixture. The LOC indicates the minimum fraction of
oxygen that must also be present to have a flammable mixture. The
limiting temperature is based on the flash point of isoprene and is
that lowest temperature at which combustion of isoprene can
propagate. These limits are specific to the concentration of
isoprene, type and concentration of oxidant, inerts present in the
system, temperature, and pressure of the system. Compositions that
fall within the limits of the flammability envelope propagate
combustion and require additional safety precautions in both the
design and operation of process equipment.
[0343] The following conditions were tested using computer
simulation and mathematical analysis and experimental testing. If
desired, other conditions (such as other temperature, pressure, and
permanent gas compositions) may be tested using the methods
described herein to determine the LFL, UFL, and LOC
concentrations.
(1) Computer Simulation and Mathematical Analysis
Test Suite 1:
[0344] isoprene: 0 wt %-14 wt %
O.sub.2: 6 wt %-21 wt %
N.sub.2: 79 wt %-94 wt %
Test Suite 2:
[0345] isoprene: 0 wt %-14 wt %
O.sub.2: 6 wt %-21 wt %
N.sub.2: 79 wt %-94 wt %
[0346] Saturated with H.sub.2O
Test Suite 3:
[0347] isoprene: 0 wt %-14 wt %
O.sub.2: 6 wt %-21 wt %
N.sub.2: 79 wt %-94 wt %
CO.sub.2: 5 wt %-30 wt %
(2) Experimental Testing for Final Determination of Flammability
Limits
Test Suite 1:
[0348] isoprene: 0 wt %-14 wt %
O.sub.2: 6 wt %-21 wt %
N.sub.2: 79 wt %-94 wt %
Test Suite 2:
[0349] isoprene: 0 wt %-14 wt %
O.sub.2: 6 wt %-21 wt %
N.sub.2: 79 wt %-94 wt %
[0350] Saturated with H.sub.2O
[0351] Simulation software was used to give an estimate of the
flammability characteristics of the system for several different
testing conditions. CO.sub.2 showed no significant affect on the
system's flammability limits. Test suites 1 and 2 were confirmed by
experimental testing. The modeling results were in-line with the
experimental test results. Only slight variations were found with
the addition of water.
[0352] The LOC was determined to be 9.5 vol % for an isoprene,
O.sub.2, N.sub.2, and CO.sub.2 mixture at 40.degree. C. and 1
atmosphere. The addition of up to 30% CO.sub.2 did not
significantly affect the flammability characteristics of an
isoprene, O.sub.2, and N.sub.2 mixture. Only slight variations in
flammability characteristics were shown between a dry and water
saturated isoprene, O.sub.2, and N.sub.2 system. The limiting
temperature is about -54.degree. C. Temperatures below about
-54.degree. C. are too low to propagate combustion of isoprene.
[0353] In some embodiments, the LFL of isoprene ranges from about
1.5 vol. % to about 2.0 vol %, and the UFL of isoprene ranges from
about 2.0 vol. % to about 12.0 vol. %, depending on the amount of
oxygen in the system. In some embodiments, the LOC is about 9.5 vol
% oxygen. In some embodiments, the LFL of isoprene is between about
1.5 vol. % to about 2.0 vol %, the UFL of isoprene is between about
2.0 vol. % to about 12.0 vol. %, and the LOC is about 9.5 vol %
oxygen when the temperature is between about 25.degree. C. to about
55.degree. C. (such as about 40.degree. C.) and the pressure is
between about 1 atmosphere and 3 atmospheres.
[0354] In some embodiments, isoprene is produced in the presence of
less than about 9.5 vol % oxygen (that is, below the LOC required
to have a flammable mixture of isoprene). In some embodiments in
which isoprene is produced in the presence of greater than or about
9.5 vol % oxygen, the isoprene concentration is below the LFL (such
as below about 1.5 vol. %). For example, the amount of isoprene can
be kept below the LFL by diluting the isoprene composition with an
inert gas (e.g., by continuously or periodically adding an inert
gas such as nitrogen to keep the isoprene composition below the
LFL). In some embodiments in which isoprene is produced in the
presence of greater than or about 9.5 vol % oxygen, the isoprene
concentration is above the UFL (such as above about 12 vol. %). For
example, the amount of isoprene can be kept above the UFL by using
a system (such as any of the cell culture systems described herein)
that produces isoprene at a concentration above the UFL. If
desired, a relatively low level of oxygen can be used so that the
UFL is also relatively low. In this case, a lower isoprene
concentration is needed to remain above the UFL.
[0355] In some embodiments in which isoprene is produced in the
presence of greater than or about 9.5 vol % oxygen, the isoprene
concentration is within the flammability envelope (such as between
the LFL and the UFL). In some embodiments when the isoprene
concentration may fall within the flammability envelope, one or
more steps are performed to reduce the probability of a fire or
explosion. For example, one or more sources of ignition (such as
any materials that may generate a spark) can be avoided. In some
embodiments, one or more steps are performed to reduce the amount
of time that the concentration of isoprene remains within the
flammability envelope. In some embodiments, a sensor is used to
detect when the concentration of isoprene is close to or within the
flammability envelope. If desired, the concentration of isoprene
can be measured at one or more time points during the culturing of
cells, and the cell culture conditions and/or the amount of inert
gas can be adjusted using standard methods if the concentration of
isoprene is close to or within the flammability envelope. In
particular embodiments, the cell culture conditions (such as
fermentation conditions) are adjusted to either decrease the
concentration of isoprene below the LFL or increase the
concentration of isoprene above the UFL. In some embodiments, the
amount of isoprene is kept below the LFL by diluting the isoprene
composition with an inert gas (such as by continuously or
periodically adding an inert gas to keep the isoprene composition
below the LFL).
[0356] In some embodiments, the amount of flammable volatiles other
than isoprene (such as one or more sugars) is at least about 2, 5,
10, 50, 75, or 100-fold less than the amount of isoprene produced.
In some embodiments, the portion of the gas phase other than
isoprene gas comprises between about 0% to about 100% (volume)
oxygen, such as between about 0% to about 10%, about 10% to about
20%, about 20% to about 30%, about 30% to about 40%, about 40% to
about 50%, about 50% to about 60%, about 60% to about 70%, about
70% to about 80%, about 90% to about 90%, or about 90% to about
100% (volume) oxygen. In some embodiments, the portion of the gas
phase other than isoprene gas comprises between about 0% to about
99% (volume) nitrogen, such as between about 0% to about 10%, about
10% to about 20%, about 20% to about 30%, about 30% to about 40%,
about 40% to about 50%, about 50% to about 60%, about 60% to about
70%, about 70% to about 80%, about 90% to about 90%, or about 90%
to about 99% (volume) nitrogen.
[0357] In some embodiments, the portion of the gas phase other than
isoprene gas comprises between about 1% to about 50% (volume)
CO.sub.2, such as between about 1% to about 10%, about 10% to about
20%, about 20% to about 30%, about 30% to about 40%, or about 40%
to about 50% (volume) CO.sub.2.
[0358] In some embodiments, an isoprene composition also contains
ethanol. For example, ethanol may be used for extractive
distillation of isoprene, resulting in compositions (such as
intermediate product streams) that include both ethanol and
isoprene. Desirably, the amount of ethanol is outside the
flammability envelope for ethanol. The LOC of ethanol is about 8.7
vol %, and the LFL for ethanol is about 3.3 vol % at standard
conditions, such as about 1 atmosphere and about 60.degree. F.
(NFPA 69 Standard on Explosion Prevention Systems, 2008 edition,
which is hereby incorporated by reference in its entirety,
particularly with respect to LOC, LFL, and UFL values). In some
embodiments, compositions that include isoprene and ethanol are
produced in the presence of less than the LOC required to have a
flammable mixture of ethanol (such as less than about 8.7% vol %).
In some embodiments in which compositions that include isoprene and
ethanol are produced in the presence of greater than or about the
LOC required to have a flammable mixture of ethanol, the ethanol
concentration is below the LFL (such as less than about 3.3 vol.
%).
[0359] In various embodiments, the amount of oxidant (such as
oxygen) is below the LOC of any fuel in the system (such as
isoprene or ethanol). In various embodiments, the amount of oxidant
(such as oxygen) is less than about 60, 40, 30, 20, 10, or 5% of
the LOC of isoprene or ethanol. In various embodiments, the amount
of oxidant (such as oxygen) is less than the LOC of isoprene or
ethanol by at least 2, 4, 5, or more absolute percentage points
(vol %). In particular embodiments, the amount of oxygen is at
least 2 absolute percentage points (vol %) less than the LOC of
isoprene or ethanol (such as an oxygen concentration of less than
7.5 vol % when the LOC of isoprene is 9.5 vol %). In various
embodiments, the amount of fuel (such as isoprene or ethanol) is
less than or about 25, 20, 15, 10, or 5% of the LFL for that
fuel.
Exemplary Production of Isoprene
[0360] The invention provides, inter alia, compositions and methods
for increasing the production of isoprene from cultured cells using
various DXP pathway enzymes in combination with iron-sulfur
cluster-interacting redox genes or polypeptides and isoprene
synthase genes or polypeptides, optionally with IDI and DXP pathway
associated genes and polypeptides. In some embodiments, the cells
are cultured in a culture medium under conditions permitting the
production of isoprene by the cells. By "peak absolute
productivity" is meant the maximum absolute amount of isoprene in
the off-gas during the culturing of cells for a particular period
of time (e.g., the culturing of cells during a particular
fermentation run). By "peak absolute productivity time point" is
meant the time point during a fermentation run when the absolute
amount of isoprene in the off-gas is at a maximum during the
culturing of cells for a particular period of time (e.g., the
culturing of cells during a particular fermentation run). In some
embodiments, the isoprene amount is measured at the peak absolute
productivity time point. In some embodiments, the peak absolute
productivity for the cells is about any of the isoprene amounts
disclosed herein.
[0361] By "peak specific productivity" is meant the maximum amount
of isoprene produced per cell during the culturing of cells for a
particular period of time (e.g., the culturing of cells during a
particular fermentation run). By "peak specific productivity time
point" is meant the time point during the culturing of cells for a
particular period of time (e.g., the culturing of cells during a
particular fermentation run) when the amount of isoprene produced
per cell is at a maximum. The specific productivity is determined
by dividing the total productivity by the amount of cells, as
determined by optical density at 600 nm (OD600). In some
embodiments, the isoprene amount is measured at the peak specific
productivity time point. In some embodiments, the peak specific
productivity for the cells is about any of the isoprene amounts per
cell disclosed herein.
[0362] By "cumulative total productivity" is meant the cumulative,
total amount of isoprene produced during the culturing of cells for
a particular period of time (e.g., the culturing of cells during a
particular fermentation run). In some embodiments, the cumulative,
total amount of isoprene is measured. In some embodiments, the
cumulative total productivity for the cells is about any of the
isoprene amounts disclosed herein.
[0363] By "relative detector response" refers to the ratio between
the detector response (such as the GC/MS area) for one compound
(such as isoprene) to the detector response (such as the GC/MS
area) of one or more compounds (such as all C5 hydrocarbons). The
detector response may be measured as described herein, such as the
GC/MS analysis performed with an Agilent 6890 GC/MS system fitted
with an Agilent HP-5MS GC/MS column (30 m.times.250 .mu.m; 0.25
.mu.m film thickness). If desired, the relative detector response
can be converted to a weight percentage using the response factors
for each of the compounds. This response factor is a measure of how
much signal is generated for a given amount of a particular
compound (that is, how sensitive the detector is to a particular
compound). This response factor can be used as a correction factor
to convert the relative detector response to a weight percentage
when the detector has different sensitivities to the compounds
being compared. Alternatively, the weight percentage can be
approximated by assuming that the response factors are the same for
the compounds being compared. Thus, the weight percentage can be
assumed to be approximately the same as the relative detector
response.
[0364] In some embodiments, the cells in culture produce isoprene
at greater than or about 1, 10, 25, 50, 100, 150, 200, 250, 300,
400, 500, 600, 700, 800, 900, 1,000, 1,250, 1,500, 1,750, 2,000,
2,500, 3,000, 4,000, 5,000, or more nmole of isoprene/gram of cells
for the wet weight of the cells/hour (nmole/g.sub.wcm/hr). In some
embodiments, the amount of isoprene is between about 2 to about
5,000 nmole/g.sub.wcm/hr, such as between about 2 to about 100
nmole/g.sub.wcm/hr, about 100 to about 500 nmole/g.sub.wcm/hr,
about 150 to about 500 nmole/g.sub.wcm/hr, about 500 to about 1,000
nmole/g.sub.wcm/hr, about 1,000 to about 2,000 nmole/g.sub.wcm/hr,
or about 2,000 to about 5,000 nmole/g.sub.wcm/hr. In some
embodiments, the amount of isoprene is between about 20 to about
5,000 nmole/g.sub.wcm/hr, about 100 to about 5,000
nmole/g.sub.wcm/hr, about 200 to about 2,000 nmole/g.sub.wcm/hr,
about 200 to about 1,000 nmole/g.sub.wcm/hr, about 300 to about
1,000 nmole/g.sub.wcm/hr, or about 400 to about 1,000
nmole/g.sub.wcm/hr.
[0365] The amount of isoprene in units of nmole/g.sub.wcm/hr can be
measured as disclosed in U.S. Pat. No. 5,849,970, which is hereby
incorporated by reference in its entirety, particularly with
respect to the measurement of isoprene production. For example, two
mL of headspace (e.g., headspace from a culture such as 2 mL of
culture cultured in sealed vials at 320 C with shaking at 200 rpm
for approximately 3 hours) are analyzed for isoprene using a
standard gas chromatography system, such as a system operated
isothermally (850 C) with an n-octane/porasil C column (Alltech
Associates, Inc., Deerfield, Ill.) and coupled to a RGD2 mercuric
oxide reduction gas detector (Trace Analytical, Menlo Park, Calif.)
(see, for example, Greenberg et al, Atmos. Environ. 27A: 2689-2692,
1993; Silver et al., Plant Physiol. 97:1588-1591, 1991, which are
each hereby incorporated by reference in their entireties,
particularly with respect to the measurement of isoprene
production). The gas chromatography area units are converted to
nmol isoprene via a standard isoprene concentration calibration
curve. In some embodiments, the value for the grams of cells for
the wet weight of the cells is calculated by obtaining the
A.sub.600 value for a sample of the cell culture, and then
converting the A.sub.600 value to grams of cells based on a
calibration curve of wet weights for cell cultures with a known
A.sub.600 value. In some embodiments, the grams of the cells is
estimated by assuming that one liter of broth (including cell
medium and cells) with an A.sub.600 value of 1 has a wet cell
weight of 1 gram. The value is also divided by the number of hours
the culture has been incubating for, such as three hours.
[0366] In some embodiments, the cells in culture produce isoprene
at greater than or about 1, 10, 25, 50, 100, 150, 200, 250, 300,
400, 500, 600, 700, 800, 900, 1,000, 1,250, 1,500, 1,750, 2,000,
2,500, 3,000, 4,000, 5,000, 10,000, 100,000, or more ng of
isoprene/gram of cells for the wet weight of the cells/hr
(ng/g.sub.wcm/h). In some embodiments, the amount of isoprene is
between about 2 to about 5,000 ng/g.sub.wcm/h, such as between
about 2 to about 100 ng/g.sub.wcm/h, about 100 to about 500
ng/g.sub.wcm/h, about 500 to about 1,000 ng/g.sub.wcm/h, about
1,000 to about 2,000 ng/g.sub.wcm/h, or about 2,000 to about 5,000
ng/g.sub.wcm/h. In some embodiments, the amount of isoprene is
between about 20 to about 5,000 ng/g.sub.wcm/h, about 100 to about
5,000 ng/g.sub.wcm/h, about 200 to about 2,000 ng/g.sub.wcm/h,
about 200 to about 1,000 ng/g.sub.wcm/h, about 300 to about 1,000
ng/g.sub.wcm/h, or about 400 to about 1,000 ng/g.sub.wcm/h. The
amount of isoprene in ng/g.sub.wcm/h can be calculated by
multiplying the value for isoprene production in the units of
nmole/g.sub.wcm/hr discussed above by 68.1 (as described in
Equation 5 below).
[0367] In some embodiments, the cells in culture produce a
cumulative titer (total amount) of isoprene at greater than or
about 1, 10, 25, 50, 100, 150, 200, 250, 300, 400, 500, 600, 700,
800, 900, 1,000, 1,250, 1,500, 1,750, 2,000, 2,500, 3,000, 4,000,
5,000, 10,000, 50,000, 100,000, or more mg of isoprene/L of broth
(mg/L.sub.broth, wherein the volume of broth includes the volume of
the cells and the cell medium). In some embodiments, the amount of
isoprene is between about 2 to about 5,000 mg/L.sub.broth, such as
between about 2 to about 100 mg/L.sub.broth, about 100 to about 500
mg/L.sub.broth, about 500 to about 1,000 mg/L.sub.broth, about
1,000 to about 2,000 mg/L.sub.broth, or about 2,000 to about 5,000
mg/L.sub.broth. In some embodiments, the amount of isoprene is
between about 20 to about 5,000 mg/L.sub.broth, about 100 to about
5,000 mg/L.sub.broth, about 200 to about 2,000 mg/L.sub.broth,
about 200 to about 1,000 mg/L.sub.broth, about 300 to about 1,000
mg/L.sub.broth, or about 400 to about 1,000 mg/L.sub.broth.
[0368] In some embodiments, the cells in culture produce at least
about 2 g/L.sub.broth, at least about 2.1 g/L.sub.broth, at least
about 2.2 g/L.sub.broth, at least about 2.3 g/L.sub.broth, at least
about 2.4 g/L.sub.broth, at least about 2.5 g/L.sub.broth, at least
about 2.6 g/L.sub.broth, at least about 2.7 g/L.sub.broth, at least
about 2.8 g/L.sub.broth, at least about 2.9 g/L.sub.broth, at least
about 3.0 g/L.sub.broth, at least about 3.2 g/L.sub.broth, at least
about 3.5 g/L.sub.broth, at least about 3.7 g/L.sub.broth, or at
least about 4.0 g/L.sub.broth.
[0369] The specific productivity of isoprene in mg of isoprene/L of
headspace from shake flask or similar cultures can be measured by
taking a 1 ml sample from the cell culture at an OD.sub.600 value
of approximately 1.0, putting it in a 20 mL vial, incubating for 30
minutes, and then measuring the amount of isoprene in the headspace
(as described, for example, in Example I, part II). If the
OD.sub.600 value is not 1.0, then the measurement can be normalized
to an OD.sub.600 value of 1.0 by dividing by the OD.sub.600 value.
The value of mg isoprene/L headspace can be converted to
mg/L.sub.broth/hr/OD.sub.600 of culture broth by multiplying by a
factor of 38. The value in units of mg/L.sub.broth/hr/OD.sub.600
can be multiplied by the number of hours and the OD.sub.600 value
to obtain the cumulative titer in units of mg of isoprene/L of
broth.
[0370] The instantaneous isoprene production rate in
mg/L.sub.broth/hr in a fermentor can be measured by taking a sample
of the fermentor off-gas, analyzing it for the amount of isoprene
(in units such as mg of isoprene per L.sub.gas) as described, for
example, in Example I, part II and multiplying this value by the
rate at which off-gas is passed though each liter of broth (e.g.,
at 1 vvm (volume of air/volume of broth/minute) this is 60
L.sub.gas per hour). Thus, an off-gas level of 1 mg/L.sub.gas
corresponds to an instantaneous production rate of 60
mg/L.sub.broth/hr at air flow of 1 vvm. If desired, the value in
the units mg/L.sub.broth/hr can be divided by the OD.sub.600 value
to obtain the specific rate in units of mg/L.sub.broth/hr/OD. The
average value of mg isoprene/L.sub.gas can be converted to the
total product productivity (grams of isoprene per liter of
fermentation broth, mg/L.sub.broth) by multiplying this average
off-gas isoprene concentration by the total amount of off-gas
sparged per liter of fermentation broth during the fermentation.
Thus, an average off-gas isoprene concentration of 0.5
mg/L.sub.broth/hr over 10 hours at 1 vvm corresponds to a total
product concentration of 300 mg isoprene/L.sub.broth.
[0371] In some embodiments, the cells in culture convert greater
than or about 0.0015, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.12,
0.14, 0.16, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.2, 1.4,
1.6, 1.8, 2.0, 2.5, 3.0, 3.5, 4.0, 5.0, 6.0, 7.0, or 8.0% of the
carbon in the cell culture medium into isoprene. In some
embodiments, the percent conversion of carbon into isoprene is
between such as about 0.002 to about 4.0%, about 0.002 to about
3.0%, about 0.002 to about 2.0%, about 0.002 to about 1.6%, about
0.002 to about 0.005%, about 0.005 to about 0.01%, about 0.01 to
about 0.05%, about 0.05 to about 0.15%, 0.15 to about 0.2%, about
0.2 to about 0.3%, about 0.3 to about 0.5%, about 0.5 to about
0.8%, about 0.8 to about 1.0%, or about 1.0 to about 1.6%. In some
embodiments, the percent conversion of carbon into isoprene is
between about 0.002 to about 0.4%, 0.002 to about 0.16%, 0.04 to
about 0.16%, about 0.005 to about 0.3%, about 0.01 to about 0.3%,
or about 0.05 to about 0.3%.
[0372] The percent conversion of carbon into isoprene (also
referred to as "% carbon yield") can be measured by dividing the
moles carbon in the isoprene produced by the moles carbon in the
carbon source (such as the moles of carbon in batched and fed
glucose and yeast extract). This number is multiplied by 100% to
give a percentage value (as indicated in Equation 1).
% Carbon Yield=(moles carbon in isoprene produced)/(moles carbon in
carbon source)*100 Equation 1
[0373] For this calculation, yeast extract can be assumed to
contain 50% w/w carbon. As an example, for the 500 liter described
in Example 7, part VIII, the percent conversion of carbon into
isoprene can be calculated as shown in Equation 2.
% Carbon Yield=(39.1 g isoprene*1/68.1 mol/g*5 C/mol)/[(181221 g
glucose*1/180 mol/g*6 C/mol)+(17780 g yeast extract*0.5*1/12
mol/g)]*100=0.042% Equation 2
[0374] For the two 500 liter fermentations described herein
(Example 7, parts VII and VIII), the percent conversion of carbon
into isoprene was between 0.04-0.06%. A 0.11-0.16% carbon yield has
been achieved using 14 liter systems as described herein.
[0375] One skilled in the art can readily convert the rates of
isoprene production or amount of isoprene produced into any other
units. Exemplary equations are listed below for interconverting
between units.
[0376] Units for Rate of Isoprene production (total and
specific)
1 g isoprene/L.sub.broth/hr=14.7 mmol isoprene/L.sub.broth/hr
(total volumetric rate) Equation 3
1 nmol isoprene/g.sub.wcm/hr=1 nmol
isoprene/L.sub.broth/hr/OD.sub.600 (This conversion assumes that
one liter of broth with an OD.sub.600 value of 1 has a wet cell
weight of 1 gram.) Equation 4
1 nmol isoprene/g.sub.wcm/hr=68.1 ng isoprene/g.sub.wcm/hr (given
the molecular weight of isoprene) Equation 5
1 nmol isoprene/L.sub.gas O.sub.2/hr=90 nmol
isoprene/L.sub.broth/hr (at an O.sub.2 flow rate of 90 L/hr per L
of culture broth) Equation 6
1 ug isoprene/L.sub.gas isoprene in off-gas=60 ug
isoprene/L.sub.broth/hr at a flow rate of 60 L.sub.gas per
L.sub.broth (1 vvm) Equation 7
Units for Titer (total and specific)
1 nmol isoprene/mg cell protein=150 nmol
isoprene/L.sub.broth/OD.sub.600 (This conversion assumes that one
liter of broth with an OD.sub.600 value of 1 has a total cell
protein of approximately 150 mg)(specific productivity) Equation
8
1 g isoprene/L.sub.broth=14.7 mmol isoprene/L.sub.broth (total
titer) Equation 9
[0377] If desired, Equation 10 can be used to convert any of the
units that include the wet weight of the cells into the
corresponding units that include the dry weight of the cells.
Dry weight of cells=(wet weight of cells)/3.3 Equation 10
[0378] If desired, Equation 11 can be used to convert between units
of ppm and ug/L. In particular, "ppm" means parts per million
defined in terms of ug/g (w/w). Concentrations of gases can also be
expressed on a volumetric basis using "ppmv" (parts per million by
volume), defined in terms of uL/L (vol/vol). Conversion of ug/L to
ppm (e.g., ug of analyte per g of gas) can be performed by
determining the mass per L of off-gas (i.e., the density of the
gas). For example, a liter of air at standard temperature and
pressure (STP; 101.3 kPa (1 bar) and 273.15K) has a density of
approximately 1.29 g/L. Thus, a concentration of 1 ppm (ug/g)
equals 1.29 ug/L at STP (equation 11). The conversion of ppm (ug/g)
to ug/L is a function of both pressure, temperature, and overall
composition of the off-gas.
1 ppm (ug/g) equals 1.29 ug/L at standard temperature and pressure
(STP; 101.3 kPa (1 bar) and 273.15K). Equation 11
[0379] Conversion of ug/L to ppmv (e.g., uL of analyte per L of
gas) can be performed using the Universal Gas Law (equation 12).
For example, an off-gas concentration of 1000 ug/L.sub.gas
corresponds to 14.7 umol/L.sub.gas. The universal gas constant is
0.082057 L.atm K.sup.-1mol.sup.-1, so using equation 12, the volume
occupied by 14.7 umol of HG at STP is equal to 0.329 mL. Therefore,
the concentration of 1000 ug/L HG is equal to 329 ppmv or 0.0329%
(v/v) at STP.
PV=nRT, where "P" is pressure, "V" is volume, "n" is moles of gas,
"R" is the Universal gas constant, and "T" is temperature in
Kelvin. Equation 12
[0380] The amount of impurities in isoprene compositions are
typically measured herein on a weight per volume (w/v) basis in
units such as ug/L. If desired, measurements in units of ug/L can
be converted to units of mg/m.sup.3 using equation 13.
1 ug/L=1 mg/m.sup.3 Equation 13
[0381] In some embodiments encompassed by the invention, a cell
comprising a heterologous nucleic acid encoding an isoprene
synthase polypeptide produces an amount of isoprene that is at
least or about 2-fold, 3-fold, 5-fold, 10-fold, 25-fold, 50-fold,
100-fold, 150-fold, 200-fold, 400-fold, or greater than the amount
of isoprene produced from a corresponding cell grown under
essentially the same conditions without the heterologous nucleic
acid encoding the isoprene synthase polypeptide.
[0382] In some embodiments encompassed by the invention, a cell
comprising a heterologous nucleic acid encoding an isoprene
synthase polypeptide and one or more heterologous nucleic acids
encoding a DXP pathway polypeptide produces an amount of isoprene
that is at least or about 2-fold, 3-fold, 5-fold, 10-fold, 25-fold,
50-fold, 100-fold, 150-fold, 200-fold, 400-fold, or greater than
the amount of isoprene produced from a corresponding cell grown
under essentially the same conditions without the heterologous
nucleic acids.
[0383] In some embodiments, the isoprene composition comprises
greater than or about 99.90, 99.92, 99.94, 99.96, 99.98, or 100%
isoprene by weight compared to the total weight of all C5
hydrocarbons in the composition. In some embodiments, the
composition has a relative detector response of greater than or
about 99.90, 99.91, 99.92, 99.93, 99.94, 99.95, 99.96, 99.97,
99.98, 99.99, or 100% for isoprene compared to the detector
response for all C5 hydrocarbons in the composition. In some
embodiments, the isoprene composition comprises between about 99.90
to about 99.92, about 99.92 to about 99.94, about 99.94 to about
99.96, about 99.96 to about 99.98, about 99.98 to 100% isoprene by
weight compared to the total weight of all C5 hydrocarbons in the
composition.
[0384] In some embodiments, the isoprene composition comprises less
than or about 0.12, 0.10, 0.08, 0.06, 0.04, 0.02, 0.01, 0.005,
0.001, 0.0005, 0.0001, 0.00005, or 0.00001% C5 hydrocarbons other
than isoprene (such as 1,3-cyclopentadiene, trans-1,3-pentadiene,
cis-1,3-pentadiene, 1,4-pentadiene, 1-pentyne, 2-pentyne,
3-methyl-1-butyne, pent-4-ene-1-yne, trans-pent-3-ene-1-yne, or
cis-pent-3-ene-1-yne) by weight compared to the total weight of all
C5 hydrocarbons in the composition. In some embodiments, the
composition has a relative detector response of less than or about
0.12, 0.10, 0.08, 0.06, 0.04, 0.02, 0.01, 0.005, 0.001, 0.0005,
0.0001, 0.00005, or 0.00001% for C5 hydrocarbons other than
isoprene compared to the detector response for all C5 hydrocarbons
in the composition. In some embodiments, the composition has a
relative detector response of less than or about 0.12, 0.10, 0.08,
0.06, 0.04, 0.02, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005, or
0.00001% for 1,3-cyclopentadiene, trans-1,3-pentadiene,
cis-1,3-pentadiene, 1,4-pentadiene, 1-pentyne, 2-pentyne,
3-methyl-1-butyne, pent-4-ene-1-yne, trans-pent-3-ene-1-yne, or
cis-pent-3-ene-1-yne compared to the detector response for all C5
hydrocarbons in the composition. In some embodiments, the isoprene
composition comprises between about 0.02 to about 0.04%, about 0.04
to about 0.06%, about 0.06 to 0.08%, about 0.08 to 0.10%, or about
0.10 to about 0.12% C5 hydrocarbons other than isoprene (such as
1,3-cyclopentadiene, trans-1,3-pentadiene, cis-1,3-pentadiene,
1,4-pentadiene, 1-pentyne, 2-pentyne, 3-methyl-1-butyne,
pent-4-ene-1-yne, trans-pent-3-ene-1-yne, or cis-pent-3-ene-1-yne)
by weight compared to the total weight of all C5 hydrocarbons in
the composition.
[0385] In some embodiments, the isoprene composition comprises less
than or about 50, 40, 30, 20, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, or
0.005 ug/L of a compound that inhibits the polymerization of
isoprene for any compound in the composition that inhibits the
polymerization of isoprene. In some embodiments, the isoprene
composition comprises between about 0.005 to about 50, such as
about 0.01 to about 10, about 0.01 to about 5, about 0.01 to about
1, about 0.01 to about 0.5, or about 0.01 to about 0.005 ug/L of a
compound that inhibits the polymerization of isoprene for any
compound in the composition that inhibits the polymerization of
isoprene. In some embodiments, the isoprene composition comprises
less than or about 50, 40, 30, 20, 10, 5, 1, 0.5, 0.1, 0.05, 0.01,
or 0.005 ug/L of a hydrocarbon other than isoprene (such as
1,3-cyclopentadiene, trans-1,3-pentadiene, cis-1,3-pentadiene,
1,4-pentadiene, 1-pentyne, 2-pentyne, 3-methyl-1-butyne,
pent-4-ene-1-yne, trans-pent-3-ene-1-yne, or cis-pent-3-ene-1-yne).
In some embodiments, the isoprene composition comprises between
about 0.005 to about 50, such as about 0.01 to about 10, about 0.01
to about 5, about 0.01 to about 1, about 0.01 to about 0.5, or
about 0.01 to about 0.005 ug/L of a hydrocarbon other than
isoprene. In some embodiments, the isoprene composition comprises
less than or about 50, 40, 30, 20, 10, 5, 1, 0.5, 0.1, 0.05, 0.01,
or 0.005 ug/L of a protein or fatty acid (such as a protein or
fatty acid that is naturally associated with natural rubber).
[0386] In some embodiments, the isoprene composition comprises less
than or about 10, 5, 1, 0.8, 0.5, 0.1, 0.05, 0.01, or 0.005 ppm of
alpha acetylenes, piperylenes, acetonitrile, or
1,3-cyclopentadiene. In some embodiments, the isoprene composition
comprises less than or about 5, 1, 0.5, 0.1, 0.05, 0.01, or 0.005
ppm of sulfur or allenes. In some embodiments, the isoprene
composition comprises less than or about 30, 20, 15, 10, 5, 1, 0.5,
0.1, 0.05, 0.01, or 0.005 ppm of all acetylenes (such as pentyne-1,
butyne-2, 2MB1-3yne, and 1-pentyne-4yne). In some embodiments, the
isoprene composition comprises less than or about 2000, 1000, 500,
200, 100, 50, 40, 30, 20, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, or 0.005
ppm of isoprene dimers, such as cyclic isoprene dimmers (e.g.,
cyclic C10 compounds derived from the dimerization of two isoprene
units).
[0387] In some embodiments, the composition comprises greater than
about 2 mg of isoprene, such as greater than or about 5, 10, 20,
30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800,
900, or 1000 mg of isoprene. In some embodiments, the composition
comprises greater than or about 2, 5, 10, 20, 30, 40, 50, 60, 70,
80, 90, 100 g of isoprene. In some embodiments, the amount of
isoprene in the composition is between about 2 to about 5,000 mg,
such as between about 2 to about 100 mg, about 100 to about 500 mg,
about 500 to about 1,000 mg, about 1,000 to about 2,000 mg, or
about 2,000 to about 5,000 mg. In some embodiments, the amount of
isoprene in the composition is between about 20 to about 5,000 mg,
about 100 to about 5,000 mg, about 200 to about 2,000 mg, about 200
to about 1,000 mg, about 300 to about 1,000 mg, or about 400 to
about 1,000 mg. In some embodiments, greater than or about 20, 25,
30, 40, 50, 60, 70, 80, 90, or 95% by weight of the volatile
organic fraction of the composition is isoprene.
[0388] In some embodiments, the composition includes ethanol. In
some embodiments, the composition includes between about 75 to
about 90% by weight of ethanol, such as between about 75 to about
80%, about 80 to about 85%, or about 85 to about 90% by weight of
ethanol. In some embodiments in which the composition includes
ethanol, the composition also includes between about 4 to about 15%
by weight of isoprene, such as between about 4 to about 8%, about 8
to about 12%, or about 12 to about 15% by weight of isoprene.
Exemplary Isoprene Purification Methods
[0389] In some embodiments, any of the methods described herein
further include recovering the isoprene. For example, the isoprene
produced using the compositions and methods of the invention can be
recovered using standard techniques, such as gas stripping,
membrane enhanced separation, fractionation, adsorption/desorption,
pervaporation, thermal or vacuum desorption of isoprene from a
solid phase, or extraction of isoprene immobilized or absorbed to a
solid phase with a solvent (see, for example, U.S. Pat. Nos.
4,703,007 and 4,570,029, which are each hereby incorporated by
reference in their entireties, particularly with respect to
isoprene recovery and purification methods). In one embodiment, the
isoprene is recovered by absorption stripping (see, e.g., U.S.
Appl. 61/288,142). In particular, embodiments, extractive
distillation with an alcohol (such as ethanol, methanol, propanol,
or a combination thereof) is used to recover the isoprene. In some
embodiments, the recovery of isoprene involves the isolation of
isoprene in a liquid form (such as a neat solution of isoprene or a
solution of isoprene in a solvent). Gas stripping involves the
removal of isoprene vapor from the fermentation off-gas stream in a
continuous manner. Such removal can be achieved in several
different ways including, but not limited to, adsorption to a solid
phase, partition into a liquid phase, or direct condensation (such
as condensation due to exposure to a condensation coil or do to an
increase in pressure). In some embodiments, membrane enrichment of
a dilute isoprene vapor stream above the dew point of the vapor
resulting in the condensation of liquid isoprene. In some
embodiments, the isoprene is compressed and condensed.
[0390] The recovery of isoprene may involve one step or multiple
steps. In some embodiments, the removal of isoprene vapor from the
fermentation off-gas and the conversion of isoprene to a liquid
phase are performed simultaneously. For example, isoprene can be
directly condensed from the off-gas stream to form a liquid. In
some embodiments, the removal of isoprene vapor from the
fermentation off-gas and the conversion of isoprene to a liquid
phase are performed sequentially. For example, isoprene may be
adsorbed to a solid phase and then extracted from the solid phase
with a solvent. In one embodiment, the isoprene is recovered by
using absorption stripping as described in U.S. Provisional Appl.
No. 61/288,142.
[0391] In some embodiments, any of the methods described herein
further include purifying the isoprene. For example, the isoprene
produced using the compositions and methods of the invention can be
purified using standard techniques. Purification refers to a
process through which isoprene is separated from one or more
components that are present when the isoprene is produced. In some
embodiments, the isoprene is obtained as a substantially pure
liquid. Examples of purification methods include (i) distillation
from a solution in a liquid extractant and (ii) chromatography. As
used herein, "purified isoprene" means isoprene that has been
separated from one or more components that are present when the
isoprene is produced. In some embodiments, the isoprene is at least
about 20%, by weight, free from other components that are present
when the isoprene is produced. In various embodiments, the isoprene
is at least or about 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%,
95%, or 99%, by weight, pure. Purity can be assayed by any
appropriate method, e.g., by column chromatography, HPLC analysis,
or GC-MS analysis.
[0392] In some embodiments, at least a portion of the gas phase
remaining after one or more recovery steps for the removal of
isoprene is recycled by introducing the gas phase into a cell
culture system (such as a fermentor) for the production of
isoprene.
[0393] In some embodiments, any of the methods described herein
further include polymerizing the isoprene. For example, standard
methods can be used to polymerize the purified isoprene to form
cis-polyisoprene or other down stream products using standard
methods. Accordingly, the invention also features a tire comprising
polyisoprene, such as cis-1,4-polyisoprene and/or
trans-1,4-polyisoprene made from any of the isoprene compositions
disclosed herein.
EXAMPLES
[0394] The examples, which are intended to be purely exemplary of
the invention and should therefore not be considered to limit the
invention in any way, also describe and detail aspects and
embodiments of the invention discussed above. Unless indicated
otherwise, temperature is in degrees Centigrade and pressure is at
or near atmospheric. The foregoing examples and detailed
description are offered by way of illustration and not by way of
limitation. All publications, patent applications, and patents
cited in this specification are herein incorporated by reference as
if each individual publication, patent application, or patent were
specifically and individually indicated to be incorporated by
reference. In particular, all publications cited herein are
expressly incorporated herein by reference for the purpose of
describing and disclosing compositions and methodologies which
might be used in connection with the invention. Although the
foregoing invention has been described in some detail by way of
illustration and example for purposes of clarity of understanding,
it will be readily apparent to those of ordinary skill in the art
in light of the teachings of this invention that certain changes
and modifications may be made thereto without departing from the
spirit or scope of the appended claims.
Example 1
Production of Isoprene in E. coli Expressing Recombinant Kudzu
Isoprene Synthase
[0395] I. Construction of Vectors for Expression of the Kudzu
Isoprene Synthase in E. coli
[0396] The protein sequence for the kudzu (Pueraria montana)
isoprene synthase gene (IspS) was obtained from GenBank (AAQ84170).
A kudzu isoprene synthase gene, optimized for E. coli codon usage,
was purchased from DNA2.0 (SEQ ID NO:1). The isoprene synthase gene
was removed from the supplied plasmid by restriction endonuclease
digestion with BspLU11I/PstI, gel-purified, and ligated into
pTrcHis2B (Invitrogen) that had been digested with NcoI/PstI. The
construct was designed such that the stop codon in the isoprene
synthase gene 5' to the PstI site. As a result, when the construct
was expressed the His-Tag is not attached to the isoprene synthase
protein. The resulting plasmid, pTrcKudzu, was verified by
sequencing (FIGS. 2 and 3).
[0397] The isoprene synthase gene was also cloned into pET16b
(Novagen). In this case, the isoprene synthase gene was inserted
into pET16b such that the recombinant isoprene synthase protein
contained the N-terminal His tag. The isoprene synthase gene was
amplified from pTrcKudzu by PCR using the primer set
pET-His-Kudzu-2F: 5'-CGTGAGATCATATGTGTGCGACCTCTTCTCAATTTAC (SEQ ID
NO:3) and pET-His-Kudzu-R:
5'-CGGTCGACGGATCCCTGCAGTTAGACATACATCAGCTG (SEQ ID NO:4). These
primers added an NdeI site at the 5'-end and a BamH1 site at the 3'
end of the gene respectively. The plasmid pTrcKudzu, described
above, was used as template DNA, Herculase polymerase (Stratagene)
was used according to manufacture's directions, and primers were
added at a concentration of 10 pMols. The PCR was carried out in a
total volume of 25 .mu.l. The PCR product was digested with
NdeI/BamH1 and cloned into pET16b digested with the same enzymes.
The ligation mix was transformed into E. coli Top10 (Invitrogen)
and the correct clone selected by sequencing. The resulting
plasmid, in which the kudzu isoprene synthase gene was expressed
from the T7 promoter, was designated pETNHisKudzu (FIGS. 4 and
5).
[0398] The kudzu isoprene synthase gene was also cloned into the
low copy number plasmid pCL1920. Primers were used to amplify the
kudzu isoprene synthase gene from pTrcKudzu described above. The
forward primer added a HindIII site and an E. coli consensus RBS to
the 5' end. The PstI cloning site was already present in pTrcKudzu
just 3' of the stop codon so the reverse primer was constructed
such that the final PCR product includes the PstI site. The
sequences of the primers were: HindIII-rbs-Kudzu F:
5'-CATATGAAAGCTTGTATCGATTAAATAAGGAGGAATAAACC (SEQ ID NO:6) and
BamH1-Kudzu R:
[0399] 5'-CGGTCGACGGATCCCTGCAGTTAGACATACATCAGCTG (SEQ ID NO:4). The
PCR product was amplified using Herculase polymerase with primers
at a concentration of 10 pmol and with 1 ng of template DNA
(pTrcKudzu). The amplification protocol included 30 cycles of
(95.degree. C. for 1 minute, 60.degree. C. for 1 minute, 72.degree.
C. for 2 minutes). The product was digested with HindIII and PstI
and ligated into pCL1920 which had also been digested with HindIII
and PstI. The ligation mix was transformed into E. coli Top10.
Several transformants were checked by sequencing. The resulting
plasmid was designated pCL-lac-Kudzu (FIGS. 6 and 7).
II. Determination of Isoprene Production
[0400] For the shake flask cultures, one ml of a culture was
transferred from shake flasks to 20 ml CTC headspace vials (Agilent
vial cat#5188 2753; cap cat#5188 2759). The cap was screwed on
tightly and the vials incubated at the equivalent temperature with
shaking at 250 rpm. After 30 minutes the vials were removed from
the incubator and analyzed as described below (see Table 1 for some
experimental values from this assay).
[0401] In cases where isoprene production in fermentors was
determined, samples were taken from the off-gas of the fermentor
and analyzed directly as described below (see Table 2 for some
experimental values from this assay).
[0402] The analysis was performed using an Agilent 6890 GC/MS
system interfaced with a CTC Analytics (Switzerland) CombiPAL
autosampler operating in headspace mode. An Agilent HP-5MS GC/MS
column (30 m.times.0.25 mm; 0.25 .mu.m film thickness) was used for
separation of analytes. The sampler was set up to inject 500 .mu.L
of headspace gas. The GC/MS method utilized helium as the carrier
gas at a flow of 1 ml/min. The injection port was held at
250.degree. C. with a split ratio of 50:1. The oven temperature was
held at 37.degree. C. for the 2 minute duration of the analysis.
The Agilent 5793N mass selective detector was run in single ion
monitoring (SIM) mode on m/z 67. The detector was switched off from
1.4 to 1.7 minutes to allow the elution of permanent gases. Under
these conditions isoprene (2-methyl-1,3-butadiene) was observed to
elute at 1.78 minutes. A calibration table was used to quantify the
absolute amount of isoprene and was found to be linear from 1
.mu.g/L to 2000 .mu.g/L. The limit of detection was estimated to be
50 to 100 ng/L using this method.
III. Production of Isoprene in Shake Flasks Containing E. coli
Cells Expressing Recombinant Isoprene Synthase
[0403] The vectors described above were introduced to E. coli
strain BL21 (Novagen) to produce strains BL21/ptrcKudzu,
BL21/pCL-lac-Kudzu and BL21/pETHisKudzu. The strains were spread
for isolation onto LA (Luria agar)+carbenicillin (50 .mu.g/ml) and
incubated overnight at 37.degree. C. Single colonies were
inoculated into 250 ml baffled shake flasks containing 20 ml Luria
Bertani broth (LB) and carbenicillin (100 .mu.g/ml). Cultures were
grown overnight at 20.degree. C. with shaking at 200 rpm. The
OD.sub.600 of the overnight cultures were measured and the cultures
were diluted into a 250 ml baffled shake flask containing 30 ml
MagicMedia (Invitrogen)+carbenicillin (100 .mu.g/ml) to an
OD.sub.600.about.0.05. The culture was incubated at 30.degree. C.
with shaking at 200 rpm. When the OD.sub.600.about.0.5-0.8, 400
.mu.M IPTG was added and the cells were incubated for a further 6
hours at 30.degree. C. with shaking at 200 rpm. At 0, 2, 4 and 6
hours after induction with IPTG, 1 ml aliquots of the cultures were
collected, the OD.sub.600 was determined and the amount of isoprene
produced was measured as described above. Results are shown in FIG.
8.
IV. Production of Isoprene from BL21/ptrcKudzu in 14 Liter
Fermentation
[0404] Large scale production of isoprene from E. coli containing
the recombinant kudzu isoprene synthase gene was determined from a
fed-batch culture. The recipe for the fermentation media (TM2) per
liter of fermentation medium was as follows: K.sub.2HPO.sub.4 13.6
g, KH.sub.2PO.sub.4 13.6 g, MgSO4*7H.sub.2O 2 g, citric acid
monohydrate 2 g, ferric ammonium citrate 0.3 g,
(NH.sub.4).sub.2SO.sub.4 3.2 g, yeast extract 5 g, 1000.times.
Modified Trace Metal Solution 1 ml. All of the components were
added together and dissolved in diH.sub.2O. The pH was adjusted to
6.8 with potassium hydroxide (KOH) and q.s. to volume. The final
product was filter sterilized with 0.22.mu. filter (only, do not
autoclave). The recipe for 1000.times. Modified Trace Metal
Solution was as follows: Citric Acids*H.sub.2O 40 g,
MnSO.sub.4*H.sub.2O 30 g, NaCl 10 g, FeSO.sub.4*7H.sub.2O 1 g,
CoCl.sub.2*6H.sub.2O 1 g, ZnSO.sub.4*7H.sub.2O 1 g,
CuSO.sub.4*5H.sub.2O 100 mg, H.sub.3BO.sub.3 100 mg,
NaMoO.sub.4*2H.sub.2O 100 mg. Each component was dissolved one at a
time in diH.sub.2O, pH to 3.0 with HCl/NaOH, then q.s. to volume
and filter sterilized with a 0.22.mu. filter.
[0405] This experiment was carried out in 14 L bioreactor to
monitor isoprene formation from glucose at the desired
fermentation, pH 6.7 and temperature 34.degree. C. An inoculum of
E. coli strain BL21/ptrcKudzu taken from a frozen vial was prepared
in soytone-yeast extract-glucose medium. After the inoculum grew to
OD.sub.550=0.6, two 600 ml flasks were centrifuged and the contents
resuspended in 70 ml supernatant to transfer the cell pellet (70 ml
of OD 3.1 material) to the bioreactor. At various times after
inoculation, samples were removed and the amount of isoprene
produced was determined as described above. Results are shown in
FIG. 9.
Example 2
Production of Isoprene in E. coli Expressing Recombinant Poplar
Isoprene Synthase
[0406] The protein sequence for the poplar (Populus
alba.times.Populus tremula) isoprene synthase (Schnitzler, J-P, et
al. (2005) Planta 222:777-786) was obtained from GenBank
(CAC35696). A gene, codon optimized for E. coli, was purchased from
DNA2.0 (p9796-poplar, FIGS. 30 and 31). The isoprene synthase gene
was removed from the supplied plasmid by restriction endonuclease
digestion with BspLU11I/PstI, gel-purified, and ligated into
pTrcHis2B that had been digested with NcoI/PstI. The construct is
cloned such that the stop codon in the insert is before the PstI
site, which results in a construct in which the His-Tag is not
attached to the isoprene synthase protein. The resulting plasmid
pTrcPoplar (FIGS. 26 and 27), was verified by sequencing.
Example 3
Production of Isoprene in Panteoa citrea Expressing Recombinant
Kudzu Isoprene Synthase
[0407] The pTrcKudzu and pCL-lac Kudzu plasmids described in
Example 1 were electroporated into P. citrea (U.S. Pat. No.
7,241,587). Transformants were selected on LA containing
carbenicillin (200 .mu.g/ml) or spectinomycin (50 .mu.g/ml)
respectively. Production of isoprene from shake flasks and
determination of the amount of isoprene produced was performed as
described in Example 1 for E. coli strains expressing recombinant
kudzu isoprene synthase. Results are shown in FIG. 10.
Example 4
Production of Isoprene in Bacillus subtilis Expressing Recombinant
Kudzu Isoprene Synthase
[0408] I. Construction of a B. subtilis Replicating Plasmid for the
Expression of Kudzu Isoprene Synthase
[0409] The kudzu isoprene synthase gene was expressed in Bacillus
subtilis aprEnprE Pxyl-comK strain (BG3594comK) using a replicating
plasmid (pBS19 with a chloramphenicol resistance cassette) under
control of the aprE promoter. The isoprene synthase gene, the aprE
promoter and the transcription terminator were amplified separately
and fused using PCR. The construct was then cloned into pBS19 and
transformed into B. subtilis.
a) Amplification of the aprE Promoter
[0410] The aprE promoter was amplified from chromosomal DNA from
Bacillus subtilis using the following primers:
TABLE-US-00001 CF 797 (+) Start aprE promoter MfeI (SEQ ID NO: 29)
5'-GACATCAATTGCTCCATTTTCTTCTGCTATC CF 07-43 (-) Fuse aprE promoter
to Kudzu ispS (SEQ ID NO: 30)
5'-ATTGAGAAGAGGTCGCACACACTCTTTACCCTCTCCTTTTA
b) Amplification of the Isoprene Synthase Gene
[0411] The kudzu isoprene synthase gene was amplified from plasmid
pTrcKudzu (SEQ ID NO:2). The gene had been codon optimized for E.
coli and synthesized by DNA 2.0. The following primers were
used:
TABLE-US-00002 CF 07-42 (+) Fuse the aprE promoter to kudzu
isoprene synthase gene (GTG start codon) (SEQ ID NO: 31)
5'-TAAAAGGAGAGGGTAAAGAGTGTGTGCGACCTCTTCTCAAT CF 07-45 (-) Fuse the
3' end of kudzu isoprene synthase gene to the terminator (SEQ ID
NO: 32) 5'-CCAAGGCCGGTTTTTTTTAGACATACATCAGCTGGTTAATC
c) Amplification of the Transcription Terminator
[0412] The terminator from the alkaline serine protease of Bacillus
amyliquefaciens was amplified from a previously sequenced plasmid
pJHPms382 using the following primers:
TABLE-US-00003 CF 07-44 (+) Fuse the 3' end of kudzu isoprene
synthase to the terminator (SEQ ID NO: 33)
5'-GATTAACCAGCTGATGTATGTCTAAAAAAAACCGGCCTTGG CF 07-46 (-) End of B.
amyliquefaciens terminator (BamHI) (SEQ ID NO: 34)
5'-GACATGACGGATCCGATTACGAATGCCGTCTC
[0413] The kudzu fragment was fused to the terminator fragment
using PCR with the following primers:
TABLE-US-00004 CF 07-42 (+) Fuse the aprE promoter to kudzu
isoprene synthase gene (GTG start codon) (SEQ ID NO: 32)
5'-TAAAAGGAGAGGGTAAAGAGTGTGTGCGACCTCTTCTCAAT CF 07-46 (-) End of B.
amyliquefaciens terminator (BamHI) (SEQ ID NO: 34)
5'-GACATGACGGATCCGATTACGAATGCCGTCTC
[0414] The kudzu-terminator fragment was fused to the promoter
fragment using PCR with the following primers:
TABLE-US-00005 CF 797 (+) Start aprE promoter MfeI (SEQ ID NO: 29)
5'-GACATCAATTGCTCCATTTTCTTCTGCTATC CF 07-46 (-) End of B.
amyliquefaciens terminator (BamHI) (SEQ ID NO: 34)
5'-GACATGACGGATCCGATTACGAATGCCGTCTC
[0415] The fusion PCR fragment was purified using a Qiagen kit and
digested with the restriction enzymes MfeI and BamHI. This digested
DNA fragment was gel purified using a Qiagen kit and ligated to a
vector known as pBS19, which had been digested with EcoRI and BamHI
and gel purified.
[0416] The ligation mix was transformed into E. coli Top 10 cells
and colonies were selected on LA+50 carbenicillin plates. A total
of six colonies were chosen and grown overnight in LB+50
carbenicillin and then plasmids were isolated using a Qiagen kit.
The plasmids were digested with EcoRI and BamHI to check for
inserts and three of the correct plasmids were sent in for
sequencing with the following primers:
TABLE-US-00006 CF 149 (+) EcoRI start of aprE promoter (SEQ ID NO:
36) 5'-GACATGAATTCCTCCATTTTCTTCTGC CF 847 (+) Sequence in pXX 049
(end of aprE promoter) (SEQ ID NO: 37) 5'-AGGAGAGGGTAAAGAGTGAG CF
07-45 (-) Fuse the 3' end of kudzu isoprene synthase to the
terminator (SEQ ID NO: 32)
5'-CCAAGGCCGGTTTTTTTTAGACATACATCAGCTGGTTAATC CF 07-48 (+)
Sequencing primer for kudzu isoprene synthase (SEQ ID NO: 38)
5'-CTTTTCCATCACCCACCTGAAG CF 07-49 (+) Sequencing in kudzu isoprene
synthase (SEQ ID NO: 39) 5'-GGCGAAATGGTCCAACAACAAAATTATC
[0417] The plasmid designated pBS Kudzu #2 (FIGS. 44 and 12) was
correct by sequencing and was transformed into BG 3594 comK, a
Bacillus subtilis host strain. Selection was done on LA+5
chloramphenicol plates. A transformant was chosen and struck to
single colonies on LA+5 chloramphenicol, then grown in LB+S
chloramphenicol until it reached an OD.sub.600 of 1.5. It was
stored frozen in a vial at -80.degree. C. in the presence of
glycerol. The resulting strain was designated CF 443.
II. Production of Isoprene in Shake Flasks Containing B. subtilis
Cells Expressing Recombinant Isoprene Synthase
[0418] Overnight cultures were inoculated with a single colony of
CF 443 from a LA+Chloramphenicol (Cm, 25 .mu.g/ml). Cultures were
grown in LB+Cm at 37.degree. C. with shaking at 200 rpm. These
overnight cultures (1 ml) were used to inoculate 250 ml baffled
shake flasks containing 25 ml Grants II media and chloramphenicol
at a final concentration of 25 .mu.g/ml. Grants II Media recipe was
10 g soytone, 3 ml 1M K.sub.2HPO.sub.4, 75 g glucose, 3.6 g urea,
100 ml 10.times.MOPS, q.s. to 1 L with H.sub.2O, pH 7.2;
10.times.MOPS recipe was 83.72 g MOPS, 7.17 g tricine, 12 g KOH
pellets, 10 ml 0.276M K.sub.2SO.sub.4 solution, 10 ml 0.528M
MgCl.sub.2 solution, 29.22 g NaCl, 100 ml 100.times.micronutrients,
q.s. to 1 L with H.sub.2O; and 100.times. micronutrients recipe was
1.47 g CaCl.sub.2*2H.sub.2O, 0.4 g FeSO.sub.4*7H.sub.2O, 0.1 g
MnSO.sub.4*H.sub.2O, 0.1 g ZnSO.sub.4*H.sub.2O, 0.05 g
CuCl.sub.2*2H.sub.2O, 0.1 g CoCl.sub.2*6H.sub.2O, 0.1 g
Na.sub.2MoO.sub.4*2H.sub.2O, q.s. to 1 L with H.sub.2O. Shake
flasks were incubated at 37.degree. C. and samples were taken at
18, 24, and 44 hours. At 18 hours the headspaces of CF443 and the
control strain were sampled. This represented 18 hours of
accumulation of isoprene. The amount of isoprene was determined by
gas chromatography as described in Example 1. Production of
isoprene was enhanced significantly by expressing recombinant
isoprene synthase (FIG. 11).
III. Production of Isoprene by CF443 in 14 L Fermentation
[0419] Large scale production of isoprene from B. subtilis
containing the recombinant kudzu isoprene synthase gene on a
replication plasmid was determined from a fed-batch culture.
Bacillus strain CF 443, expressing a kudzu isoprene synthase gene,
or control stain which does not express a kudzu isoprene synthase
gene were cultivated by conventional fed-batch fermentation in a
nutrient medium containing soy meal (Cargill), sodium and potassium
phosphate, magnesium sulfate and a solution of citric acid, ferric
chloride and manganese chloride. Prior to fermentation the media is
macerated for 90 minutes using a mixture of enzymes including
cellulases, hemicellulases and pectinases (see, WO95/04134). 14-L
batch fermentations are fed with 60% wt/wt glucose (Cargill DE99
dextrose, ADM Versadex greens or Danisco invert sugar) and 99%
wt/wt oil (Western Family soy oil, where the 99% wt/wt is the
concentration of oil before it was added to the cell culture
medium). Feed was started when glucose in the batch was
non-detectable. The feed rate was ramped over several hours and was
adjusted to add oil on an equal carbon basis. The pH was controlled
at 6.8-7.4 using 28% w/v ammonium hydroxide. In case of foaming,
antifoam agent was added to the media. The fermentation temperature
was controlled at 37.degree. C. and the fermentation culture was
agitated at 750 rpm. Various other parameters such as pH, D0%,
airflow, and pressure were monitored throughout the entire process.
The DO % is maintained above 20. Samples were taken over the time
course of 36 hours and analyzed for cell growth (OD.sub.550) and
isoprene production. Results of these experiments are presented in
FIGS. 45A and 45B.
IV. Integration of the Kudzu Isoprene Synthase (ispS) in B.
subtilis.
[0420] The kudzu isoprene synthase gene was cloned in an
integrating plasmid (pJH101-cmpR) under the control of the aprE
promoter. Under the conditions tested, no isoprene was
detected.
Example 5
Production of Isoprene in Trichoderma
[0421] I. Construction of Vectors for Expression of the Kudzu
Isoprene Synthase in Trichoderma reesei
[0422] The Yarrowia lipolytica codon-optimized kudzu IS gene was
synthesized by DNA 2.0 (SEQ ID NO:8) (FIG. 13). This plasmid served
as the template for the following PCR amplification reaction: 1
.mu.l plasmid template (20 ng/ul), 1 .mu.l Primer EL-945 (10 uM)
5'-GCTTATGGATCCTCTAGACTATTACACGTACATCAATTGG (SEQ ID NO:9), 1 .mu.l
Primer EL-965 (10 uM) 5'-CACCATGTGTGCAACCTCCTCCCAGTTTAC (SEQ ID
NO:10), 1 .mu.l dNTP (10 mM), 5 .mu.l 10.times. PfuUltra II Fusion
HS DNA Polymerase Buffer, 1 .mu.l PfuUltra II Fusion HS DNA
Polymerase, 40 .mu.l water in a total reaction volume of 50 .mu.l.
The forward primer contained an additional 4 nucleotides at the
5'-end that did not correspond to the Y lipolytica codon-optimized
kudzu isoprene synthase gene, but was required for cloning into the
pENTR/D-TOPO vector. The reverse primer contained an additional 21
nucleotides at the 5'-end that did not correspond to the Y.
lipolytica codon-optimized kudzu isoprene synthase gene, but were
inserted for cloning into other vector backbones. Using the MJ
Research PTC-200 Thermocycler, the PCR reaction was performed as
follows: 95.degree. C. for 2 minutes (first cycle only), 95.degree.
C. for 30 seconds, 55.degree. C. for 30 seconds, 72.degree. C. for
30 seconds (repeat for 27 cycles), 72.degree. C. for 1 minute after
the last cycle. The PCR product was analyzed on a 1.2% E-gel to
confirm successful amplification of the Y. lipolytica
codon-optimized kudzu isoprene synthase gene.
[0423] The PCR product was then cloned using the TOPO pENTR/D-TOPO
Cloning Kit following manufacturer's protocol: 1 .mu.l PCR
reaction, 1 .mu.l Salt solution, 1 .mu.l TOPO pENTR/D-TOPO vector
and 3 .mu.l water in a total reaction volume of 6 .mu.l. The
reaction was incubated at room temperature for 5 minutes. One
microliter of TOPO reaction was transformed into TOP10 chemically
competent E. coli cells. The transformants were selected on LA+50
.mu.g/ml kanamycin plates. Several colonies were picked and each
was inoculated into a 5 ml tube containing LB+50 .mu.g/ml kanamycin
and the cultures grown overnight at 37.degree. C. with shaking at
200 rpm. Plasmids were isolated from the overnight culture tubes
using QIAprep Spin Miniprep Kit, following manufacturer's protocol.
Several plasmids were sequenced to verify that the DNA sequence was
correct.
[0424] A single pENTR/D-TOPO plasmid, encoding a Y. lipolytica
codon-optimized kudzu isoprene synthase gene, was used for Gateway
Cloning into a custom-made pTrex3g vector. Construction of pTrex3g
is described in WO 2005/001036 A2. The reaction was performed
following manufacturer's protocol for the Gateway LR Clonase II
Enzyme Mix Kit (Invitrogen): 1 .mu.l Y. lipolytica codon-optimized
kudzu isoprene synthase gene pENTR/D-TOPO donor vector, 1 .mu.l
pTrex3g destination vector, 6 .mu.l TE buffer, pH 8.0 in a total
reaction volume of 8 .mu.l. The reaction was incubated at room
temperature for 1 hour and then 1 .mu.l proteinase K solution was
added and the incubation continued at 37.degree. C. for 10 minutes.
Then 1 .mu.l of reaction was transformed into TOP10 chemically
competent E. coli cells. The transformants were selected on LA+50
.mu.g/ml carbenicillin plates. Several colonies were picked and
each was inoculated into a 5 ml tube containing LB+50 .mu.lg/ml
carbenicillin and the cultures were grown overnight at 37.degree.
C. with shaking at 200 rpm. Plasmids were isolated from the
overnight culture tubes using QIAprep Spin Miniprep Kit (Qiagen,
Inc.), following manufacturer's protocol. Several plasmids were
sequenced to verify that the DNA sequence was correct.
[0425] Biolistic transformation of Y. lipolytica codon-optimized
kudzu isoprene synthase pTrex3g plasmid (FIG. 14) into a quad
delete Trichoderma reesei strain was performed using the Biolistic
PDS-1000/HE Particle Delivery System (see WO 2005/001036 A2).
Isolation of stable transformants and shake flask evaluation was
performed using protocol listed in Example 11 of patent publication
WO 2005/001036 A2.
II. Production of Isoprene in Recombinant Strains of T. reesei
[0426] One ml of 15 and 36 hour old cultures of isoprene synthase
transformants described above were transferred to head space vials.
The vials were sealed and incubated for 5 hours at 30.degree. C.
Head space gas was measured and isoprene was identified by the
method described in Example 1. Two of the transformants showed
traces of isoprene. The amount of isoprene could be increased by a
14 hour incubation. The two positive samples showed isoprene at
levels of about 0.5 .mu.g/L for the 14 hour incubation. The
untransformed control showed no detectable levels of isoprene. This
experiment shows that T. reesei is capable of producing isoprene
from endogenous precursor when supplied with an exogenous isoprene
synthase.
Example 6
Production of Isoprene in Yarrowia
[0427] I. Construction of Vectors for Expression of the Kudzu
Isoprene Synthase in Yarrowia lipolytica.
[0428] The starting point for the construction of vectors for the
expression of the kudzu isoprene synthase gene in Yarrowia
lipolytica was the vector pSPZ1(MAP29Spb). The complete sequence of
this vector (SEQ ID No:11) is shown in FIG. 15.
[0429] The following fragments were amplified by PCR using
chromosomal DNA of a Y. lipolytica strain GICC 120285 as the
template: a promotorless form of the URA3 gene, a fragment of 18S
ribosomal RNA gene, a transcription terminator of the Y. lipolytica
XPR2 gene and two DNA fragments containing the promoters of XPR2
and ICL1 genes. The following PCR primers were used:
TABLE-US-00007 ICL1 3 (SEQ ID NO: 40)
5'-GGTGAATTCAGTCTACTGGGGATTCCCAAATCTATATATACTGCA GGTGAC ICL1 5 (SEQ
ID NO: 41) 5'-GCAGGTGGGAAACTATGCACTCC XPR 3 (SEQ ID NO: 42)
5'-CCTGAATTCTGTTGGATTGGAGGATTGGATAGTGGG XPR 5 (SEQ ID NO: 43)
5'-GGTGTCGACGTACGGTCGAGCTTATTGACC XPRT3 (SEQ ID NO: 44)
5'-GGTGGGCCCGCATTTTGCCACCTACAAGCCAG XPRT 5 (SEQ ID NO: 45)
5'-GGTGAATTCTAGAGGATCCCAACGCTGTTGCCTACAACGG Y18S3 (SEQ ID NO: 46)
5'-GGTGCGGCCGCTGTCTGGACCTGGTGAGTTTCCCCG Y18S 5 (SEQ ID NO: 47)
5'-GGTGGGCCCATTAAATCAGTTATCGTTTATTTGATAG YURA3 (SEQ ID NO: 48)
5'-GGTGACCAGCAAGTCCATGGGTGGTTTGATCATGG YURA 50 (SEQ ID NO: 49)
5'-GGTGCGGCCGCCTTTGGAGTACGACTCCAACTATG YURA 51 (SEQ ID NO: 50)
5'-GCGGCCGCAGACTAAATTTATTTCAGTCTCC
[0430] For PCR amplification the PfuUltraII polymerase
(Stratagene), supplier-provided buffer and dNTPs, 2.5 .mu.M primers
and the indicated template DNA were used as per the manufacturer's
instructions. The amplification was done using the following cycle:
95.degree. C. for 1 min; 34.times. (95.degree. C. for 30 sec;
55.degree. C. for 30 sec; 72.degree. C. for 3 min) and 10 min at
72.degree. C. followed by a 4.degree. C. incubation.
[0431] Synthetic DNA molecules encoding the kudzu isoprene synthase
gene, codon-optimized for expression in Yarrowia, was obtained from
DNA 2.0 (FIG. 16; SEQ ID NO:12). Full detail of the construction
scheme of the plasmids pYLA(KZ1) and pYLI(KZ1) carrying the
synthetic kudzu isoprene synthase gene under control of XPR2 and
ICL1 promoters respectively is presented in FIG. 18. Control
plasmids in which a mating factor gene (MAP29) is inserted in place
of an isoprene synthase gene were also constructed (FIGS. 18E and
18F).
[0432] A similar cloning procedure can be used to express a poplar
(Populus alba.times.Populus tremula) isoprene synthase gene. The
sequence of the poplar isoprene is described in Miller B. et al.
(2001) Planta 213, 483-487 and shown in FIG. 17 (SEQ ID NO:13). A
construction scheme for the generation the plasmids pYLA(POP1) and
pYLI(POP1) carrying synthetic poplar isoprene synthase gene under
control of XPR2 and ICL1 promoters respectively is presented in
FIGS. 18A and B.
II. Production of Isoprene by Recombinant Strains of Y.
lipolytica.
[0433] Vectors pYLA(KZ1), pYLI(KZ1), pYLA(MAP29) and pYLI(MAP29)
were digested with SacII and used to transform the strain Y.
lipolytica CLIB 122 by a standard lithium acetate/polyethylene
glycol procedure to uridine prototrophy. Briefly, the yeast cells
grown in YEPD (1% yeast extract, 2% peptone, 2% glucose) overnight,
were collected by centrifugation (4000 rpm, 10 min), washed once
with sterile water and suspended in 0.1 M lithium acetate, pH 6.0.
Two hundred .mu.l aliquots of the cell suspension were mixed with
linearized plasmid DNA solution (10-20 .mu.g), incubated for 10
minutes at room temperature and mixed with 1 ml of 50% PEG 4000 in
the same buffer. The suspensions were further incubated for 1 hour
at room temperature followed by a 2 minutes heat shock at
42.degree. C. Cells were then plated on SC his leu plates (0.67%
yeast nitrogen base, 2% glucose, 100 mg/L each of leucine and
histidine). Transformants appeared after 3-4 days of incubation at
30.degree. C.
[0434] Three isolates from the pYLA(KZ1) transformation, three
isolates from the pYLI(KZ1) transformation, two isolates from the
pYLA(MAP29) transformation and two isolates from the pYLI(MAP29)
transformation were grown for 24 hours in YEP7 medium (1% yeast
extract, 2% peptone, pH 7.0) at 30.degree. C. with shaking. Cells
from 10 ml of culture were collected by centrifugation, resuspended
in 3 ml of fresh YEP7 and placed into 15 ml screw cap vials. The
vials were incubated overnight at room temperature with gentle (60
rpm) shaking. Isoprene content in the headspace of these vials was
analyzed by gas chromatography using mass-spectrometric detector as
described in Example 1. All transformants obtained with pYLA(KZ1)
and pYLI(KZ1) produced readily detectable amounts of isoprene (0.5
.mu.g/L to 1 .mu.g/L, FIG. 20). No isoprene was detected in the
headspace of the control strains carrying phytase gene instead of
an isoprene synthase gene.
Example 7
Production of Isoprene in E. coli Expressing Kudzu Isoprene
Synthase and idi, or dxs, or idi and dxs
[0435] I. Construction of Vectors Encoding Kudzu Isoprene Synthase
and idi, or dxs, or idi and dxs for the Production of Isoprene in
E. coli i) Construction of pTrcKudzuKan
[0436] The bla gene of pTrcKudzu (described in Example 1) was
replaced with the gene conferring kanamycin resistance. To remove
the bla gene, pTrcKudzu was digested with BspHI, treated with
Shrimp Alkaline Phosphatase (SAP), heat killed at 65.degree. C.,
then end-filled with Klenow fragment and dNTPs. The 5 kbp large
fragment was purified from an agarose gel and ligated to the
kan.sup.r gene which had been PCR amplified from pCR-Blunt-II-TOPO
using primers MCM22 5'-GATCAAGCTTAACCGGAATTGCCAGCTG (SEQ ID NO:14)
and MCM23 5'-GATCCGATCGTCAGAAGAACTCGTCAAGAAGGC (SEQ ID NO:15),
digested with HindIII and PvuI, and end-filled. A transformant
carrying a plasmid conferring kanamycin resistance (pTrcKudzuKan)
was selected on LA containing kanamycin 50 .mu.g/ml.
ii) Construction of pTrcKudzu yIDI Kan
[0437] pTrcKudzuKan was digested with PstI, treated with SAP, heat
killed and gel purified. It was ligated to a PCR product encoding
idi from S. cerevisiae with a synthetic RBS. The primers for PCR
were NsiI-YIDI 1 F 5'-CATCAATGCATCGCCCTTAGGAGGTAAAAAAAAATGAC (SEQ
ID NO:16) and PstI-YIDI 1 R 5'-CCTTCTGCAGGACGCGTTGTTATAGC (SEQ ID
NO:17); and the template was S. cerevisiae genomic DNA. The PCR
product was digested with NsiI and PstI and gel purified prior to
ligation. The ligation mixture was transformed into chemically
competent TOP10 cells and selected on LA containing 50 .mu.g/ml
kanamycin. Several transformants were isolated and sequenced and
the resulting plasmid was called pTrcKudzu-yIDI(kan) (FIGS. 28 and
29).
iii) Construction of pTrcKudzu DXS Kan
[0438] Plasmid pTrcKudzuKan was digested with PstI, treated with
SAP, heat killed and gel purified. It was ligated to a PCR product
encoding dxs from E. coli with a synthetic RBS. The primers for PCR
were MCM13
5'-GATCATGCATTCGCCCTTAGGAGGTAAAAAAACATGAGTTTTGATATTGCCAAATACCCG
(SEQ ID NO:18) and MCM14 5'-CATGCTGCAGTTATGCCAGCCAGGCCTTGAT (SEQ ID
NO:19); and the template was E. coli genomic DNA. The PCR product
was digested with NsiI and PstI and gel purified prior to ligation.
The resulting transformation reaction was transformed into TOP10
cells and selected on LA with kanamycin 50 .mu.g/ml. Several
transformants were isolated and sequenced and the resulting plasmid
was called pTrcKudzu-DXS(kan) (FIGS. 30 and 31).
iv) Construction of pTrcKudzu-yIDI-Dxs (Kan)
[0439] pTrcKudzu-yIDI(kan) was digested with PstI, treated with
SAP, heat killed and gel purified. It was ligated to a PCR product
encoding E. coli dxs with a synthetic RBS (primers MCM 13
5'-GATCATGCATTCGCCCTTAGGAGGTAAAAAAACATGAGTTTTGATATTGCCAAATACCCG
(SEQ ID NO:18) and MCM14 5'-CATGCTGCAGTTATGCCAGCCAGGCCTTGAT (SEQ ID
NO:19); template TOP10 cells) which had been digested with NsiI and
PstI and gel purified. The final plasmid was called
pTrcKudzu-yIDI-dxs (kan) (FIGS. 21 and 22).
v) Construction of pCL PtrcKudzu
[0440] A fragment of DNA containing the promoter, structural gene
and terminator from Example 1 above was digested from pTrcKudzu
using SspI and gel purified. It was ligated to pCL1920 which had
been digested with PvuII, treated with SAP and heat killed. The
resulting ligation mixture was transformed into TOP10 cells and
selected in LA containing spectinomycin 50 .mu.g/ml. Several clones
were isolated and sequenced and two were selected. pCL PtrcKudzu
and pCL PtrcKudzu (A3) have the insert in opposite orientations
(FIGS. 32-35).
vi) Construction of pCL PtrcKudzu yIDI
[0441] The NsiI-PstI digested, gel purified, IDI PCR amplicon from
(ii) above was ligated into pCL PtrcKudzu which had been digested
with PstI, treated with SAP, and heat killed. The ligation mixture
was transformed into TOP10 cells and selected in LA containing
spectinomycin 50 .mu.g/ml. Several clones were isolated and
sequenced and the resulting plasmid is called pCL PtrcKudzu yIDI
(FIGS. 36 and 37).
vii) Construction of pCL PtrcKudzu DXS
[0442] The NsiI-PstI digested, gel purified, DXS PCR amplicon from
(iii) above was ligated into pCL PtrcKudzu (A3) which had been
digested with PstI, treated with SAP, and heat killed. The ligation
mixture was transformed into TOP10 cells and selected in LA
containing spectinomycin 50 .mu.g/ml. Several clones were isolated
and sequenced and the resulting plasmid is called pCL PtrcKudzu DXS
(FIGS. 38 and 39).
II. Measurement of Isoprene in Headspace from Cultures Expressing
Kudzu Isoprene synthase, idi, and/or dxs at Different Copy
Numbers.
[0443] Cultures of E. coli BL21(.lamda.DE3) previously transformed
with plasmids pTrcKudzu(kan) (A), pTrcKudzu-yIDI kan (B),
pTrcKudzu-DXS kan (C), pTrcKudzu-yIDI-DXS kan (D) were grown in LB
kanamycin 50 .mu.g/mL. Cultures of pCL PtrcKudzu (E), pCL
PtrcKudzu, pCL PtrcKudzu-yIDI (F) and pCL PtrcKudzu-DXS (G) were
grown in LB spectinomycin 50 .mu.g/mL. Cultures were induced with
400 .mu.M IPTG at time 0 (OD.sub.600 approximately 0.5) and samples
taken for isoprene headspace measurement (see Example 1). Results
are shown in FIG. 23A-23G.
[0444] Plasmid pTrcKudzu-yIDI-dxs (kan) was introduced into E. coli
strain BL21 by transformation. The resulting strain BL21/pTrc Kudzu
IDI DXS was grown overnight in LB containing kanamycin (50
.mu.g/ml) at 20.degree. C. and used to inoculate shake flasks of
TM3 (13.6 g K.sub.2PO.sub.4, 13.6 g KH.sub.2PO.sub.4, 2.0 g
MgSO.sub.4*7H.sub.2O), 2.0 g citric acid monohydrate, 0.3 g ferric
ammonium citrate, 3.2 g (NH.sub.4).sub.2SO.sub.4, 0.2 g yeast
extract, 1.0 ml 1000.times. Modified Trace Metal Solution, adjusted
to pH 6.8 and q.s. to H.sub.2O, and filter sterilized) containing
1% glucose. Flasks were incubated at 30.degree. C. until an
OD.sub.600 of 0.8 was reached, and then induced with 400 .mu.M
IPTG. Samples were taken at various times after induction and the
amount of isoprene in the head space was measured as described in
Example 1. Results are shown in FIG. 23H.
III. Production of Isoprene from Biomass in E. coli/pTrcKudzu yIDI
DXS
[0445] The strain BL21 pTrcKudzuIDIDXS was tested for the ability
to generate isoprene from three types of biomass; bagasse, corn
stover and soft wood pulp with glucose as a control. Hydrolysates
of the biomass were prepared by enzymatic hydrolysis (Brown, L and
Torget, R., 1996, NREL standard assay method Lap-009 "Enzymatic
Saccharification of Lignocellulosic Biomass") and used at a
dilution based upon glucose equivalents. In this example, glucose
equivalents were equal to 1% glucose. A single colony from a plate
freshly transformed cells of BL21 (DE3) pTrcKudzu yIDI DXS (kan)
was used to inoculate 5 ml of LB plus kanamycin (50 .mu.g/ml). The
culture was incubated overnight at 25.degree. C. with shaking. The
following day the overnight culture was diluted to an OD.sub.600 of
0.05 in 25 ml of TM3+0.2% YE+1% feedstock. The feedstock was corn
stover, bagasse, or softwood pulp. Glucose was used as a positive
control and no glucose was used as a negative control. Cultures
were incubated at 30.degree. C. with shaking at 180 rpm. The
culture was monitored for OD.sub.600 and when it reached an
OD.sub.600 of .about.0.8, cultures were analyzed at 1 and 3 hours
for isoprene production as described in Example 1. Cultures are not
induced. All cultures containing added feedstock produce isoprene
equivalent to those of the glucose positive control. Experiments
were done in duplicate and are shown in FIG. 40.
IV. Production of Isoprene from Invert Sugar in E.
coli/pTrcKudzuIDIDXS
[0446] A single colony from a plate freshly transformed cells of
BL21 (.lamda.DE3)/pTrcKudzu yIDI DXS (kan) was used to inoculate 5
mL of LB+kanamycin (50 .mu.g/ml). The culture was incubated
overnight at 25.degree. C. with shaking. The following day the
overnight culture was diluted to an OD.sub.600 of 0.05 in 25 ml of
TM3+0.2% YE+1% feedstock. Feedstock was glucose, inverted glucose
or corn stover. The invert sugar feedstock (Danisco Invert Sugar)
was prepared by enzymatically treating sucrose syrup. AFEX corn
stover was prepared as described below (Part V). The cells were
grown at 30.degree. C. and the first sample was measured when the
cultures reached an OD.sub.600.about.0.8-1.0 (0 hour). The cultures
were analyzed for growth as measured by OD.sub.600 and for isoprene
production as in Example 1 at 0, 1 and 3 hours. Results are shown
in FIG. 41.
V. Preparation of Hydrolysate from AFEX Pretreated Corn Stover
[0447] AFEX pretreated corn stover was obtained from Michigan
Biotechnology Institute. The pretreatment conditions were 60%
moisture, 1:1 ammonia loading, and 90.degree. C. for 30 minutes,
then air dried. The moisture content in the AFEX pretreated corn
stover was 21.27%. The contents of glucan and xylan in the AFEX
pretreated corn stover were 31.7% and 19.1% (dry basis),
respectively. The saccharification process was as follows; 20 g of
AFEX pretreated corn stover was added into a 500 ml flask with 5 ml
of 1 M sodium citrate buffer pH 4.8, 2.25 ml of Accellerase 1000,
0.1 ml of Grindamyl H121 (Danisco xylanase product from Aspergillus
niger for bread-making industry), and 72.65 ml of DI water. The
flask was put in an orbital shaker and incubated at 50.degree. C.
for 96 hours. One sample was taken from the shaker and analyzed
using HPLC. The hydrolysate contained 38.5 g/1 of glucose, 21.8 g/1
of xylose, and 10.3 g/1 of oligomers of glucose and/or xylose.
VI. The Effect of Yeast Extract on Isoprene Production in E. coli
Grown in Fed-Batch Culture
[0448] Fermentation was performed at the 14-L scale as previously
described with E. coli cells containing the pTrcKudzu yIDI DXS
plasmid described above. Yeast extract (Bio Springer, Montreal,
Quebec, Canada) was fed at an exponential rate. The total amount of
yeast extract delivered to the fermentor was varied between 70-830
g during the 40 hour fermentation. Optical density of the
fermentation broth was measured at a wavelength of 550 nm. The
final optical density within the fermentors was proportional to the
amount of yeast extract added (FIG. 42A). The isoprene level in the
off-gas from the fermentor was determined as previously described.
The isoprene titer increased over the course of the fermentation
(FIG. 42B). The amount of isoprene produced was linearly
proportional to the amount of fed yeast extract (FIG. 42C).
VII. Production of Isoprene in 500 L Fermentation of pTrcKudzu DXS
yIDI
[0449] A 500 liter fermentation of E. coli cells with a kudzu
isoprene synthase, S. cerevisiae IDI, and E. coli DXS nucleic acids
(E. coli BL21 (.lamda.DE3) pTrc Kudzu dxs yidi) was used to produce
isoprene. The levels of isoprene varied from 50 to 300 .mu.g/L over
a time period of 15 hours. On the basis of the average isoprene
concentrations, the average flow through the device and the extent
of isoprene breakthrough, the amount of isoprene collected was
calculated to be approximately 17 g.
VIII. Production of Isoprene in 500 L Fermentation of E. coli Grown
in Fed-Batch Culture
Medium Recipe (Per Liter Fermentation Medium):
[0450] K.sub.2HPO.sub.4 7.5 g, MgSO.sub.4*7H.sub.2O 2 g, citric
acid monohydrate 2 g, ferric ammonium citrate 0.3 g, yeast extract
0.5 g, 1000.times. Modified Trace Metal Solution 1 ml. All of the
components were added together and dissolved in diH.sub.2O. This
solution was autoclaved. The pH was adjusted to 7.0 with ammonium
gas (NH.sub.3) and q.s. to volume. Glucose 10 g, thiamine*HCl 0.1
g, and antibiotic were added after sterilization and pH
adjustment.
1000.times. Modified Trace Metal Solution:
[0451] Citric Acids*H.sub.2O 40 g, MnSO.sub.4*H.sub.2O 30 g, NaCl
10 g, FeSO.sub.4*7H.sub.2O 1 g, CoCl.sub.2*6H.sub.2O 1 g,
ZnSO.sub.4*7H.sub.2O 1 g, CuSO.sub.4*5H.sub.2O 100 mg,
H.sub.3BO.sub.3 100 mg, NaMoO.sub.4*2H.sub.2O 100 mg. Each
component is dissolved one at a time in DI H.sub.2O, pH to 3.0 with
HCl/NaOH, then q.s. to volume and filter sterilized with 0.22
micron filter.
[0452] Fermentation was performed in a 500-L bioreactor with E.
coli cells containing the pTrcKudzu yIDI DXS plasmid. This
experiment was carried out to monitor isoprene formation from
glucose and yeast extract at the desired fermentation pH 7.0 and
temperature 30.degree. C. An inoculum of E. coli strain taken from
a frozen vial was prepared in soytone-yeast extract-glucose medium.
After the inoculum grew to OD 0.15, measured at 550 nm, 20 ml was
used to inoculate a bioreactor containing 2.5-L soytone-yeast
extract-glucose medium. The 2.5-L bioreactor was grown at
30.degree. C. to OD 1.0 and 2.0-L was transferred to the 500-L
bioreactor.
[0453] Yeast extract (Bio Springer, Montreal, Quebec, Canada) and
glucose were fed at exponential rates. The total amount of glucose
and yeast extract delivered to the bioreactor during the 50 hour
fermentation was 181.2 kg and 17.6 kg, respectively. The optical
density within the bioreactor over time is shown in FIG. 43A. The
isoprene level in the off-gas from the bioreactor was determined as
previously described. The isoprene titer increased over the course
of the fermentation (FIG. 43B). The total amount of isoprene
produced during the 50 hour fermentation was 55.1 g and the time
course of production is shown in FIG. 43C.
Example 8
Overexpression of Flavodoxin I (MA) Increase Isoprene Production in
a Strain Expressing Over-Expressing E. coli dxs, Saccharomyces idi,
and Kudzu Isoprene Synthase
[0454] BL21 (DE3) strain harboring pTrcKudzuDXSyIDI produced more
isoprene under non-inducing conditions compared to IPTG induction
conditions, and was observed to accumulate HMBPP
((E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate), the substrate of
HDS (GcpE or IspG). Using the BL21 (DE3) strain harboring
pTrcKudzuDXSyIDI as the parental host strain, the introduction of
an additional plasmid-born copy of the Kudzu isoprene synthase gene
alone and in combination with the fldA gene encoding flavodoxin I
were assessed for the effects on isoprene production by the strains
under non-inducing conditions relative to the empty vector control
strain.
[0455] These experiments investigated whether an additional copy of
the isoprene synthase improves isoprene production under
non-inducing conditions in the BL21 (DE3) strain harboring the
pTrcKudzuDXSyIDI construct. Under the non-inducing conditions,
isoprene synthase may be limiting and an additional copy of the
Kudzu enzyme may be able to improve the specific productivity of
isoprene generation by the strain. The experiments also
investigated whether other factor(s) contributed to the modest
level of isoprene produced by the strain and whether a plasmid-born
copy of fldA could increase isoprene production by the BL21 (DE3)
strain that harbors the pTrcKudzuDXSyIDI construct under
non-inducing conditions. The flavodoxin I encoded by fldA was
intended to be expressed ectopically from the pTrcHgSfldA/pBAD33
construct at a level surpassing that generated from the endogenous
fldA locus. An increased amount of flavodoxin I may increase the
activity demonstrated by the DXP pathway enzymes GcpE (HDS or IspG)
and LytB (HDR or IspH) in vivo, as was previously seen in vitro
(Seemann, M. et al. Agnew. Chem. Int. Ed., 41: 4337-4339, 2002;
Wolff, M. et al. FEBS Letters, 541: 115-120, 2003), and possibly
improve carbon flux to isoprene synthesis in the strain of interest
over that of the comparable pTrcKudzuDXSyIDI-containing BL21 (DE3)
control strain.
[0456] Bacterial transformation and molecular biology techniques
were performed using standard protocols (Sambrook et al), which is
hereby incorporated by reference in its entirety, particularly with
respect to bacterial transformation. The E. coli strains BL21 (DE3)
and TOP10 were obtained from Invitrogen. TOP10 cells were used
during the preparation of the pTrcHgS/pBAD33 and pTrcHgSfldA/pBAD33
constructs described below. Vector constructs were moved via
chemical transformation into the BL21 (DE3) strain for the
subsequent assessment of isoprene production.
Constructs
TABLE-US-00008 [0457] Forward primer Name: 5' fldA NsiI SpeI rbs
(SEQ ID NO: 54) Sequence: GG ATGCAT ACTAGT TTCA AGAGG TATTTCACTC
ATG Features: NsiI SpeI rbs start A G Region homologous MG1655 fldA
locus
[0458] Primers were purchased from Integrated DNA Technologies
(Coralville, Iowa). PCR reactions were performed with Herculase II
Fusion (Stratagene) according to manufacturer's specifications.
TABLE-US-00009 Reverse primer Name: 3' fldA PstI stop (SEQ ID NO:
55) Sequence: ATC CTGCAG TCA GGCATTGAGAATTTCGTC Features: PstI stop
T C Region homologous to MG1655 fldA locus
[0459] Primers were purchased from Integrated DNA Technologies
(Coralville, Iowa). PCR reactions were performed with Herculase II
Fusion (Stratagene) according to manufacturer's specifications.
[0460] E. coli 12 MG1655 (world wide web at
genome.wisc.edu/resources/strains.htm) was the source of genomic
template used to amplify the fldA locus; cells were added directly
to the PCR reaction using a sterile toothpick.
[0461] The fldA PCR product was cleaned utilizing the MinElute PCR
Purification Kit (Qiagen). pBAD33 is described, for example, in
Luz-Maria, G. et al., J. Bacteriology, 77: 4121-4130, 1995, which
is hereby incorporated by reference in its entirety, particularly
with respect to pBAD33. pTrcKudzu, and pTrcKudzuDXSyIDI kan, were
described, for example, in U.S. application Ser. No. 12/335,071 and
PCT/US2008/086809, which are hereby incorporated by reference in
their entireties, particularly with respect to Examples 1 and
7.
[0462] pTrcHgS/pBAD33 was constructed here by cloning the SspI-PstI
(1934 bp) fragment containing the Trc promoter region, rbs, and the
coding sequence of the Kudzu isoprene synthase derived from
pTrcKudzu into the SmaI-PstI sites of pBAD33.
[0463] pTrcHgSfldA/pBAD33 was constructed here. The NsiI-PstI (1471
bp) digested PCR amplified fldA fragment encompassing 22 bp
upstream of the fldA start, including the endogenous rbs, through
the stop codon of the fldA gene was cloned into the PstI site
located just downstream of the isoprene synthase open reading frame
in pTrcHgS/pBAD33.
[0464] Constructs were verified by sequencing that was performed by
Sequetech (Mountain View, Calif.).
Culture Conditions
[0465] Bacteria were grown at 25.degree. C. and 30.degree. C. on LB
1.5% agar plates and in TM3 liquid media (see description of TM3,
for example, U.S. application Ser. No. 12/335,071 and
PCT/US2008/086809, which are hereby incorporated by reference in
their entireties, particularly with respect to TM3 liquid media)
supplemented to a final concentration with 0.1% yeast extract and
1.0% glucose. When appropriate, kanamycin (Kan) and/or
chloramphenicol (Cmp) were added to the growth media at 50 .mu.g/ml
and 10 .mu.g/ml, respectively; pTrc-based constructs encode
Kan.sup.R and pBAD33-based constructs encode Cmp.sup.R. Bacterial
growth was monitored by optical density measured at 600 nm.
Assessment of Isoprene Production
[0466] Headspace assay for isoprene production was described in
Example 1. The specific productivity of each strain was reported as
.mu.g/LODhour; note ratio of 1900 .mu.l headspace:100 .mu.l broth
in assay vials. Graphs depicting the growth rate and specific
productivity of each strain were generated using Microsoft Office
Excel 2003 software.
Construction of BL21 (DE3) Strains and Assessment of the Isoprene
Production
[0467] The following BL21 (DE3) strains were constructed and
assessed for the production of isoprene relative to one another:
BL21 (DE3) harboring the pTrcKudzuDXSyIDI vector and either 1)
empty pBAD33 vector (also referred to as "empty vector"); 2)
pTrcHgS/pBAD33 construct (also referred to as "HgS"), or 3)
pTrcHgSfldA/pBAD33 construct (as referred to as "HgS-FldA").
[0468] All three BL21 (DE3) test strains harbor Kan.sup.R and
Cmp.sup.R and were grown under appropriate selection for both
plasmid constructs. The empty vector strain represented the
parental control strain; the HgS strain represented the parental
strain harboring an addition plasmid-born copy of the Kudzu
isoprene synthase gene; the HgS-FldA strain represented the
parental strain harboring the addition plasmid-born copies of
flavodoxin I and isoprene synthase genes.
[0469] The bacteria strains were grown overnight shaking (250 rpm)
at 25.degree. C. in 10 ml of supplemented TM3 media containing
antibiotics; here and for the following experiments 50 .mu.g/ml of
kanamycin and 10 .mu.g/ml of chloramphenicol were present in the
growth media. The cultures were then diluted into fresh
supplemented TM3 media containing antibiotics to an optical density
at 600 nm of approximately 0.05 and allowed to grow shaking (250
rpm) at 30.degree. C. in 12.5-25 ml of supplemented TM3 media
containing antibiotics in 250 ml Erlenmeyer flasks. Strains were
typically assessed for isoprene production once the optical density
at 600 nm of the culture reached 0.4. In the most densely sampled
experiments, once isoprene measurements commenced the isoprene
production for each culture was monitored in 45 min. intervals. The
results from two independent experiments depicting growth rate and
specific productivity of isoprene generation for the empty vector
(control), HgS, and HgS-FldA strains are shown in the FIGS.
46A-46D. The strains were grown under non-inducing conditions;
meaning that IPTG-induced expression from the Trc promoter
regulated gene constructs was not performed. All plasmid-born genes
of interest in the experiments described here were governed by the
IPTG-inducible Trc promoter. The Trc promoter is well known in the
art to be active in the absence of the IPTG inducer.
[0470] Under the non-inducing conditions tested, the results
obtained from the isoprene headspace assays performed on the empty
vector, HgS, and HgS-FldA strains indicate that an additional copy
of fldA present on the pTrcHgSfldA/pBAD33 construct substantially
increases isoprene production in the HgS-FldA strain over that
produced by both the HgS and empty vector control strains. The
HgS-FldA strain was observed to exhibit increased specific
productivity of isoprene generation ranging from 1.5- to 1.9-fold
and 1.3- to 1.8-fold higher than the control strain over a
3.75-hour and 2.5-hour time course, respectively, during two
independent experiments. The observed effect on isoprene production
appears to be specific to the presence of the fldA-containing
construct, as the HgS strain produces comparable levels of isoprene
under the non-inducing conditions to that produced by the empty
vector control strain.
Example 9
Expression of Alternative ispG (gcpE or HDS) and ispH (lytB or HDR)
and their Corresponding Reducing Shuttle System, from
Thermosynechococcus elongatus BP-1 in an Isoprene-Producing E. coli
to Improve Isoprene Production
[0471] In this example, we demonstrated that the
ferredoxin/ferredoxin-NADP oxidoreductase/NADPH reducing system
together with the GcpE and LytB enzymes from T. elongates improve
isoprene production in E. coli BL21(DE3).
[0472] T. elongatus, like E. coli, synthesizes isoprenoids via the
DXP pathway, but does not harbor any genes coding for a flavodoxin
protein. It was previously shown that the plant GcpE enzyme is a
ferredoxin-dependent enzyme, and that flavodoxin could not support
the enzymatic conversion of cMEPP (ME-CPP) into HDMAPP (HMBPP) by
this enzyme (see Seemann et al., FEBS Lett., 580(6):1547-52 (2006),
which is hereby incorporated by reference in its entirety). It was
also demonstrated in vitro that GcpE of T. elongatus together with
PetF (ferredoxin), Pet H (ferredoxin-NADP.sup.+ oxidoreductase),
and NADPH could convert cMEPP into HDMAPP (Okada and Hase, J Biol
Chem, 280(21):20627-9 (2005)), which is hereby incorporated by
reference in its entirety). With the lack of other small electron
carrier proteins besides ferredoxin in the genome, it is likely
that LytB of T. elongatus also utilizes the same reducing shuttle
system as GcpE.
[0473] Demonstration of increased isoprene production and elevated
cMEPP levels in REM23-26 by overexpression of GcpE, PetF, and PetH
from T. elongatus BP-1
[0474] We have previously demonstrated that increased expression of
dxs increases flux through the DXP pathway in E. coli.
Isoprene-producing strains (REM19-22) harboring increased and
varied levels of dxs expression were constructed by integrating the
GI 1.X-promoter series immediately upstream of the dxs locus within
the E. coli BL21(DE3) genome. Subsequently, the test set of
strains, REM23-26 were created by transformation with plasmids
expressing the T. elongatus GcpE and its corresponding reducing
shuttle system encoded by petF and petH. The parental and test
strains were evaluated for growth, isoprene production, and the
presence of DXP pathway metabolites. The results are presented in
FIGS. 47-49.
Construction of MCM16 MCM640, MCM639, MCM641, and the Parental
Strains to REM19-22
[0475] The GI 1.X-promoter insertions and subsequent loopout of the
antibiotic resistance markers described in this example were
carried out using the Red/ET system from Gene Bridges GmbH
according to the manufacturer's instructions. The strain BL21(DE3)
(Invitrogen) was used.
TABLE-US-00010 Primer Sequences MCM319: (SEQ ID NO: 57)
5'-ctctctttcggcaacagtcgtaactcctgggtggagtcgaccagtg
ccagggtcgggtatttggcaatatcaaaactcatatattccaccagcta
tttgttagtgaataaaagtggttgaattatttgctcaggatgtggcatN
gtcaagggctaatacgactcactatagggc tc.
degenerate N base: A base yields GI 1.6-, T base yields GI 1.5-, G
base yields GI1.2-, and C base yields GI 1.0-promoter.
TABLE-US-00011 MCM320: (SEQ ID NO: 58)
5'-tcgatacctcggcactggaagcgctagcggactacatcatccagc
gtaataaataaacaataagtattaataggcccctgaattaaccctcac taaagggcgg.
MCM327: (SEQ ID NO: 59) 5'-TTGTAGACATAGTGCAGCGCCA. GB-DW: (SEQ ID
NO: 60) 5'-aaagaccgaccaagcgacgtctga.
Strategy for Creating the MCM638-641 Strains
[0476] The strategy for inserting the GI1.X-promoter series in
front of dxs is shown in FIG. 50. The antibiotic resistance
cassette GB-NeoR was amplified by PCR using primer sets
MCM319/MCM320. The primers contain 50 bases of homology to the
region immediately 5' to the dxs coding region to allow
recombination at the specific locus upon electroporation of the PCR
product in the presence of the pRed-ET plasmid.
Amplification of the Deletion Cassettes
[0477] To amplify the GB-NeoR cassette for inserting the GI
1.X-promoters immediately upstream of the dxs locus the following
PCR reactions were set up:
1 ul (100 ng GB-NeoR)
10 ul HerculaseII Buffer
[0478] 0.5 ul dNTP's (100 mM) 1.25 ul primer (10 uM) MCM319 1.25 ul
primer (10 uM) MCM320 35 ul diH2O +1 ul of HerculaseII fusion from
Stratagene
Cycle Parameter
[0479] 95.degree. C..times.2 minutes, [95.degree. C..times.20
seconds, 55.degree. C..times.20 seconds, 72.degree. C..times.50
seconds].times.30 cycles; 72.degree. C..times.3 minutes, 4.degree.
C. until cool (BioRadPCR machine).
[0480] The resulting PCR fragments were separated on a 1.2% E-gel
(Invitrogen) for verification of successful amplification, and
purified using the QIAquick PCR Purification kits according to
manufacturer's instructions. The resulting stock was GB-NeoR-GI
1.X-dxs fragment.
Integration of GB-NeoR-GI 1.X-Dxs PCR Product into
BL21(DE3)/pRed-ET Strain
[0481] The pRed-ET vector (Gene Bridges kit) was transformed into
BL21(DE3) by electroporation resulting in strain MCM327
(BL21(DE3)/pRed-ET). Approximately 500 ng of the GB-NeoR-GI 1.x-dxs
PCR fragment was electroporated into MCM327. The transformants were
recovered in L Broth for 1 hour with shaking at 200 rpm at
37.degree. C. and then plated on L agar containing kanamycin (10
ug/ml). Kanamycin resistant colonies were analyzed by PCR for the
presence of the GB-NeoR cassette and the GI 1.X-promoters using
primers GB-DW/MCM327. The PCR fragments from a number of
transformants (MCM617-625) were sequenced using the MCM327 and
GB-DW primers (Quintara; Berkeley, Calif.) and the various GI
1.X-dxs strains of interest identified. The correct strains were
designated MCM617 (FRT-neo-FRT-GI 1.0-dxs), MCM618 (FRT-neo-FRT-GI
1.5-dxs), MCM623 (FRT-neo-FRT-GI 1.2-dxs), and MCM625
(FRT-neo-FRT-GI 1.6-dxs). The kanamycin resistance cassette was
looped out of the strains using pCP20 from the RED/ET kit according
to the manufacturer's instructions. Transformants were verified by
loss of resistance to kanamycin (10 ug/ml) and PCR demonstrating
loopout of the GB-NeoR cassette. The resulting strains were
designated MCM638 (BL21(DE3) GI 1.0-dxs), MCM639 (BL21(DE3) GI
1.5-dxs), MCM640 (BL21(DE3) GI 1.2-dxs) and MCM641 (BL21(DE3) GI
1.6-dxs).
Construction of the Parental Strains REM19-22 from MCM638, MCM640,
MCM639, and MCM641, Respectively
[0482] The construction of the T7-MEARR alba/pBBR1MCS-5 described
in this example was carried out using standard molecular biology
techniques (Sambrook et al., 1989, which is hereby incorporated by
reference in its entirety). The pBBR1MCS-5 plasmid has been
previously described (Kovach et al., Biotechniques, 16(5):800-2
(1994), which is hereby incorporated by reference in its entirety,
particularly with respect to cloning of the pBBR1MCS). A picture
illustrating the resulting plasmid construct is shown in FIG. 51.
The MCM638-641 strains were used for the transformations described
here.
TABLE-US-00012 Primer Sequences 5' KpnI to lacI MEARR T7 frag: (SEQ
ID NO: 61) 5'-GCTGGGTACCCTGCCCGCTTTCCAG TCGGGAAACCT 3' SpeI to T7
terminator MEARR T7 frag: (SEQ ID NO: 62) 5'-TAGAACTAGTCAAAAAACCCC
TCAAGACCCGTTTAG M13 Forward (-20): (SEQ ID NO: 63)
5'-GTAAAACGACGGCCAGT EL-1000: (SEQ ID NO: 64)
5'-GCACTGTCTTTCCGTCTGCTGC A-rev: (SEQ ID NO: 65)
5'-CTCGTACAGGCTCAGGATAG A-rev2: (SEQ ID NO: 66)
5'-TTACGTCCCAACGCTCAACT
Strategy for Creating the REM19-22 Strains
[0483] Electroporation of T7-MEARR alba/pBBR1MCS-5 into strains
MCM638-641. The vector construct harboring the T7 polymerase
governed MEARR alba allele, MD09-173 (BL21(DE3)pLysS, pET24a-P.
alba (MEA) Untagged (pDu39)), was used as the PCR template.
Amplification of the T7-MEARR alba Fragment
[0484] To amplify the T7-MEARR alba fragment for cloning into the
pBBR1MCS-5 plasmid the following PCR reaction was performed:
1 ul (approx. 120 ng MDO9-173)
10 ul HerculaseII Buffer
[0485] 0.5 ul dNTP's (100 mM) 1.25 ul primer (10 uM) 5' KpnI to lad
MEARR T7 frag 1.25 ul primer (10 uM) 3' SpeI to T7 terminator MEARR
T7 frag 35 ul diH2O +1 ul of HerculaseII fusion from
Stratagene.
Cycle Parameter:
[0486] 95.degree. C..times.2 minutes, [95.degree. C..times.30
seconds, 63.degree. C..times.30 seconds, 72.degree. C..times.3
minutes].times.29 cycles; 72.degree. C..times.5 minutes, 4.degree.
C. until cool (Biometra T3000 Combi Thermocycler).
[0487] The resulting PCR fragment was separated on a 1.2% E-gel
(Invitrogen) for verification of successful amplification, and
purified using the QIAquick PCR Purification kits (Qiagen)
according to manufacturer's instructions. The resulting stock was
T7-MEARR alba fragment.
Cloning of the T7-MEARR alba Fragment into pBBR1MCS-5
[0488] Approximately 600 ng of the T7-MEARR alba fragment and 200
ng of the pBBR1MCS-5 plasmid were digested with KpnI and SpeI
(Roche) according to the manufacturer's specifications. The digests
were subsequently combined and cleaned using the Qiagen QiaQuick
Gel Extraction Kit. Approximately a fourth to a third of the
cleaned cut DNA was ligated using T4 DNA Ligase (New England
Biolabs) according to the manufacturer's suggested protocol.
Chemically competent TOP10 cells (Invitrogen) was transformed with
the ligation reaction using a standard heat-shock protocol (See,
e.g., Sambrook et al., 1989, which is hereby incorporated by
reference in its entirety), recovered in L broth for 1 hour at
37.degree. C. and then plated on L agar containing gentamycin (10
ug/ml) and 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside
(X-GAL at 40 ug/ml; Sigma). White, gentamycin resistant colonies
were selected, grown overnight in L broth containing gentamycin (10
ug/ml), and harvested for plasmid preparation the following day.
Plasmid constructs were isolated using Qiagen Qiaprep Spin Miniprep
Kit and first analyzed by restriction enzyme digestion and
electrophoresis (as described above) for the putative presence of
the T7-MEARR alba fragment. Plasmid preparations of interest were
sequenced (Sequetech; Mountain View, Calif.) using primers M13
Forward (-20), EL-1000, A-rev, and A-rev2, and the correct T7-MEARR
alba/pBBR1MCS-5 clone identified.
Transformation of T7-MEARR alba/pBBR1MCS-5 into MCM638-641
[0489] To build the isoprene-producing strains REM19-22 the
T7-MEARR alba/pBBR1MCS-5 plasmid was transformed by electroporation
into MCM638-641. Transformants were recovered in L broth and plated
on L agar containing gentamycin (10 ug/ml). The resulting strains
were designated as such: REM19 (MCM638/T7-MEARR alba/pBBR1MCS-5),
REM20 (MCM640/T7-MEARR alba/pBBR1MCS-5), REM21 (MCM639/T7-MEARR
alba/pBBR1MCS-5), and REM22 (MCM641/T7-MEARR alba/pBBR1MCS-5).
Construction of the Test Strains REM23-26
[0490] REM23-26 were constructed by transformation of the
Ptac-gcpE-petF-petH/pK184 construct into MCM638, MCM640, MCM639,
and MCM641. The plasmid Ptac-gcpE-petF-petH/pK184 described in this
example was synthesized by Gene Oracle, Inc. (Mountain View,
Calif.) with codon optimization of gcpE, petF, and petH for
expression in E. coli. The Ptac promoter and aspA terminator
sequences have been previously described (Genbank accession #
E02927 and CP001164, respectively). The pK184 cloning vector has
been described, for example, by Jobling and Holmes, Nucleic Acids
Res. 18(17):5315-6 (1990), which is hereby incorporated by
reference in its entirety, particularly with respect to the pK184
cloning vector. A picture illustrating the resulting plasmid
construct is shown in FIG. 52. The REM19-22 strains were used for
the transformations described herein.
Strategy for Creating the REM23-26 Strains
[0491] Electroporation of Ptac-gcpE-petF-petH/pK184 into strains
REM19-22. A plasmid preparation of Ptac-gcpE-petF-petH/pK184 was
provided by Gene Oracle, Inc.
Transformation of Ptac-gcpE-petF-petH/pK184 into REM19-22
[0492] To build the isoprene-producing test strains, REM23-26, the
Ptac-gcpE-petF-petH/pK184 plasmid was transformed by
electroporation into REM19-22. Transformants were recovered in L
broth and plated on L agar containing kanamycin (10 ug/ml) and
gentamycin (10 ug/ml). The resulting strains were designated as
such: REM23 (REM19/Ptac-gcpE-petF-petH/pK184), REM24
(REM20/Ptac-gcpE-petF-petH/pK184), REM25
(REM21/Ptac-gcpE-petF-petH/pK184), and REM26
(REM22/Ptac-gcpE-petF-petH/pK184).
Analysis of REM19-26 for Growth, Isoprene Production, and DXP
Metabolite Generation
[0493] The parental strains REM19-22 were compared against the test
strains REM23-26 in a shake flask isoprene headspace assay as well
as in a DXP metabolite determination study. The benefits of
expressing the T. elongatus GcpE enzyme on DXP metabolite
generation and isoprene production from the E. coli host is
illustrated in FIGS. 47 and 48.
Growth
[0494] Strains REM19-26 were grown at 30.degree. C. in TM3 liquid
media (13.6 g K.sub.2PO.sub.4, 13.6 g KH.sub.2PO.sub.4, 2.0 g
MgSO.sub.4*7H.sub.2O), 2.0 g citric acid monohydrate, 0.3 g ferric
ammonium citrate, 3.2 g (NH.sub.4).sub.2SO.sub.4, 0.2 g yeast
extract, 1.0 ml 1000.times. Modified Trace Metal Solution, adjusted
to pH 6.8 and q.s. to H.sub.2O, and filter sterilized) supplemented
to a final concentration with 0.1% yeast extract and 1.0% glucose
and including kanamycin (10 ug/ml) and gentamycin (10 ug/ml).
Growth was monitored periodically by recording each of the
culture's optical density measured at 600 nm using an Eppendorf
Biophotometer spectrometer (Eppendorf).
Isoprene Production
[0495] Isoprene production was analyzed using a headspace assay.
For the shake flask cultures, one ml of a culture was transferred
from shake flasks to 20 ml CTC headspace vials (Agilent vial
cat#5188 2753; cap cat#5188 2759). The cap was screwed on tightly
and the vials incubated at the equivalent temperature with shaking
at 250 rpm. After 30 minutes the vials were removed from the
incubator and analyzed. The analysis was performed using an Agilent
6890 GC/MS system interfaced with a CTC Analytics (Switzerland)
CombiPAL autosampler operating in headspace mode. An Agilent HP-5MS
GC/MS column (30 m.times.0.25 mm; 0.25 .mu.m film thickness) was
used for separation of analytes. The sampler was set up to inject
500 .mu.L of headspace gas. The GC/MS method utilized helium as the
carrier gas at a flow of 1 ml/minutes The injection port was held
at 250.degree. C. with a split ratio of 50:1. The oven temperature
was held at 37.degree. C. for the 2 minute duration of the
analysis. The Agilent 5793N mass selective detector was run in
single ion monitoring (SIM) mode on m/z 67. The detector was
switched off from 1.4 to 1.7 minutes to allow the elution of
permanent gases. Under these conditions isoprene
(2-methyl-1,3-butadiene) was observed to elute at 1.78 minutes. A
calibration table was used to quantify the absolute amount of
isoprene and was found to be linear from 1 .mu.g/L to 200 .mu.g/L.
The limit of detection was estimated to be 50 to 100 ng/L using
this method. The specific productivity of each strain is reported
as ug/L OD Hr. Note, ratio of 1900 ul headspace:100 ul broth in
assay vials for 30 min. incubation results in the following
conversion of isoprene ug/L of culture to specific productivity:
(isoprene/L determined by GC-MS).times.(38)/(OD 600 nm of the
culture).
DXP Metabolite Accumulation
[0496] The DXP metabolites of the isoprene-producing parental and
test strains, REM19-22 and REM23-26, respectively, described above
and depicted in FIG. 48 were isolated and quantified as
follows:
Metabolite Quantification
[0497] Cell metabolism was rapidly inactivated by withdrawing 3.5
mL of the culture into a tube filled with 3.5 mL of dry ice-cold
methanol. Cell debris was pelleted by centrifugation and the
supernatant was loaded onto Strata-X-AW anion exchange column
(Phenomenex) containing 30 mg of sorbent. The pellet was
re-extracted twice, first with 3 mL of 50% MetOH containing 1 mM
NH.sub.4HCO.sub.3 buffer (pH=7.0) and then with 3 mL of 75% MetOH/1
mM NH.sub.4HCO.sub.3 buffer (pH=7.0). After each extraction, cell
debris was pelleted by centrifugation and the supernatants were
consecutively loaded onto the same anion exchange column. During
the extraction and centrifugation steps the samples were kept at
below +4.degree. C. Prior to metabolite elution, the anion exchange
columns were washed with water and methanol (1 mL of each) and the
analytes were eluted by adding 0.35 mL of concentrated
NH.sub.4OH/methanol (1:14, v/v) and then 0.35 mL of concentrated
NH.sub.4OH/water/methanol (1:2:12, v/v/v) mixtures. The eluant was
neutralized with 30 .mu.L of glacial acetic acid and cleared by
centrifugation in a microcentrifuge.
Metabolite Quantification
[0498] Metabolites were analyzed using a Thermo Scientific TSQ
Quantum Access mass spectrometer (Thermo Electron Corporation, San
Jose, Calif.). All system control, data acquisition, and mass
spectral data evaluation were performed using XCalibur and LCQuan
software (Thermo Electron Corp). For the LC-ESI-MS/MS method, a
chiral Nucleodex .beta.-OH 5 .mu.M HPLC column (100.times.2 mm,
Macherey-Nagel, Germany) equipped with a CC 8/4 Nucleodex beta-OH
guard cartridge was eluted with a mobile phase gradient shown in
Table 1 (flow rate of 0.4 mL/min). The sample injection volume was
10 .mu.L.
TABLE-US-00013 TABLE 1 HPLC gradient used to elute metabolites.
Mobile phase, % B Time, A (100 mM ammonium C min (water)
bicarbonate, pH = 8.0) (acetonitrile) 0.0 0.0 20.0 80.0 0.5 15.0
5.0 80.0 4.5 37.5 12.5 50.0 6.5 37.5 12.5 50.0 7.0 49.5 0.5 50.0
12.0 34.9 0.1 65.0 12.5 0.0 20.0 80.0 13.0 0.0 20.0 80.0
[0499] Mass detection was carried out using electrospray ionization
in the negative mode. The following m/z values for precursor ions
were selected to detect the metabolites of interest in SRM mode:
245.0 for IPP and DMAPP, 381.1 for FPP, 213.0 for DXP, 215.0 for
MEP, 260.0 for HDMAPP, and 277.0 for cMEPP. Concentrations of
metabolites were determined based on the integrated intensities of
peaks generated by PO.sub.3.sup.- product ion (m/z=79.0).
Calibration curves obtained by injection of corresponding standards
purchased from Echelon Biosciences Inc. Intracellular
concentrations of metabolites were calculated based on the
assumption that in 1 mL of the culture at OD=200 the integrated
volume of all cells is 50 .mu.L.
Demonstration of Increased Isoprene Production in REM31 and REM29
by Overexpression of GcpE, LytB PetF and PetH of T. elongatus
BP-1
[0500] We have demonstrated that increased expression of dxs
permits increased flux through the DXP pathway within E. coli,
while the additional overexpression of an idi gene increases the
production of downstream isoprenoids significantly. To demonstrate
the benefits of expressing the non-flavodoxin-dependent GcpE and
LytB enzymes on carbon flux through the endogenous E. coli DXP
pathway to isoprene synthesis, E. coli BL21(DE3) isoprene-producing
strains with constitutive expression of dxs and the yeast IDI
enzyme were constructed. The BL21(DE3) GI 1.6-dxs strain MCM641 is
described above. The construction of the vector construct harboring
the yeast IDI enzyme, pDU9-pET-16b rev-yIDI, is described herein.
The T7-(-3) alba/pBBR1MCS-5 and T7-MTE alba/pBBR1MCS-5 P. alba
isoprene synthase-containing constructs are described below. A set
of parental isoprene-producing, IDI-overexpressing strains derived
from MCM641 were created (REM H76 and REMH86) to compare to the
newly generated test set of strains (REM31 and REM29) which harbor
the T. elongatus GcpE, LytB, and their corresponding reducing
shuttle system (described below). The parental and test strains
were evaluated for growth, isoprene production, and the presence of
DXP pathway metabolites. The results are depicted in FIG. 49.
Construction of pDU-9
[0501] The IPP isomerase from Saccharomyces cerevisiae (yIDI) was
cloned into the vector pET16b (Invitrogen). The primer set
Hg-yIDI-R2/Hg-yIDI-F2 was used for PCR with the template DNA
pTrcKudzu yIDI Kan. The PCR cycle conditions:
PCR Reaction
[0502] 1 ul of template (pMVK1--Fernando's template) [0503] 5 ul of
10.times. PfuII Ultra buffer [0504] 1 ul of dNTP [0505] 1 ul of
primer (50 uM) Hg-MVK-F2-NdeI
[0506] 1 ul of primer (50 uM) Hg-yIDI-R2
[0507] 40 ul of DiH2O
[0508] +1 ul of Pfu UltraII Fusion DNA Polymerase from
Stratagene
Cycle Parameter:
(95.degree. C. 2 min., 95.degree. C. 20 sec., 55.degree. C. 20
sec., 72.degree. C. 21 sec., 29.times., 72 C 3 min.,
[0509] 4.degree. C. until cool, use Eppendorf Mastercycler Gradient
Machine)
[0510] The PCR product was purified using the QiaQuick PCR
purification kit according to the manufacturer's suggestion. An
aliquot of 5 uL purified of the PCR product was ligated to
Invitrogen pET-16b Vector that was previously digested with
NdeI-SAP (Shrimp Alkaline Phosphatase) treated using T4 ligase
enzyme (NEB). The ligation was carried out overnight at 16.degree.
C.
[0511] 5 uL of overnight ligation mixture was introduced into
Invitrogen TOP10 cells and transformants were selected on L agar
containing Carbenicillin (50 ug/ml) incubated at 37.degree. C.
Plasmids from transformants were isolated using QiaQuick spin
miniprep kit. The insert is sequenced with T7 promoter and T7
terminator (Use Quintara Bio Sequencing Service). The resulting
plasmid r is called pDu-9.
[0512] Once the sequence is verified, 1 ul of plasmid pDu-9 was
transformed into BL21(DE3) pLysS hst strain according to
manufacturer's protocol. Transformants are selected on L agar
containing Carbenicillin (50 ug/ml) plate and incubated at
37.degree. C.
TABLE-US-00014 Primer sequences Hg-yIDI-R2 (SEQ ID NO: 111)
5'...cagcagcagGGATCCgacgcgttgttatagca Hg-yIDI-F2 (SEQ ID NO: 112)
5'...cagcagcagCATATGactgccgacaacaatag
Construction of REMD76 (MCM641/pDU9-pET-16b Rev-yIDI), REMH76 and
REMH86 (REMD76/T7-(-3) alba/pBBR1MCS-5 and REMD76/T7-MTE
alba/pBBR1MCS-5, Respectively)
Strategy for Creating the REMD76
[0513] pDU9-pET-16b rev-yIDI was electroporated into MCM641.
Transformation of pDU9-pET-16b rev-yIDI into MCM641
[0514] To build the BL21(DE3) GI 1.6-dxs yIDI-overexpressing strain
REMD76, the pDU9-pET-16b rev-yIDI plasmid expressing a yeast IDI
(yIDI) allele was transformed by electroporation into MCM641.
Transformants were recovered in L broth and plated on L agar
containing carbinicillin (50 ug/ml). A carbinicillin resistant
colony was selected and designated REMD76.
Generation of the Parental Strains REMH76 and REMH86
(REMD76/T7-(-3) alba/pBBR1MCS-5 and REMD76/T7-MTE alba/pBBR1MCS-5,
Respectively
[0515] The construction of the T7-(-3) alba/pBBR1MCS-5 and T7-MTE
alba/pBBR1MCS-5 constructs described in this example were carried
out using standard molecular biology techniques (ee, e.g., Sambrook
et al., 1989). The pBBR1MCS-5 plasmid has been previously described
(see, Kovach et al., Biotechniques, 16(5):800-2 (1994), which is
hereby incorporated by reference in its entirety, particularly with
respect to the pBBR1MCS-5 plasmid). The pictures illustrating the
resulting plasmid constructs are shown in FIG. 53. The REMD76
strain was used for the transformations described herein.
Strategy for Creating the REMH76 and REMH86 Strains
[0516] Electroporation of T7-(-3) alba/pBBR1MCS-5 and T7-MTE
alba/pBBR1MCS-5 into strain REMD76. The vector constructs harboring
the T7 polymerase governed (-3) and MTE alba alleles,
pDU47-3-pET24a-P. alba (-3) and pDU42 pET24a-P. alba-MTE untagged,
were used as the PCR templates.
TABLE-US-00015 Primer Sequences 5' KpnI to lacI MEARR T7 frag: (SEQ
ID NO: 61) 5'-GCTGGGTACCCTGCCCGCTTTCCAG TCGGGAAACCT 3' SpeI to T7
terminator MEARR T7 frag: (SEQ ID NO: 62) 5'-TAGAACTAGTCAAAAAACCCC
TCAAGACCCGTTTAG M13 Forward (-20): (SEQ ID NO: 63)
5'-GTAAAACGACGGCCAGT EL-1000: (SEQ ID NO: 64)
5'-GCACTGTCTTTCCGTCTGCTGC A-rev: (SEQ ID NO: 65)
5'-CTCGTACAGGCTCAGGATAG A-rev2: (SEQ ID NO: 66)
5'-TTACGTCCCAACGCTCAACT
Amplification of the T7-(-3) and T7-MTE alba Fragments
[0517] To amplify the T7-(-3) and T7-MTE alba fragments for cloning
into the pBBR1MCS-5 plasmid the following PCR reactions were
performed: 1 ul (approx. 100 ng pDU47-3-pET24a-P. alba (-3) or
pDU42 pET24a-P. alba-MTE untagged)
10 ul HerculaseII Buffer
[0518] 0.5 ul dNTP's (100 mM) 1.25 ul primer (10 uM) 5' KpnI to lad
MEARR T7 frag 1.25 ul primer (10 uM) 3' SpeI to T7 terminator MEARR
T7 frag 35 ul diH2O +1 ul of HerculaseII fusion from Stratagene
Cycle Parameter:
[0519] 95.degree. C..times.2 minutes, [95.degree. C..times.30
seconds, 63.degree. C..times.30 seconds, 72.degree. C..times.3
minutes.].times.29 cycles; 72.degree. C..times.5 minutes, 4.degree.
C. until cool (Biometra T3000 Combi Thermocycler)
[0520] The resulting PCR fragments were separated on a 1.2% E-gel
(Invitrogen) for verification of successful amplification, and
purified using the QIAquick PCR Purification kits according to
manufacturer's instructions. The resulting stocks were T7-(-3) alba
fragment and T7-MTE alba fragment.
Cloning of the T7-(-3) alba and T7-MTE alba Fragments into
pBBR1MCS-5
[0521] Approximately 600 ng of the T7-(-3) alba fragment or T7-MTE
alba fragment and 200 ng of the pBBR1MCS-5 plasmid were digested
with KpnI and SpeI from Roche according to the manufacturer's
specifications. The digests were subsequently combined and cleaned
using the Qiagen QiaQuick Gel Extraction Kit. Approximately a
fourth to a third of the cleaned cut DNA was ligated using T4 DNA
Ligase from New England Biolabs according to the manufacturer's
suggested protocol.
[0522] Chemically competent TOP10 cells (Invitrogen) were
transformed with the ligation reaction using a standard heat-shock
protocol (Sambrook et al., 1989, which is hereby incorporated by
reference in its entirety), recovered in L broth for 1 hour at
37.degree. C. and then plated on L agar containing gentamycin (10
ug/ml) and 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside
(X-GAL at 40 ug/ml; Sigma). White gentamycin resistant colonies
were selected, grown overnight in L broth containing gentamycin (10
ug/ml), and harvested for plasmid preparation the following day.
Plasmid constructs were isolated using Qiagen Qiaprep Spin Miniprep
Kit and first analyzed by restriction digest and electrophoresis
(as described above) for the putative presence of the T7-(-3) alba
fragment or T7-MTE alba fragment. Plasmid preparations of interest
identified were sequenced (Sequetech; Mountain View, Calif.) using
primers M13 Forward (-20), EL-1000, A-rev, and A-rev2, and the
correct T7-(-3) alba/pBBR1MCS-5 and T7-MTE alba/pBBR1MCS-5 clones
identified.
Construction of the Test Strains REM31 and REM29
[0523] To create strains REM31 and 29 the plasmid
Ptac-gcpE-lytB-petF-petH/pK184 was transformed into REMH76 and
REMH86. The synthesis and codon optimization for E. coli of the
Ptac-gcpE-lytB-petF-petH/pK184 described in this example was
performed by Gene Oracle, Inc. (Mopuntain View, Calif.). The Ptac
promoter and aspA terminator sequences have been previously
described (Genbank accession # E02927 and CP001164, respectively)
and were also constructed synthetically. The pK184 cloning vector
has been described previously (see, Jobling and Holmes, Nucleic
Acids Res. 18(17):5315-6 (1990), which is hereby incorporated by
reference in its entirety, particularly with respect to the pK184
cloning vector). A picture illustrating the resulting plasmid
construct is shown in FIG. 54. The REMH76 and REMH86 strains were
used for the transformations described herein.
Strategy for Creating the REM31 and REM29 Strains
[0524] Electroporation of Ptac-gcpE-lytB-petF-petH/pK184 into
strains REMH76 and REMH86 strains: A plasmid preparation of
Ptac-gcpE-lytB-petF-petH/pK184 was provided by Gene Oracle,
Inc.
Transformation of Ptac-gcpE-lytB-petF-petH/pK184 into REMH76 and
REMH86
[0525] To build the isoprene-producing test strains (REM31 and
REM29) which harbor the T. elognatus GcpE and LytB enzymes to
assess against the parental strains (REMH76 and REMH86) for
benefits in DXP pathway flux and isoprene production, the
Ptac-gcpE-lytB-petF-petH/pK184 plasmid was transformed by
electroporation into REMH76 and REMH86. Transformants were
recovered in L broth and plated on L agar containing carbinicillin
(50 ug/ml), kanamycin (10 ug/ml), and gentamycin (10 ug/ml). The
resulting strains are designated as such: REM31
(REMH76/Ptac-gcpE-lytB-petF-petH/pK184) and REM29
(REMH86/Ptac-gcpE-lytB-petF-petH/pK184).
Comparing REMH76 and REMH86 to REM31 and REM29 for Growth and
Isoprene Production
[0526] The parental strains REMH76 and REMH86 were compared against
the test strains REM31 and REM29, respectively, in a shake flask
isoprene headspace assay as well as in a DXP metabolite
determination study. The benefit of expressing the T. elongatus
GcpE and LytB enzymes on isoprene production from the E. coli host
is illustrated in FIG. 48.
Growth
[0527] Parental strains REMH76 and REMH86 and test strains REM31
and REM29 were grown at 30.degree. C. in TM3 liquid media (13.6 g
K.sub.2PO.sub.4, 13.6 g KH.sub.2PO.sub.4, 2.0 g
MgSO.sub.4*7H.sub.2O), 2.0 g citric acid monohydrate, 0.3 g ferric
ammonium citrate, 3.2 g (NH.sub.4).sub.2SO.sub.4, 0.2 g yeast
extract, 1.0 ml 1000.times. Modified Trace Metal Solution, adjusted
to pH 6.8 and q.s. to H.sub.2O, and filter sterilized) supplemented
to a final concentration with 0.1% yeast extract and 1.0% glucose
and including carbinicillin, (50 ug/ml) kanamycin (10 ug/ml) and
gentamycin (10 ug/ml). Growth was monitored periodically by
recording each of the culture's optical density measured at 600 nm
using an Eppendorf Biophotometer spectrometer (Eppendorf).
Isoprene Production
[0528] Isoprene production was analyzed using a headspace assay.
For the shake flask cultures, one ml of a culture was transferred
from shake flasks to 20 ml CTC headspace vials (Agilent vial
cat#5188 2753; cap cat#5188 2759). The cap was screwed on tightly
and the vials incubated at the equivalent temperature with shaking
at 250 rpm. After 30 minutes the vials were removed from the
incubator and analyzed. The analysis was performed using an Agilent
6890 GC/MS system interfaced with a CTC Analytics (Switzerland)
CombiPAL autosampler operating in headspace mode. An Agilent HP-5MS
GC/MS column (30 m.times.0.25 mm; 0.25 .mu.m film thickness) was
used for separation of analytes. The sampler was set up to inject
500 .mu.L of headspace gas. The GC/MS method utilized helium as the
carrier gas at a flow of 1 ml/minutes The injection port was held
at 250.degree. C. with a split ratio of 50:1. The oven temperature
was held at 37.degree. C. for the 2 minute duration of the
analysis. The Agilent 5793N mass selective detector was run in
single ion monitoring (SIM) mode on m/z 67. The detector was
switched off from 1.4 to 1.7 minutes to allow the elution of
permanent gases. Under these conditions isoprene
(2-methyl-1,3-butadiene) was observed to elute at 1.78 minutes. A
calibration table was used to quantify the absolute amount of
isoprene and was found to be linear from 1 .mu.g/L to 200 .mu.g/L.
The limit of detection was estimated to be 50 to 100 ng/L using
this method.
[0529] The specific productivity of each strain was reported as
ug/L OD Hr. Ratio of 1900 ul headspace:100 ul broth in assay vials
for 30 min. incubation resulted in the following conversion of
isopreneug/L of culture to specific productivity: (isoprene/L
determined by GC-MS).times.(38)/(OD 600 nm of the culture).
DXP Metabolite Accumulation
[0530] The DXP metabolites of the isoprene-producing parental
(REMH76 and REMH86) and test strains (REM31 and REM29) described
above were isolated and quantified as described below. The
resulting data is discussed in the legend to FIG. 48.
Metabolite Extraction
[0531] Cell metabolism was rapidly inactivated by withdrawing 3.5
mL of the culture into a tube filled with 3.5 mL of dry ice-cold
methanol. Cell debris was pelleted by centrifugation and the
supernatant was loaded onto Strata-X-AW anion exchange column
(Phenomenex) containing 30 mg of sorbent. The pellet was
re-extracted twice, first with 3 mL of 50% MetOH containing 1 mM
NH.sub.4HCO.sub.3 buffer (pH=7.0) and then with 3 mL of 75% MetOH/1
mM NH.sub.4HCO.sub.3 buffer (pH=7.0). After each extraction, cell
debris was pelleted by centrifugation and the supernatants were
consecutively loaded onto the same anion exchange column. During
the extraction and centrifugation steps the samples were kept at
below +4.degree. C. Prior to metabolite elution, the anion exchange
columns were washed with water and methanol (1 mL of each) and the
analytes were eluted by adding 0.35 mL of concentrated
NH.sub.4OH/methanol (1:14, v/v) and then 0.35 mL of concentrated
NH.sub.4OH/water/methanol (1:2:12, v/v/v) mixtures. The eluant was
neutralized with 30 .mu.L of glacial acetic acid and cleared by
centrifugation in a microcentrifuge.
Metabolite Quantification
[0532] Metabolites were analyzed using a Thermo Scientific TSQ
Quantum Access mass spectrometer (Thermo Electron Corporation, San
Jose, Calif.). All system control, data acquisition, and mass
spectral data evaluation were performed using XCalibur and LCQuan
software (Thermo Electron Corp). For the LC-ESI-MS/MS method, a
chiral Nucleodex .beta.-OH 5 .mu.M HPLC column (100.times.2 mm,
Macherey-Nagel, Germany) equipped with a CC 8/4 Nucleodex beta-OH
guard cartridge was eluted with a mobile phase gradient shown in
Table 1 (flow rate of 0.4 mL/min). The sample injection volume was
10 .mu.L.
[0533] Mass detection was carried out using electrospray ionization
in the negative mode. The following m/z values for precursor ions
were selected to detect the metabolites of interest in SRM mode:
245.0 for IPP and DMAPP, 381.1 for FPP, 213.0 for DXP, 215.0 for
MEP, 260.0 for HDMAPP, and 277.0 for cMEPP. Concentrations of
metabolites were determined based on the integrated intensities of
peaks generated by PO.sub.3.sup.- product ion (m/z=79.0).
Calibration curves obtained by injection of corresponding standards
purchased from Echelon Biosciences Inc. Intracellular
concentrations of metabolites were calculated based on the
assumption that in 1 mL of the culture at OD=200 the integrated
volume of all cells is 50 .mu.L.
Example 10
Deletion of iscR in E. coli BL21(DE3) Genotype to Improve Isoprene
Production
[0534] Previous studies suggest that repair of damaged Fe--S
centers and the turnover or regeneration of active 4Fe-4S centers
within GcpE is partially contributable to the perceived bottleneck
in DXP-mediated isoprenoid biosynthesis at the catalytic step
carried out by GcpE. Increased levels of the related enzyme LytB
have been obtained from E. coli engineered to overexpress the isc
operon (Grawert et al., J Am Chem Soc. 126(40):12847-55 (2004),
which is hereby incorporated by reference in its entirety). The
enzymes encoded by the E. coli isc operon have been shown to play a
role in Fe--S cluster biogenesis and maintenance (Tokumoto and
Takahashi, J. Biochem., 130: 63-71 (2001); Djaman et al., J. of
Biol. Chem., 279(43):44590-44599 (2004), which are each hereby
incorporated by reference in their entireties). An alternative
approach to overexpressing the isc operon in E. coli to generate
increased levels of active 4Fe-4S cluster containing enzymes such
as GcpE and LytB is to remove the IscR transcriptional repressor
that inhibits expression of the isc operon (Schwartz et al., PNAS,
98(26):14751-3 (2001), which is hereby incorporated by reference in
its entirety). Such an approach was recently proved successful for
a group expressing Clostridial hydrogenase in E. coli BL21(DE3)
(Akhtar and Jones, Appl. Microbiol. Biotechnol. 78(5):853-62
(2008), which is hereby incorporated by reference in its
entirety).
[0535] In this example, we demonstrated that the removal of iscR
from the E. coli BL21(DE3) genome significantly improves isoprene
production over that produced from the corresponding wild-type
strain.
Deletion of iscR from BL21(DE3)/pRed/ET
[0536] The gene deletions and subsequent loopout of the antibiotic
resistance markers described in this example were carried out using
the Red/ET system from Gene Bridges GmbH according to the
manufacturer's instructions. The strain BL21(DE3) (Invitrogen) was
used.
Primer Sequences Used
TABLE-US-00016 [0537] top iscR deletion: (SEQ ID NO: 80)
5'-GGGCGAGTTTGAGGTGAAGTAAGACATGAGACTGACA
TCTGAACCCTCACTAAAGGGCGGCCGC bottom iscR deletion: (SEQ ID NO: 81)
5'-TTCTTTTTATTAAGCGCGTAACTTAACGTCGATCGC
GTCTTGAAGTTCCTATACTTTCTAGAGAATAGGAACTTCTTACGCCCCG CCCTGCCACTCATCGCA
5' screen up of up iscR: (SEQ ID NO: 82) 5'-AGCCAGGAGTTGAATATCCTG
3' down of down iscR: (SEQ ID NO: 83) 5'-TGATGGACACGAGGATGGTGT
Strategy for Creating the Deletion Strains
[0538] The strategy for the deletion of iscR is shown in FIG. 61.
The antibiotic resistance cassette GB-CmR was amplified by PCR
using primer sets top iscR deletion/bottom iscR deletion for
deletion of the iscR locus. The primers contain 50 bases of
homology to the region flanking the iscR gene to allow
recombination at the specific locus upon electroporation of the PCR
product in the presence of the pRed-ET plasmid.
Amplification of the Deletion Cassettes
[0539] To amplify the GB-CmR cassette for deletion of iscR the
following PCR reactions were set up:
1 ul (100 ng GB-CmR)
10 ul HerculaseII Buffer
[0540] 0.5 ul dNTP's (100 mM) 1.25 ul primer (10 uM) top iscR
deletion 1.25 ul primer (10 uM) bottom iscR deletion 35 ul diH2O +1
ul of HerculaseII fusion from Stratagene
Cycle Parameter:
[0541] 95.degree. C..times.2 min., [95.degree. C..times.30 sec.,
63.degree. C..times.30 sec., 72.degree. C..times.3 min].times.29
cycles; 72.degree. C..times.5 min, 4.degree. C. until cool
(Biometra T3000 Combi Thermocycler).
[0542] The resulting PCR fragment was separated on a 1.2% E-gel
(Invitrogen) to verify successful amplification, and purified using
QIAquick PCR Purification kit according to manufacturer's
instructions. The resulting stock was designated GB-CmR-iscR
fragment.
Integration of GB-CmR-iscR Product into the BL21(DE3) Genome
[0543] The pRed-ET vector (Gene Bridges) was transformed into
electrocompetent BL21(DE3) (Invitrogen) by electroporation
resulting in strain BL21(DE3)/pRed-ET. Approximately 500 ng of
GB-CmR-iscR PCR fragment was electroporated into BL21(DE3)/pRed-ET.
The transformants were recovered in L Broth for 1 hour at
37.degree. C. and then plated on L agar containing chloramphenical
(10 ug/ml). Chloramphenicol resistant colonies were analyzed by PCR
for the replacement of the iscR by the GB-CmR-iscR fragment using
primers 5' screen up of up iscR/3' screen down of down iscR. The
correct strain was designated REM14::CMP. The chloramphenicol
resistance cassette was looped out of the strain using pCP20 from
the RED/ET kit according to the manufacturer's instructions.
Transformants were verified by loss of resistance to
chloramphenicol (10 ug/ml) and PCR demonstrating loopout of the
GB-CmR cassette. The resulting strain was designated REM14.
Creation of Strains REM65-1 and REM4, the Parental Strains to REM12
and REM13
[0544] The wild-type BL21(DE3) (Invitrogen) and .DELTA.iscR strain
REM14 were transformed with the T7-MEARR alba/pBBR1MCS-5 construct
to create the isoprene-producing strains REM65-1 and REM4 strains,
respectively. A picture of the isoprene synthase containing vector,
T7-MEARR alba/pBBR1MCS-5, is shown in FIG. 61. The construction of
T7-MEARR alba/pBBR1MCS-5 is described in the Example: Expression of
alternative ispG (gcpE) and ispH (lytB) and their corresponding
reducing shuttle system, from Thermosynechococcus elongatus BP-1 in
an isoprene-producing E. coli to improve isoprene production.
Transformation of T7-MEARR alba/pBBR1MCS-5 into BL21(DE3) and
REM14
[0545] To build the isoprene-producing strains REM65-1 and REM4
strains, the T7-MEARR alba/pBBR1MCS-5 plasmid was transformed by
electroporation into BL21(DE3) (Invitrogen) and REM14.
Transformants were recovered in L broth and plated on L agar
containing gentamycin (10 ug/ml). The resulting strains are
designated as such: REM65-1 (BL21(DE3)/T7-MEARR alba/pBBR1MCS-5 and
REM4 (REM14/T7-MEARR alba/pBBR1MCS-5).
Construction of the Test Strains REM12 and REM13
[0546] The entire DXP pathway from E. coli was synthesized by
DNA2.0 (Menlo Park, Calif.) and cloned into pET24a (see FIG.
63).
[0547] To build the higher flux DXP pathway isoprene-producing
REM12 and REM13 strains, the DXP operon pET24a plasmid was
transformed by electroporation into REM65-1 and REM4. A picture of
the DXP pathway enzyme containing vector, DXP operon pET24a
plasmid, is shown in FIG. 63.
Transformation of DXP Operon pET24a into REM65-1 and REM4
[0548] To build the test strains REM12 and REM13 strains, the DXP
operon pET24a plasmid was transformed by electroporation into
REM65-1 and REM4. Transformants were recovered in L broth and
plated on L agar containing gentamycin (10 ug/ml) and kanamycin (10
ug/ml). The resulting strains are designated as such: REM12
(REM65-1/DXP operon pET24a) and REM13 (REM4//DXP operon
pET24a).
Analysis of REM12 and REM13 for Growth and Isoprene Production
[0549] The wild-type strain REM12 and otherwise isogenic
.DELTA.iscR strain REM13 were compared in a shake flask isoprene
headspace assay. The benefits on isoprene production and effect on
growth rate the loss of iscR causes in the E. coli host are
illustrated in FIG. 60.
Growth
[0550] Strains REM12 and REM13 were grown at 30.degree. C. in TM3
liquid media (13.6 g K.sub.2PO.sub.4, 13.6 g KH.sub.2PO.sub.4, 2.0
g MgSO.sub.4*7H.sub.2O), 2.0 g citric acid monohydrate, 0.3 g
ferric ammonium citrate, 3.2 g (NH.sub.4).sub.2SO.sub.4, 0.2 g
yeast extract, 1.0 ml 1000.times. Modified Trace Metal Solution,
adjusted to pH 6.8 and q.s. to H.sub.2O, and filter sterilized)
supplemented to a final concentration with 0.1% yeast extract and
1.0% glucose and including kanamycin (10 ug/ml) and gentamycin (10
ug/ml). Growth was monitored periodically by recording each of the
culture's optical density measured at 600 nm using an Eppendorf
Biophotometer spectrometer (Eppendorf). 50 uM isopropyl
.beta.-D-1-thiogalactopyranoside (IPTG) was added to the cultures
to induce expression of the isoprene synthase and DXP enzymes
harbored by the strains at time zero, as indicated in the legend to
FIG. 60.
Isoprene Production
[0551] Isoprene production was analyzed using a headspace assay.
For the shake flask cultures, one ml of a culture was transferred
from shake flasks to 20 ml CTC headspace vials (Agilent vial
cat#5188 2753; cap cat#5188 2759). The cap was screwed on tightly
and the vials incubated at the equivalent temperature with shaking
at 250 rpm. After 30 minutes the vials were removed from the
incubator and analyzed. The analysis was performed using an Agilent
6890 GC/MS system interfaced with a CTC Analytics (Switzerland)
CombiPAL autosampler operating in headspace mode. An Agilent HP-5MS
GC/MS column (30 m.times.0.25 mm; 0.25 .mu.m film thickness) was
used for separation of analytes. The sampler was set up to inject
500 .mu.L of headspace gas. The GC/MS method utilized helium as the
carrier gas at a flow of 1 ml/minutes The injection port was held
at 250.degree. C. with a split ratio of 50:1. The oven temperature
was held at 37.degree. C. for the 2 minute duration of the
analysis. The Agilent 5793N mass selective detector was run in
single ion monitoring (SIM) mode on m/z 67. The detector was
switched off from 1.4 to 1.7 minutes to allow the elution of
permanent gases. Under these conditions isoprene
(2-methyl-1,3-butadiene) was observed to elute at 1.78 minutes. A
calibration table was used to quantify the absolute amount of
isoprene and was found to be linear from 1 .mu.g/L to 200 .mu.g/L.
The limit of detection was estimated to be 50 to 100 ng/L using
this method. The specific productivity of each strain is reported
as ug/L OD Hr. Note, ratio of 1900 ul headspace:100 ul broth in
assay vials for 30 min. incubation results in the following
conversion of isopreneug/L of culture to specific productivity:
(isoprene/L determined by GC-MS).times.(38)/(OD 600 nm of the
culture).
Example 11
Evaluation of Alternative ispG (gcpE) and ispH (lytB) Alleles from
Different Organisms by Complementation of dispG and/or dispH
Strains of BL21(DE3)PL.2 mKKDyI::FRT
[0552] We constructed an E. coli strain expressing the lower
mevalonic acid pathway (mevalonate kinase, phosphomevalonate
kinase, diphosphomevalonte decarboxylase and IPP isomerase from
yeast) as a base strain for testing the functionality of DXP
pathway enzymes from heterologous organisms. This strain produces
IPP and DMAPP from the lower mevalonate pathway if it is grown in
the presence of mevalonate. Deletions of enzymes of the DXP pathway
can be rescued by growing the stain in the presence of mevalonate.
Therefore, functionality of heterologous DXP pathway genes can be
expressed in the E. coli containing the lower MVA pathway and
looking for growth in the absence of mevalonate.
Construction of MD09-170 (BL21(DE3)PL.2 mKKDyI::FRT
TABLE-US-00017 Primer Sequences MCM 161: (SEQ ID NO: 84)
5'-CACCATGGTATCCTGTTCTGCG MCM162: (SEQ ID NO: 85)
5'-TTAATCTACTTTCAGACCTTGC MCM143: (SEQ ID NO: 86)
5'-aggaggtggtctcaaATGACTGCCGACAACAATAGTA MCM144: (SEQ ID NO: 87)
5'-aggaggtggtctcagcgctctgcagTTATAGCATTCTATGAATTTG CCTG
[0553] A P1 phage lysate was generated from MCM521 (BL21
neo-PL.2-mKKDyI) and transduced into BL21(DE3) (according to
Procedure 12-Genetic Transduction Using P1vir protocol). The
transductants were selected on L agar plates containing kanamycin
(20 ug/ml), with incubation at 37.degree. C. overnight. Four
colonies were verified by PCR to be correct transductants. One of
these colonies was selected and designated MD09-169 (BL21(DE3)PL.2
mKKDyI::Kan). The kanamycin resistance marker was looped out of
this strain using pCP20 from the Red/ET system from Gene Bridges
GmbH according to the manufacturer's instructions. The correct
loopout was confirmed by testing for sensitivity to kanamycin (20
ug/ml) and then loss of the kanamycin resistance cassette was
verified by PCR. The correct strain was designated MD09-170
(BL21(DE3)PL.2 mKKDyI::FRT).
Deletion of ispG and ispH from MD09-170
[0554] The gene deletions and subsequent loopout of the antibiotic
resistance markers described in this example were carried out using
the Red/ET system from Gene Bridges GmbH according to the
manufacturer's instructions. The strain MD09-170 was used.
Primer Sequences Used
TABLE-US-00018 [0555] MQ09-18F (SEQ ID NO: 88)
5'-GAACAATCACCGGCGCAGTAACAGACGGGTAACGCGGGAGATTTTT
CATGaattaaccctcactaaagggcgg MQ09-18R (SEQ ID NO: 89)
5'-CGGGAAGCGAGGCGCTTCCCATCACGTTATTATTTTTCAACCTGCT
GAACTAATACGACTCACTATAGGGCTCG MQ09-19F (SEQ ID NO: 90)
5'-TTTTGATATTGAAGTGCTGGAAATCGATCCGGCACTGGAGGCGTAA
CATGaattaaccctcactaaagggcgg MQ09-19R (SEQ ID NO: 91)
5'-ATTTTCGCATAACTTAGGCTGCTAATGACTTAATCGACTTCACGAA
TATCTAATACGACTCACTATAGGGCTCG MQ09-20F (SEQ ID NO: 92)
5'-cggcgcagtaacagacgggtaacgcgggagatttttcatg MQ09-20R (SEQ ID NO:
93) 5'-cgcttcccatcacgttattatttttcaacctgctgaac MQ09-21F (SEQ ID NO:
94) 5'-gaagtgctggaaatcgatccggcactggaggcgtaacatg MQ09-21R (SEQ ID
NO: 95) 5'-cttaggctgctaatgacttaatcgacttcacgaatatc
Strategy for Creating the Deletion Strains
[0556] The strategy for the deletion of ispG and ispH is shown in
FIG. 66. The antibiotic resistance cassette GB-CmR was amplified by
PCR using primer sets MQ09-18F/MQ09-18R or MQ09-19F/MQ09-19R for
deletion of ispG or ispH respectively. The primers contain 50 bases
of homology to the region flanking the ispG or ispH genes to allow
recombination at the specific locus upon electroporation of the PCR
product in the presence of the pRed-ET plasmid.
Amplification of the Deletion Cassettes
[0557] To amplify the GB-CmR cassette for deletion of ispG or ispH
the following PCR reactions were set up:
2 ul (100 ng GB-CmR)
10 ul HerculaseII Buffer
[0558] 0.5 ul dNTP's (100 mM) 1.25 ul primer (10 uM) MQ09-18F/19F
1.25 ul primer (10 uM) MQ09-18R/19R
2 ul DMSO
[0559] 32 ul diH2O +1 ul of HerculaseII fusion from Stratagene
Cycle Parameter:
[0560] 95.degree. C..times.2 min., [95.degree. C..times.20 sec.,
55.degree. C..times.20 sec., 72.degree. C..times.50 sec].times.29
cycles; 72.degree. C..times.3 min, 4.degree. C. until cool
(Eppendorf Mastercycler PCR machine)
[0561] The resulting PCR fragments were separated on a 1.2% E-gel
(Invitrogen), and purified using the Qiagen QiaQuick Gel Extraction
and QIAquick PCR Purification kits according to manufacturer's
instructions. The resulting stocks were: GB-CmR-ispG fragment
(1.593 kb)-180 ng/ul, and GB-CmR-ispH fragment (1.593 kb)-165
ng/ul.
Integration of GB-CmR-ispG or GB-CmR-ispH PCR Products into
MD09-170/pRed-ET Strain
[0562] The pRed-ET vector (Gene Bridges kit) was transformed into
MD09-170 by electroporation resulting in strain MD09-170/pRed-ET.
Approximately 300-500 ng of GB-CmR-ispG or GB-CmR-ispH PCR
fragments were electroporated into MD09-170/pRed-ET. The
transformants were recovered in L Broth containing 500 uM mevalonic
acid (Sigma) for 1 hour at 37.degree. C. and then plated on L agar
containing chloramphenical (5 ug/ml) and mevalonic acid (MVA) (500
uM). Chloramphenicol resistant colonies were analyzed by PCR for
the presence of the GB-CmR cassette and the absence of the ispG or
ispH genes using primers MQ09-20F/MQ09-20R or MQ09-21F/MQ09-21R
respectively. The correct strains were designated
MD09-209(BL21(DE3)PL.2 mKKDyI::FRT-.DELTA.ispG::Cm) and MD09-210
(BL21(DE3)PL.2 mKKDyI::FRT-.DELTA.ispH::Cm). The chloramphenicol
resistance cassette was looped out of both strains using pCP20 from
the RED/ET kit according to the manufacturer's instructions.
Transformants were verified by loss of resistance to
chloramphenicol (5 ug/ml) and PCR demonstrating loopout of the
GB-CmR cassette.
[0563] The resulting strains were designated MD09-219
(BL21(DE3)PL.2 mKKDyI::FRT-.DELTA.ispG::FRT) and MD09-220
(BL21(DE3)PL.2 mKKDyI::FRT-.DELTA.ispH::FRT).
Complementation of MD09-219 and MD09-220 with Alleles from
Thermosynechococcus elongatus BP-1
[0564] To test the functionality of the gcpE and lytB genes
(annotated) from T. elongates, the following plasmids expressing
these constructs or gcpE and lytB from E. coli were transformed by
electroporation into MD09-219 and MD09-220: [0565] 1. E. coli: GI
1.6-gcpE-lytB-yidi/pCR-Blunt II-TOPO (Kan) (positive control)
[0566] 2. T. elong: Ptac-gcpE-petF-petH/pK184 (Kan) [0567] 3. T.
elong: Ptac-gcpE-lytB-petF-petH/pK184 (Kan)
[0568] Transformants from 1. (E. coli) were recovered in L broth
containing MVA (500 uM) and plated on L agar containing kanamycin
(50 ug/ml). The resulting strain is designated
MD09-219/GI1.6-gcpE-lytB-yidi/pCRII-TOPO (Kan).
[0569] Transformants from 2. (T. elong) or 3. (T. elong) were
recovered in L broth containing MVA (500 uM) and IPTG (200 uM) and
then plated on L agar containing on kanamycin (50 ug/ml) and IPTG
(200 uM). The resulting strains were designated
MD09-219/Ptac-gcpE-petF-petH/pK184 (Kan) and
MD09-219/Ptac-gcpE-lytB-petF-petH/pK184 (Kan) respectively.
[0570] Several transformants were obtained on all of the plates
suggesting that the T. elongatus gcpE and lytB were functional in
E. coli. To confirm this, transformants were grown in L broth
containing kanamycin (50 ug/ml) with and without IPTG (200 uM).
Construction of GI 1.6-gcpE-lytB-yidi/pCR-Blunt II-TOPO
[0571] The construction of the GI 1.6-gcpE-lytB-yidi/pCR-Blunt
II-TOPO described in this example was carried out using standard
molecular biology techniques (see, for example, Sambrook et al.,
Molecular Cloning: A Laboratory Manual, 2.sup.nd ed., Cold Spring
Harbor, 1989, which is hereby incorporated by reference in its
entirety, particularly with respect to cloning techniques). A
picture illustrating the resulting plasmid construct is shown in
FIG. 67. The MD09-219 and MD09-220 strains were used for the
transformations described herein.
TABLE-US-00019 Primer sequences 5' EcoRI-GI 1.X-BamHI gcpE DXP
oper: (SEQ ID NO: 96) 5'-GAG GAA TTC GCG AGC CGT CAC GCC CTT GAC
NAT GCC ACA TCC TGA GCA AAT AAT TCA ACC ACT AAA CAA ATC AAC CGC GTT
TCC CGG AGG TAA CCG GAT CCA AGG AGA TAT ACC ATG CAT AAC CAG GCT CCA
ATT CAA CGT AGA 3' PstI idi DXP operon: (SEQ ID NO: 97) 5'-ATA TCC
TGC AGT TAT AGC ATT CTA TGA ATT TGC CTG TC M13 Forward (-20): (SEQ
ID NO: 63 and 69) 5'-GTAAAACGACGGCCAGT M13 Reverse (-27): (SEQ ID
NO: 99) 5'-CAGGAAACAGCTATGAC
degenerate N base: A base yields GI 1.6-, T base yields GI 1.5-, G
base yields GI1.2-, and C base yields GI 1.0-promoter Strategy for
Constructing GI 1.6-gcpE-lytB-yidi/pCR-Blunt II-TOPO
[0572] The vector construct harboring the T7 polymerase governed
synthetic DXP operon, DXP operon pET24a, was used as the PCR
template.
Amplification of the GI 1.6-gcpE-lytB-yidi Fragment
[0573] To amplify the GI 1.6-gcpE-lytB-yidi fragment (among the
other GI 1.X-possibilities) for cloning into the pCR-Blunt II-TOPO
vector the following PCR reaction was performed:
1 ul (approx. 100 ng DXP operon pET24a)
10 ul HerculaseII Buffer
[0574] 0.5 ul dNTP's (100 mM) 1.25 ul primer (10 uM) 5' EcoRI-GI
1.X-BamHI gcpE DXP oper 1.25 ul primer (10 uM) 3' PstI idi DXP
operon 35 ul diH2O +1 ul of HerculaseII fusion from Stratagene
Cycle Parameter:
[0575] 95.degree. C..times.2 min., [95.degree. C..times.30 sec.,
63.degree. C..times.30 sec., 72.degree. C..times.3.5 min.].times.29
cycles; 72.degree. C..times.5 min, 4.degree. C. until cool
(Biometra T3000 Combi Thermocycler).
[0576] The resulting PCR fragment was separated on a 1.2% E-gel
(Invitrogen) for verification of successful amplification, and
purified using the QIAquick PCR Purification kits (Qiagen)
according to manufacturer's instructions. The resulting stock was
GI 1.X-gcpE-lytB-yidi fragments.
Cloning of the GI 1.6-gcpE-lytB-yidi Fragment into pCR-Blunt
II-TOPO
[0577] The GI 1.X-gcpE-lytB-yidi fragments were cloned into
pCR-Blunt II-TOPO using Invitrogen's Zero Blunt.RTM. TOPO.RTM. PCR
Cloning Kit using the suggested protocol. Chemically competent
TOP10 cells (Invitrogen) were transformed with 2 ul of the ligation
reaction using a standard heat-shock protocol, and recovered in L
broth for 1 hour at 37.degree. C. and then plated on L agar
containing kanamycin (10 ug/ml). Resulting colonies were selected,
grown overnight in L broth containing kanamycin (10 ug/ml), and
harvested for plasmid preparation the following day. Plasmid
constructs were isolated using Qiagen Qiaprep Spin Miniprep Kit. A
number of plasmid preparations were sequenced (Quintara; Mountain
View, Calif.) using primers M13 Forward (-20) and M13 Reverse (-27)
and the correct GI 1.6-gcpE-lytB-yidi/pCR-Blunt II-TOPO clone
identified.
Example 12
Improving Isoprene Production in E. coli by Deregulating Glucose
Uptake
[0578] In Escherichia coli, glucose is transported using the
phosphoenolpyruvate transport system (PTSg1c), which consists of
PtsHICRR and the transporter PtsG (see Tchieu et al., J. Mol.
Microbiol. Biotechnol. 3(3):329-46 (2001), which is hereby
incorporated by reference in its entirety). Glucose is
phosphorylated as it is transported into the cell, with the
phosphate originating from phosphoenol pyruvate. The resulting
glucose-6-phosphate is metabolized via glycolysis regenerating the
PEP. Glucose transport continues through exponential growth but is
down-regulated as cells enter stationary phase. For commercial
purposes it is desirable to maximize production time and yield of
the desired molecule, which is difficult to achieve if the
feedstock transporter is downregulated. To solve this problem, the
PTSglc system is deleted by deleting ptsHIcrr, and in some
embodiments, ptsG, and constitutively express galP and glk,
encoding the galactose permease and glucokinase respectively. The
galactose permease transports glucose without phosphorylation so it
is necessary to express the glucokinase (see US Patent Application
No. 20050079617, which is hereby incorporated by reference in its
entirety).
[0579] The ptsHIcrr operon is deleted in BL21 using the Red/ET
system from Gene Bridges. Electrocompetent BL21 (Invitrogen) are
transformed with the pRed/ET plasmid and the resulting cells are
made electrocompetent by washing 3-4.times. in ice cold dH.sub.2O.
The GB-cmR cassette is amplified using forward and reverse primers
have at least 50 bases of homology to the regions immediately
upstream of ptsH or immediately downstream of crr. The resulting
PCR product is used to transform BL21/pRED and transformants are
plated on MacConkey agar containing glucose (1%) and
chloramphenical (5 ug/ml). Transformants that grown and are white
in color will be the correct genotype. The ptsHIcrr knockout is
transduced into the desired isoprene-producing hosts using P1
transduction.
[0580] The Ptrc-galP-cat and Ptrc-glk-cat cassettes are amplified
by PCR from strains KLpts::gal-trc::Cm or KLgalPglk-trc-cat S (see
U.S. Patent Application No. 20050079617, which is hereby
incorporated by reference in its entirety) with at least 50 base
pairs (bp) of homology on the 5' and 3' ends to allow homologous
recombination into BL21 with either the DXP or the MVA or both
pathways and isoprene synthase (example ispS from P. alba or a
variant thereof) expressed and the ptsHIcrr and/or ptsG deleted.
The desired strain is made competent and transformed with the
pRed/ET plasmid, and after being made competent, the new strain is
transformed with the galP-trc-cat cassette. Transformants are
selected on MacConkey agar containing 1% glucose and
chloramphenicol (5 ug/ml). Colonies which are slightly pink have
the correct genotype. The CAT markers in these cassettes are
flanked by loxP sites and can be looped out by standard methods
(Palmeros et al., Gene 18; 247(1-2):255-64 (2000)) which is hereby
incorporated by reference in its entirety). The strain expressing
galP from Ptrc is then transformed with the glk-trc-cat cassette
and transformants are select on MacConkey agar containing 1%
glucose and chloramphenicol (5 ug/ml). Colonies which are deep red
in color are the correct colonies.
[0581] The resulting strains have the full MVA pathway, with or
without the DXP pathway constitutively expressed, an isoprene
synthase (example P. alba IspS or a variant thereof), a deletion of
the ptsHIcrr and/or ptsG, and constitutive expression of the
galactose permease and glucokinase. To demonstrate that isoprene
production is enhanced and/or prolonged in these strains compared
to the parent which transports glucose via the PTSglc system, the
strains are tested in shake flask (TM3 containing 1% glucose, 0.1%
yeast extract), microfermentor (TM3 containing 1% glucose, 0.1%
yeast extract), and in 14-Liter fermentation. These strains are
also tested using pretreated and saccharified biomass, for example
corn fiber, corn stover, switch grass, forage sorghum, softwood
pulp, hardwood pulp or other suitable biomass.
[0582] Isoprene production is enhanced and/or prolonged in the
strains with ptsHIcrr and/or ptsG deletion and constitutive
expression of the galactose permease and glucokinase compared to
the compared to the parent strains without the deletion of ptsHIcrr
and/or ptsG and constitutive expression of the galactose permease
and glucokinase.
Example 13
Expression of Monoterpene and Sesquiterpene Synthases in
Combination with the Expression of Isoprene Synthase Increases the
Specific Productivity of Isoprene in E. coli
[0583] Isopentenyl pyrophosphate (IPP) and dimethyl allyl
pyrophosphate (DMAPP) are biosynthesized by the DXP pathway (also
called the non-mevalonate pathway and MEP pathway) in E. coli. IPP
and DMAPP can be condensed to form geranyl pyrophosphate (GPP) and
subsequently farnesyl pyrophosphate (FPP) by farnesene synthase
(IspA). FPP can be converted to octaprenyl pyrophosphate (OPP) and
undecaprenyl pyrophosphate (UPP) by extension of FPP with IPP.
These products serve a variety of functions in E. coli including
prenylation of tRNA (protein synthesis component) with DMAPP,
formation of quinones (respiratory chain component) with OPP, and
peptidoglycan formation (cell wall component) with UPP.
[0584] The products of the DXP pathway may be regulated by the
production of IPP and DMAPP. Accordingly, the example shows that
the introduction of a terpene synthase that utilizes downstream
products of the DXP pathway in combination with isoprene synthase
in E. coli results in increased flux through the DXP pathway and
increased specific productivity of isoprene.
Methods
Strain Construction
[0585] The following strains are constructed.
[0586] Ocimene synthase, farnesene synthase and artemesinin
synthase are cloned into pTrchis2A plasmids to give pTrcFPP,
pTrcAS, or pTrcOS. Isoprene synthase (for example IspS from P. alba
or variants thereof) is cloned into pBBR under control of the Ptrc
promoter to give pBBRPtrcalba.
Strain set 1) BL21GI1.6yIDI/pBBRPtrcalba itself or combined with
PtrcFPP or pTrcAS or pTrcOS. Strain set 2)
BL21GI1.6yIDIGI1.6DXS/pBBRPtrcalba itself or combined with PtrcFPP
or pTrcAS or pTrcOS.
[0587] The strains in strain set 1) or 2) are grown in shake flask
or in the microfermentor in TM3 containing 0.1% yeast extract and
1% glucose. The specific productivity of isoprene is measured over
time.
[0588] The specific productivity of isoprene from strains in strain
set 1) are compared. The specific productivity of isoprene in the
strains containing FPP, OS, or AS is higher than in the strain
without FPP, OS, or AS.
[0589] The specific productivity of isoprene from strains in strain
set 2) are compared. The specific productivity of isoprene in the
strains containing FPP, OS, or AS is higher than in the strain
without FPP, OS, or AS.
Example 14
Deletion or Reduction of Carbon into Thiamine and Pyridoxine Paths
for Relief of Inhibition
[0590] 1-deoxy-D-xylulose-5-phosphate (DXP) is a substrate in three
essential anabolic pathways in E. coli, namely isoprenoids,
thiamine and pyridoxal synthesis. In order to avoid any feedback
regulation from thiamine or pyridoxal pathways, which could then
decrease the flux in the DXP pathway for isoprenoid production, we
build strains mutated in the thiamine and/or pyridoxal
pathways.
A: Construction of an E. coli Strain Deleted in the Thiamine
Synthesis Pathway
[0591] Several enzymes are involved in the biosynthesis of thiamine
from DXP. ThiG and ThiH combine to form a complex containing an
iron-sulfur cluster (Leonardi et al. FEBS Lett. 539(1-3):95-9
(2003), PMID: 12650933, which is hereby incorporated by reference
in its entirety). Together, they are required for the synthesis of
4-methyl-5-(.beta.-hydroxyethyl)thiazole phosphate, which is the
rate-limiting step in thiamine synthesis (Leonardi et al. J Biol.
Chem. 279(17):17054-62 (2004), PMID: 14757766; Vander et al., J.
Bacteriol. 175(4):982-92 (1993), PMID: 8432721; which are hereby
incorporated by reference in their entireties). Since it is in the
rate-limiting step, and it is the first enzyme after
1-deoxy-D-xylulose-5-phosphate, thiG was chosen as the gene to be
deleted.
[0592] A PCR product was obtained using primers GB400thiGF
(caggagccagaacgcaactgc (SEQ ID NO:100) and GB400thiGR
(CACTTTCGCCTGATGTTCACC (SEQ ID NO:101), and genomic DNA of strain
JW5549 from the Keio collection (Baba et al., Mol. Syst. Biol.
2006.008 (2006), which is hereby incorporated by reference in its
entirety). The PCR product contains a kanamycin cassette replacing
most of the thiG gene and around 400 bp flanking regions of both
sides of the thiG gene.
[0593] A BL21(DE3) thiG::Kan mutant is then obtained by Red/ET
recombineering (Gene Bridges, Dresden, Germany) using the PCR
product mentioned above. It is proven correct by amplification and
sequencing. The strain is named CMP179.
B: Construction of an E. coli Strain Deleted in the Pyridoxal
Synthesis Pathway
[0594] PdxJ catalyses the formation of pyridoxine-5-phosphate
(precursor of pyridoxal-5-phosphate then pyridoxal) from
1-deoxy-D-xylulose-5-phosphate and
1-amino-propan-2-one-3-phosphate. The latter is produced by a
sequence of reactions coming from erythrose-4-phsophate, the first
one catalyzed by D-erythrose 4-phosphate dehydrogenase (epd). Thus
both pdxJ and epd are good candidates for deleting the production
of pyridoxal. However, epd has been reported not to be required for
glycolysis or for synthesis of pyridoxal (Seta et al., J.
Bacteriol. 179(16):5218-21 (1997), which is hereby incorporated by
reference in its entirety). Thus, pdxJ is chosen as the target for
mutation.
[0595] A PCR product is obtained using primers GB400pdxJF (CAT TCA
GTC TCT TGC AGG GGT C (SEQ ID NO:102) and GB400pdxJR
(gcatagtgccgctcatctgcc (SEQ ID NO:103)), and genomic DNA of strain
JW2548 from the Keio collection (Baba et al. 2006). The PCR product
contains a kanamycin cassette replacing most of the pdxJ gene and
around 400 bp flanking regions of both sides of the pdxJ gene.
[0596] A BL21(DE3) pdxJ::Kan mutant is then obtained by Red/ET
recombineering (Gene Bridges, Dresden, Germany) using the PCR
product mentioned above. It is proven correct by amplification and
sequencing. The strain is named CMP180.
C: Construction of an E. coli Strain Deleted in the Thiamine and
Pyridoxal Synthesis Pathways
[0597] The kanamycin cassette is removed from CMP179 and/or CMP180
by Flp-mediated excision, using plasmid 706-Flp from Gene Bridges
(Dresden, Germany). Then the PCR product described in section A is
used to mutate BL21(DE3) pdxJ through Red/ET recombineering.
D: Production of Isoprene Via the DXP Pathway, in a thiG and/or a
pdxJ Mutant
[0598] The effect of the thiG, pdxJ or thiG pdxJ mutations on the
production of isoprene through the DXP pathway is assessed in
different constructs enhancing DXP pathway flux and expressing IspS
(isoprene synthase) from Populus alba, such as MCM597
(BL21(DE3)pLysS pET24(MEA)alba-DXS-yIDI) or MCM719 (BL21 gi1.6-yIDI
gi1.6-dxs, pTrc(MEA)alba)).
[0599] Strains are grown overnight at 30.degree. C., 200 RPM, in
HM1 medium (Table 2) plus appropriate antibiotics. The morning
after, they are resuspended to an OD=0.2 in fresh HM1
medium+appropriate antibiotics. Flasks are incubated at 30.degree.
C., 200 RPM, and regularly sampled for OD and isoprene
productivity.
TABLE-US-00020 TABLE 2 HM1 medium composition Compounds
Concentration (g/L) K2HPO4 13.6 KH2PO4 13.6 MgSO4 * 7H2O 2 Citric
Acid Monohydrate 2 Ferric Ammonium Citrate 0.3 (NH4)2SO4 3.2 Trace
metal solution 1 ml
[0600] Specific productivity (ug isoprene/OD.h) is increased when
strains MCM597 or MCM719 contains thiG, pdxJ, or thiG pdxJ
mutations.
Example 15
Balancing Pyruvate and G-3-P (Glyceraldehyde-3-Phosphate) to
Increase Isoprene Production
[0601] Flux to the DXP pathway may be positively (more flux)
effected to increase isoprene production by maximizing the balance
between the two precursors required for the DXP pathway, pyruvate
and G-3-P (glyceraldehyde-3-phosphate). Accordingly, adjusting the
expression level of enzymes that determine flux into glycolysis,
into the pentose phosphate pathway (PPP) and into the
Entner-Doudoroff (ED) pathway (FIG. 68). In Sections B-D, flux of
pyruvate and G-3-P are affected simultaneously. Optimal balance of
the two precursors to the DXP pathway may also be achieved by
redirecting flux with the effect of elevating or lowering pyruvate
or G-3-P separately. Section E demonstrates this approach with the
coexpression of the mevalonate pathway. In addition it is proposed
that desired flux balance can be achieved by choice of feed stock,
e.g., feeding a mixture of glucose+gluconic acid; Section A shows
this approach. A combination of these approaches may prove to be
additive in achieving precursor balance and maximize yield of
isoprene; this is tested in Section F.
Section A
[0602] Cells that have been constructed by procedures known to
practitioners of the art and as exemplified in this application to
overexpress the DXP pathway or wild type cells are fed with various
carbon sources, but more specifically cells are fed glucose plus
gluconic acid or gluconic acid alone. The culture is sampled and
analyzed for improved evolution of isoprene. This analysis is
accomplished by monitoring the head space of the culture with a
mass spectrometer either continuously or at specific time points
during the cultivation of cells with different concentrations of
the carbon sources.
Section B
[0603] Cells in Section A harboring the overexpressed DXP pathway
or wild type cells are genetically engineered to overexpress
glucose-6-phosphate dehydrogenase to redirect flux to PPP and ED.
Effect and benefit of these mutations can be assessed by measuring
isoprene specific productivity.
Section C
[0604] Cells in Section A harboring the overexpressed DXP pathway
or wild type cells are genetically engineered to limit expression
of glucose-6-phosphate isomerase to redirect flux to PPP and ED.
Effect and benefit of these mutations can be assessed by measuring
isoprene specific productivity.
Section D
[0605] Cells in Section A harboring the overexpressed DXP pathway
or wild type cells are genetically engineered to limit expression
of Gluconate-6-phosphate dehydrogenase (gnd) to limit flux to
pentose phosphate and maximize flux to ED. Effect and benefit of
these mutations can be assessed by measuring isoprene specific
productivity.
Section E
[0606] In this section, the DXP precursor pyruvate is adjusted by
the level of expression of the mevalonic acid pathway for which
pyruvate is the sole precursor. Cells are constructed to
overexpress the DXP pathway enzymes as well as the mevalonic acid
pathway enzymes and expression of both pathways is adjusted, by
choosing the appropriate promoter strengths, such that pyruvate
flux is balanced with G-3-P flux and neither precursor accumulates
in the cell. Similar, approaches in the presence of zwf, gnd, and
pgi mutations, singly or in all possible combination, have
potential for improved performance.
Section F
[0607] The strains created in Sections B-E, are combined for
potential additivity. Combination of zwf and gnd in a overexpressed
DXP pathway strain is tested for improved performance of the
strain. Similarly, the combination of pgi and gnd is envisaged to
provide similar results.
Example 16
Improved Carbon Flux Through the DXP Pathway in Strains Containing
PDH E1 E636Q Subunit Variants
[0608] This example describes methods for the construction of E.
coli BL21 strains containing pyruvate dehydrogenase E1 subunit
(PDH) variants that increase carbon flux through the DXP pathway.
In particular, these strains contain a mutant aceE gene, encoding
for a PDH variant with an E636Q point mutation which possesses a
reduced activity (26% of wild-type PDH activity) for the conversion
of pyruvate to acetyl-CoA. In addition, the PDH E636Q variant is
thought to have a dxs-like activity that results in the production
of 1-deoxyxylulose-5-phosphate (DXP) from the aldol condensation of
pyruvate and glyceraldehyde-3-phosphate. The carboligase activity
of the pyruvate dehydrogenase E1 E636Q mutant has been reported by
Nemeria et al. (J. Biol. Chem., 280(22), 21473-21482 (2005), which
is hereby incorporated by reference in its entirety). The net
effect is increased carbon flux into the DXP pathway, and reduced
carbon flux to acetyl-CoA relative to strains containing wild-type
PDH E1 activity.
[0609] The construction of E. coli BL21 strains containing the PDH
E1 E636Q mutant was as described by Sauret-Giieto et al. (FEBS
Lett., 580, 736-740 (2006)), which is hereby incorporated by
reference in its entirety. Briefly, the chromosomal copy of the dxs
gene is disrupted by the insertion of a chloramphenicol acetyl
transferase (CAT) containing cassette into the dxs locus of an E.
coli BL21 strain that contains one or more plasmids encoding a
heterologous mevalonic acid pathway (MVA). The resulting E. coli
BL21 MVA+(dxs::CAT) strain requires mevalonic acid for normal
growth. When the strain is cultured in the absence of mevalonic
acid, a suppressor mutation aceE gene arises at a low to moderate
frequency that rescues the surviving clones from the otherwise
lethal dxs-phenotype. Sequencing of the aceE gene and associated
promoter region is performed in order to confirm the presence of
the missense mutation that results in the PDH E636Q mutant.
[0610] The resulting E. coli BL21 dxs::CAT PDH E1 E636Q MVA+strain
is complemented with one or more functional copies of the dxs gene
derived from E. coli or from a heterologous source as described
herein. The resulting strains exhibits improved flux into the DXP
pathway relative to strains that do not possess the PDH E1 E636Q
variant.
[0611] Additionally, the resulting E. coli BL21 dxs::CAT PDH E1
E636Q MVA+strain can be further complemented with one or more
functional copies of a DXP pathway gene, a DXP pathway associated
gene, an iron-sulfur cluster-interacting redox gene (e.g., fldA or
fpr), and/or an IDI gene derived from E. coli or from a
heterologous source as described herein.
[0612] The strains can also be transformed with one or more copies
of genes encoding isoprene synthases, for example IspS from P. alba
or variants thereof as described herein. These strains produce
isoprene by both the DXP and MVA pathways where a greater
proportion of isoprene is derived from the DXP pathway relative to
the MVA pathway, as compared to strains that do not possess the PDH
E636Q variant. The ratio the DXP to MVA carbon flux is determined
using isotope-labeling techniques known to those skilled in the
art.
[0613] The strains can be optionally cured of the MVA pathway
encoding plasmids (e.g., CHL18 or any other MVA pathway strains as
described in U.S. Patent Application Nos. 61/097,186, 61/097,189,
and 61/125,336, which are each hereby incorporated by reference in
their entireties) if desired using techniques known to those
skilled in the art.
Example 17
Mutation of CRP Increases Flux to the DXP Pathway and Increases the
Production of Isoprene
[0614] Catabolite repression, in which the transcription of
sensitive operons is reduced by certain carbon sources, could be a
major restriction to flux in the DXP pathway, thereby reducing the
amount of isoprene which could be produced.
[0615] A CRP (cAMP Receptor Protein)-delete mutant is available
from the Keio collection and could easily be assessed for the
production of isoprene through the DXP pathway. Impact of its
global transcriptional regulation has been studied (Perrenoud and
Sauer, J. Bact. 187:3171-3179 (2005). which is hereby incorporated
by reference in its entirety). Other types of CRP mutants could
also be beneficial to the process. One such example is the CRP
mutant described by Eppler and Boos (Eppler and Boos, Mol.
Microbiol. 33:1221-1231 (1999), which is hereby incorporated by
reference in its entirety). CRP* is a cAMP-independent CRP
variant.
A: Construction of an Isoprene-Producing Crp* Mutant of E. coli
[0616] CRP* mutation is introduced by P1 transduction (lysate
prepared from E. coli strain ET25 (to be obtained from W. Boos)) in
an isoprene-producing strain, such as MCM597 (BL21(DE3)pLysS
pET24(MEA)alba-DXS-yIDI) or MCM719 (BL21 gi1.6-yIDI gi1.6-dxs,
pTrc(MEA)alba)) to form strains CMP220 and CMP221 respectively.
B: Production of Isoprene in a Crp* Mutant of E. coli, Via the DXP
Pathway
[0617] Strains CMP220 and CMP221, and strains MCM597 and MCM719,
are grown overnight at 30 C, 200 RPM, in HM1 medium (Table 3) plus
appropriate antibiotics+10 g/L glucose+1 g/L yeast extract. The
morning after, they are resuspended to an OD=0.2 in fresh HM1
medium+appropriate antibiotics+5 g/L glucose+1 g/L yeast extract.
Flasks are incubated at 30.degree. C., 200 RPM, and regularly
sampled for OD.sub.600 and isoprene productivity.
TABLE-US-00021 TABLE 3 HM1 medium composition Compounds
Concentration (g/L) K2HPO4 13.6 KH2PO4 13.6 MgSO4 * 7H2O 2 Citric
Acid Monohydrate 2 Ferric Ammonium Citrate 0.3 (NH4)2SO4 3.2 Trace
metal solution 1 ml
[0618] Specific productivity (ug isoprene/OD.h) is increased in
strains CMP220 and CMP221 in comparison to strains MCM597 or
MCM719.
C: Production of Isoprene in a Crp* Mutant of E. coli, Via the DXP
Pathway, when the Strain is Grown on a Glucose/Xylose Mixture
[0619] Pretreated biomass samples contain a mixture of glucose,
xylose and acetate as the main components. Xylose consumption by E.
coli is usually prevented in the presence of glucose. The CRP*
mutation should be helpful to enhance glucose and xylose
coconsumption (Cirino et al. biotech. Bioeng. 95:1167-1176 (2006),
which is hereby incorporated by reference in its entirety).
[0620] Strains CMP220 and CMP221, and strains MCM597 and MCM719,
are grown overnight at 30.degree. C., 200 RPM, in HM1 medium (Table
3) plus appropriate antibiotics+10 g/L glucose+1 g/L yeast extract.
The morning after, they are resuspended to an OD.sub.600=0.2 in
fresh HM1 medium+appropriate antibiotics+2.5 g/L xylose and 2.5 g/L
glucose+1 g/L yeast extract. Flasks are incubated at 30.degree. C.,
200 RPM, and regularly sampled for Mao, isoprene productivity and
carbohydrate concentration. Carbohydrate concentration is
determined by HPLC (Ion exclusion column Aminex HPX-87H, 300
mm.times.7.8 mm, 0.005 M H2504, 0.6 mL/min as the mobile
phase).
[0621] While strains MCM597 and MCM719 show a diauxic growth curve,
co-consumption of xylose and glucose is increased in strains CMP220
and CMP221. This allows the fermentation to be completed in a
shorter time.
Example 18
Increased Isoprene Production in an E. coli Strain with LytBG120D
Mutation
[0622] The primary issues of this concept involve the biochemical
determination of the mutant DXP pathway enzyme LytBG120D and
whether or not the anticipated function of the LytBG10D enzyme can
help serve a relevant aspect of our target DXP pathway strain to be
used for BioIsoprene production. In this example, the desired DXP
pathway strain is to produce a majority (if not as close to all as
possible) of isoprene via the dimethylallyl pyrophosphate (DMAPP)
molecule derived directly from the LytBG120D catalysis of
(E)-4-hydroxy-3-methylbutyl-2-enyl pyrophosphate (HMBPP); as
opposed to DMAPP generated via the IDI enzyme, which isomerizes
isopentenyl pyrophosphate (IPP) into DMAPP.
[0623] The wild-type LytB of E. coli and the LytB enzyme common to
a number of other organisms, including plants and algae as well as
other bacteria, have been reported to produce both DMAPP and IPP in
ratios typically ranging from 1:4 to 1:6 (DMAPP:IPP). The work by
Kia-Joo Puan et al. (FEBS Letters, 579:3802-3806 (2005), which is
hereby incorporated by reference in its entirety) provides in vivo
data that supports the hypothesis that the LytBG120D mutant enzyme
can produce DMAPP, but can not generate sufficient levels of IPP to
support the viability of an E. coli deficient for IDI. No in vitro
data supporting the suggested activity for LytBG120D has been
introduced to the field yet.
[0624] Currently, isoprenoid production systems derive the majority
of their products from IPP. If the LytBG120D is determined to
solely generate DMAPP or a majority of DMAPP relative to IPP, then
the use of the lytBG120D allele in a DXP pathway-mediated isoprene
production strain may allow the unique generation of an isoprenoid
product that is derived almost entirely from DMAPP.
[0625] The lytBG12D is generated via PCR-based methods using E.
coli MG1655 as a template and cloned into an expression vector
(pET-15b). For comparison, the wild-type lytB is cloned into the
same pET-15b expression vector backbone. Each construct is moved
into BL21(DE3), or a comparable expression host, once the sequence
of the construct has been verified. From the expression strains,
LytB and LytBG120D is produced and subsequently purified using
standard affinity purification procedures. The protein may need to
be reconstituted under anaerobic conditions prior to activity
assessment (protocols exist in the literature) for robust enzymatic
function to be determined. LytB is a 4Fe-4S cluster containing
enzyme and is known to be sensitive to oxygen. Alternatively, LytB
and LytBG120D may be able to be assayed directly from cell lysates
prior to purification if sufficient activity of each enzyme can be
supported under those conditions and if an absence of significant
Idi activity can be achieved. Expression of each enzyme is
determined and quantified by gel electrophoresis and/or
immuno-blot. Activity assays are described in the literature, but
briefly may include incubation of each enzyme (purified or
contained within a cell extract) in a previously described buffer
including the substrate HMBPP and in the absence of Idi activity.
After a defined time(s) the ratio of DMAPP to IPP is determined
using HPLC methods. The resulting data are the first in vitro
results for LytBG120D available to us.
[0626] If LytBG120D is found to solely produce DMAPP, or at least
produce DMAPP in vast abundance to IPP, then the use of the
lytBG120D allele is incorporated in the DXP pathway isoprene
production strains. Initially, this is accomplished by
overexpressing the lytBG120D gene relative to the wild-type allele
under isoprene-production phases within a host background that
supports carbon flux through the DXP pathway to isoprene synthase.
As a control to assess, any benefits specific to generating
increased DMAPP levels relative to IPP that are expected to
accompany the overexpression of lytBG120D, a similar strain
overexpressing the wild-type lytB gene is also constructed and
assessed. The levels of DMAPP and IPP generated by these strains,
as well as isoprene and other downstream isoprenoids, are
determined by HPLC and/or GC-MS methods.
[0627] Our past findings indicate that increased IPP levels are not
tolerated well by E. coli. Further more, we have seen that
increased IPP levels accompanied by a significantly active Idi
result in the synthesis of larger downstream isoprenoid products,
which also cause a significant decrease in viability. Because LytB
produces a majority of IPP to DMAPP, and because the endogenous IdI
activity of E. coli is minimal, and because DMAPP is the substrate
for isoprene synthase, our current D.times.P system relies on the
use of an IdI derived from yeast. The use of LytBG120 in a DXP
production strain removes the dependence our current system has on
the yeast IdI (if LytBG120D is determined to produce mostly DMAPP).
The use of LytBG120 is also expected to reduce the levels of
downstream isoprenoid synthesis since IPP, the major subunit of
larger isoprenoids, is not abundantly available.
Example 19
Host Change for Relief of Endogenous Regulation of DXP Pathway
[0628] The DXP pathway, required for isoprenoids production in most
Prokaryotes, is a strongly regulated pathway. Indeed, it is
essential but also needed in small amount, as it diverts carbon
from the central metabolism intermediates glyceraldehyde-3-P and
pyruvate. As such, it might be difficult to escape regulation when
working with endogenous genes.
[0629] A solution to this problem may be to express the whole DXP
pathway from one organism into another host organism, the latter
organism being close or far on the phylogenetic tree. These host
organisms include, but not limited to industrial organisms, such as
Escherichia coli, Pseudomonas fluorescens, Zymomonas mobilis,
Bacillus sp., Saccharomyces cerevisiae, Clostridium sp.,
Corynebacterium glutamicum, and Saccharomyces cerevisiae. The fact
that all the genes involved in the pathway are cloned from one
organism guarantees that the enzymes produced by those genes can
work together to produce the end product DMAPP.
A: Construction of a DXP Pathway-Expressing Plasmid by Cloning E.
coli DXP Genes
[0630] A Ptrc promoter, PCR-amplified from plasmid pTrcHis2A
(Invitrogen, Carlsbad, Calif.) is cloned into pBBR1-MCS4 plasmid
(Kovach et al, Gene, 166:175-176 (1995), which is hereby
incoporated by reference in its entirety) multiple cloning site,
leaving a PstI site downstream of the promoter. This plasmid is
named pBBR4Ptrc. E. coli genes yajP (dxs), ispC (dxr), ispD, ispE,
ispF, gcpE, lytB and idi are amplified from genomic DNA of E. coli
MG1655 with primers containing an NsiI site and a RBS on the
upstream primer, and a PstI site on the downstream primer. Genes
are added one by one to the plasmid. Restriction digestion is used
to check and select clones with the right orientation.
Alternatively, a terminator is introduced after ispF and a new
promoter (e.g. Ptrc) has been introduced in front of an operon
constituted from gcpE, lytB and idi. The plasmid thus generated is
named pBBR4PtrcDXPc and pBBR4PtrcDXPc2.
B: Construction of a Codon-Optimized DXP Pathway-Expressing Plasmid
by Synthetic DNA Synthesis
[0631] A synthetic operon similar to the one described above is
designed and ordered, codon-optimized for Pseudomonas fluorescens,
from GeneArt (Regensburg, Germany). It is subcloned in plasmid
pBBR4Ptrc to generate plasmid pBBR4PtrcDXPa.
C: Expression of E. coli DXP Pathway in Pseudomonas fluorescens,
and its Effect on Isoprene Production
[0632] An ispS (isoprene synthase from Populus) gene codon
optimized for Pseudomonas (see other Pseudomonas patent example) is
cloned into plasmid pHRP309 (gentamycin resistant) (Parales and
Harwood, Gene 133:23-30 (1993), which is hereby incorporated by
reference in it entirety), and transformed by biparental mating
into Pseudomonas fluorescens ATCC 13525. Plasmids pBBR4PtrcDXPc,
PBBR4PtrcDXPc2 and pBBR4PtrcDXPa are transformed in E. coli S17-1
by electroporation and selection of transformants on LB+kanamycin
50 ug/ml. The plasmids are then transformed into Pseudomonas
fluorescens with IspS-expressing pHRP309 by biparental mating and
selection on M9 medium+16 mM sodium citrate+kanamycin 50
.mu.g/ml+gentamycin 50 ug/ml, to form strain CMP222, CMP223 and
CMP224 respectively.
[0633] When strains CMP222, CMP223 and CMP224 are grown in HM1
medium+10 g/L glucose, isoprene specific productivity is higher
than for the Pseudomonas fluorescens strain devoid of the DXP
pathway-expressing plasmids.
Example 20
Identification of Compounds Affecting Production of Isoprene Via
the DXP Pathway
[0634] Isoprene production and growth by a strain of E. coli that
over-expresses DXP pathway enzymes and isoprene synthase was
investigated using 96-well microtiter plates with a range of
different carbon, nitrogen or phosphate sources. A number of
compounds that affected production of isoprene to a significant
degree either positively or negatively were surprisingly
identified. Compounds positively or negatively affecting the
specific productivity of isoprene may help identify metabolic
pathways that affect isoprene production. Such pathways may be
implicated directly in the production of isoprene or they may have
regulatory roles. The identified compounds or metabolic pathways
may be modified for example by genetic modification to optimize the
production of isoprene. The identified carbon, nitrogen or
phosphate sources may also be supplemented directly to the media
for increased production of isoprene.
Experimental Procedure:
TM3 Media Recipe (Per Liter Fermentation Media):
[0635] K.sub.2HPO.sub.4 13.6 g, KH.sub.2PO.sub.4 13.6 g, citric
acid monohydrate 2 g, ferric ammonium citrate 0.3 g,
(NH.sub.4).sub.2SO.sub.4 3.2 g, 1000.times. Trace Metal Solution 1
ml. All of the components were dissolved sequentially in
diH.sub.2O. The pH was adjusted to 6.8 with ammonium hydroxide
(30%) and brought to volume. Media was filter sterilized with a
0.22 micron filter. Before use, MgSO.sub.4*7H.sub.2O 2 g, yeast
extract 0.2 g was added to the media. Carbon source was added to a
final concentration of 0.5% if needed. Required antibiotics were
added after sterilization and pH adjustment.
1000.times. Trace Metal Solution (Per Liter Fermentation
Media):
[0636] Citric Acids*H.sub.2O 40 g, MnSO.sub.4*H.sub.2O 30 g, NaCl
10 g, FeSO.sub.4*7H.sub.2O 1 g, CoCl.sub.2*6H.sub.2O 1 g,
ZnSO.sub.4*7H.sub.2O 1 g, CuSO.sub.4*5H.sub.2O 100 mg,
H.sub.3BO.sub.3 100 mg, NaMoO.sub.4*2H.sub.2O 100 mg. Each
component was dissolved one at a time in diH.sub.2O, pH to 3.0 with
HCl/NaOH, and then brought to volume and filter sterilized with
0.22 micron filter.
Strain:
MCM597
[0637] (i) Construction of MCM597 (BL21(DE3) pLysS
pet24(MEA)albadxsyIDI Construction of pDU-39
TABLE-US-00022 Primer sequences: Alba TRC(MEA)-NdeI-F (SEQ ID NO:
104) 5'-gaaactgaaaccCATATGgaagctcgtcgttctgc Alba FLTRC (-) TEV-R
(SEQ ID NO: 105)
5'-cccgcgcttaCTCGAGgcgttcaaacggcagaatcggttcagtg
[0638] A truncated version of the Populus alba isoprene synthase
was created by amplifying the gene using the primer set Alba
TRC(MEA)-NdeI-F/Alba FLTRC(-) TER-R and the template pET24 alba HGS
(described in Example 10, U.S. patent application Ser. No.
12/335,071, which is hereby incorporated in its entirety). The PCR
reaction was set up as follows:
1 ul (pET24a-P. alba) 5 ul 10.times. PfuUltraII Fusion buffer 1 ul
dNTP's (10 mM) 1 ul primer (50 uM) Set #1 forward 1 ul primer (50
uM) Set #1 reverse 41 ul diH2O +1 ul of PfuUltra II Fusion DNA
Polymerase from Stratagene
Cycle Parameter:
[0639] 95.degree. C. 1 min. [95.degree. C. 30 sec., 55.degree. C.
20 sec., 72.degree. C. 25 sec].times.29 cycles, 72.degree. C. 3
min, 4.degree. C. until cool, (Eppendorf Mastercycler)
[0640] The PCR products were digested with NdeI-XhoI restriction
endonucleases (Roche) and gel purified using the QIAquick Gel
Extraction Kit (Qiagen) according to the manufacturer's
instructions. An aliquot of 3 ul of the purified product was
ligated using T4 ligase (New England BioLabs) to pET-24a vector
(Invitrogen) that was previously digested with NdeI-XhoI, gel
purified and treated with Shrimp Alkaline Phosphatase (SAP, Roche).
The ligation was carried out overnight at 16.degree. C.
[0641] An aliquot of 5 uL of the overnight ligation mixture was
transformed into TOP10 cells (Invitrogen) and transformants were
selected on L agar containing kanamycin (50 ug/ml) at 37.degree. C.
overnight.
[0642] Plasmids were isolated from a few of the transformants using
the QiaQuick Spin Kit (Qiagen) according to the manufacturer's
instructions. The insert was verified by digestion NdeI-XhoI
restriction endonucleases and the clones were sequenced with the
commercially available T7 promoter and T7 terminator (Quintara Bio
Sequencing Service, Berkeley, Calif.).
The Correct Plasmid was Designated pDu-39 (FIG. 69)
Construction of MCM597
TABLE-US-00023 [0643] Primer Sequences MCM270 (SEQ ID NO: 106)
5'-GATCGGATCCATTCGCCCTTAGGAGGTAAA MCM271 (SEQ ID NO: 107)
5'-GATCGCGGCCGCCAGCTGCAGGACGCGTTGTTATAGCATT
[0644] The DXS-yIDI genes were amplified by PCR using primers
MCM270/MCM271 and the template pMCM72 (described in Example 7 U.S.
patent application Ser. No. 12/335,071, which is hereby
incorporated by reference in its entirety). Two identical PCR
reactions were set up according to the manufacturer's protocol for
Herculase II Fusion (Stratagene). 35 uL water, 10 uL buffer, 1.25
uL each primer, 0.5 uL dNTPs, 1 uL polymerase. Reactions were
cycled: 95 C, 2:00; (95 C 0:15, 55 C 0:15, 72 C 1:45).times.30; 72
C 3:00, 4 C until cold.
[0645] The resulting PCR fragment was digested with BamHI and NotI
(Roche), and then ligated using Roche Rapid Ligation Kit into pDu39
that had been digested with the same restriction endonucleases. The
ligation reaction was set up in 10 uL containing 5 uL Buffer 1, 1
uL vector, 3 uL insert and 1 uL ligase and incubated for 1 hour at
room temperature. An aliquot of 5 uL was transformed into E. coli
Top10 chemically competent cells (Invitrogen). Transformants were
selected on L agar containing kanamycin (50 ug/ml) at 37.degree. C.
overnight. Plasmids were purified from a few transformants and
screened for the presence of insert using Herculase II Fusion
(Stratagene). 17.5 uL water, 5 uL buffer, 0.625 uL each primer,
0.25 uL dNTPs, 0.5 uL polymerase. Reactions were cycled: 95 C,
2:00; (95 C 0:15, 52 C 0:15, 72 C 0:45).times.30; 72 C 3:00, 4 C
until cold. Clones with a PCR product near 1.5 kbp were sequenced
(Quintara Biosciences, Berkeley Calif.). A correct plasmid was
designated MCM596. The plasmid was then transformed into
electrocompetent BL21(DE3)pLysS cells (Invitrogen) and
transformants were selected on L agar containing kanamycin (50
ug/ml) and chloramphenicol (35 ug/mL). One colony was selected and
designated MCM597.
Experimental Protocol
[0646] An inoculum of the E. coli strain MCM597 over-expressing the
DXP pathway enzymes dxs from E. coli and idi from Saccharomyzes
cereviciae and the isoprene synthase ispS from Populus alba was
taken from a frozen vial and streaked onto an LB broth agar plate
(with antibiotics) and incubated at 30.degree. C. overnight. A
single colony was inoculated into TM3 media containing glucose as
the only carbon source and grown overnight at 30.degree. C. The
overnight cultures were washed by centrifugation and resuspended
into fresh TM3 media containing no glucose or yeast extract. The
bacteria were then diluted into 20 mL of TM3 media to reach an
optical density of 0.05 measured at 600 nm. For experiments testing
the effect of different nitrogen sources using the Biolog PM3B
microtiter plates (Biolog, USA), the bacteria were diluted into
media containing 0.5% glucose and no yeast extract. For experiments
testing the effect of different carbon sources using the Biolog PM1
and PM2A microtiter plates (Biolog, USA), the bacteria were diluted
into media containing 0.2% yeast extract and either no or 0.5%
glucose. A total of 120 .mu.L of culture was dispensed into each
well of the Biolog plates and the plate was incubated on an orbital
shaker (250 rpm) at 30.degree. C. The optical density was measured
in the wells at 600 nm using a 384 well microtiter plate reader
(Molecular Devices, Spectramax Plus 384) in the beginning of the
experiment and every hour thereafter to follow growth of the
bacteria. None of the compounds in the biolog plates were found to
interfere with the optical density measurement at 600 nm. After
four to six hours of growth, the optical density was measured again
and two times 50 .mu.L was transferred to two 96 well quartz glass
blocks (Zinsser, Germany) and sealed with Biomek aluminum foil tape
lids (Beckman Coulter, USA). The glass blocks were shaken at 450
rpm at 30.degree. C. for 30 minutes and then heat treated for 12
minutes at 70.degree. C. The produced isoprene was measured using a
GC-MS (GC 7889A and MSD 5975C, Agilent Technologies, USA). To
account for differences from glass block to glass block, the
isoprene measurement was normalized to the block average. The
specific isoprene productivity was calculated by dividing the
isoprene production with the optical density for each well. Each
Biolog experiment was performed in duplicate. Statistical analysis
(students T-test) was used to identify compounds in the microtiter
plates that affected specific isoprene productivity with
statistical significance (p<0.1).
Results
Nitrogen Sources Affecting Isoprene Production:
[0647] When E. coli harboring the DXP pathway and isoprene synthase
was grown on 0.5% glucose as the sole carbon source in media
lacking yeast extract, a number of nitrogen containing compounds
were found to either positively or negatively affect the production
of isoprene through the DXP pathway. The PM3B plates from Biolog
were used for these experiments. Statistical analysis was used to
identify compounds that most significantly affect isoprene
production (Table 4). The addition of nitrite, nitrate, ammonia and
urea did not significantly change the specific isoprene production,
suggesting the bacteria were not directly lacking nitrogen in the
fermentation media. Compounds increasing the specific production of
isoprene surprisingly include L-glutamic acid, L-aspartic acid, the
purines inosine and guanosine, L-threonine, L-serine, L-tryptophan
and L-asparagine. Compounds negatively affecting specific isoprene
productivity particularly include adenine, and L-methionine, and
L-tyrosine among others. Some of these compounds are involved in
purine and thiamine biosynthesis, which are related to the DXP
pathway, and may as such play important roles in the regulation of
the DXP pathway.
Carbon Sources Affecting Isoprene Production During Growth on
Glucose:
[0648] When E. coli harboring the DXP pathway and isoprene synthase
was grown on 0.5% glucose in fermentation media containing 0.2%
yeast extract, a range of carbon sources were found to affect the
specific productivity of isoprene through the DXP pathway to a
surprisingly high degree. The PM1 and PM2A carbon source plates
from Biolog were used for these experiments. Statistical analysis
was used to identify compounds that most significantly affect
isoprene production (Table 5). Compounds most significantly
increasing specific productivity of isoprene include, but are not
limited to, phenylethylamine, propionic acid, D-galacturonic acid,
inosine, L-galactonic acid-.gamma.-lactone, D-psicose,
glucuronamide, 2-aminoethanol, D-cellobiose, sucrose, mucic acid,
L-malic acid, L-phenylalanine, 2,3-butanediol, L-ornithine,
D-gluconic acid, D-glucosaminic acid, D-mannose. It is to be
expected that the addition of these compounds to glucose fed
fermentations would increase the specific productivity of isoprene.
A range of other compounds were found to negatively affect specific
productivity (Table 5). These effects may be caused by regulatory
roles of the compounds or associated metabolic pathways, making
these pathways interesting for genetic modification.
Identification of Carbon Sources Useful for the Production of
Isoprene:
[0649] When E. coli harboring the DXP pathway and isoprene synthase
was grown in media containing 0.2% yeast extract in micro titer
plates containing a range of different carbon sources, it was
possibly to identify carbon sources that lead to the production of
isoprene with a surprisingly high specific productivity. The PM1
carbon source plate from Biolog was used for these experiments. A
range of compounds that lead to a very high specific isoprene
productivity is shown in Table 6. Compounds most significantly
increasing specific productivity of isoprene include, but is not
limited to, D-galacturonic acid, D-trehalose,
N-acetyl-D-glucosamine, D-mannitol, D-fructose,
D-glucose-6-phosphate, .alpha.-D-glucose. The final optical
densities of the cultures grown on the different compounds are
shown in Table 6. Some of these carbon sources may be used for the
production of isoprene.
TABLE-US-00024 TABLE 4 Nitrogen sources affecting specific
production of isoprene through the DXP pathway in E. coli. Only
compounds affecting the specific isoprene production with
statistical significance (p < 0.1) are shown. Nitrate, nitrite,
ammonia and urea have been included to illustrate that the addition
of general nitrogen sources does not affect specific productivity
of isoprene in the fermentation media (marked with grey). Isoprene
production normalized to P-value Compound negative control (T-test)
L-Glutamic Acid 2.13 0.003 Gly-Gln 1.80 0.008 Gly-Glu 1.48 0.008
Ala-Gln 1.46 0.045 Ala-Glu 1.45 0.030 L-Aspartic Acid 1.42 0.007
.delta.-Amino-N-Valeric Acid 1.40 0.012 Inosine 1.37 0.013
Guanosine 1.33 0.023 Gly-Asn 1.26 0.092 L-Threonine 1.22 0.022
Ethanolamine 1.22 0.055 L-Serine 1.21 0.059 L-Tryptophan 1.21 0.071
Ala-Asp 1.20 0.056 L-Asparagine 1.16 0.023 Nitrate 1.08 0.360
Nitrite 1.06 0.110 Ammonia 1.01 0.837 Negative Control 1.00 1.000
Urea 0.99 0.919 D-Alanine 0.84 0.046 N-Phthaloyl-L-Glutamic Acid
0.81 0.021 N-Acetyl-D-Mannosamine 0.81 0.091 Histamine 0.80 0.036
D-Valine 0.80 0.061 Tyramine 0.76 0.037 Ala-Thr 0.72 0.013
.beta.-Phenylethylamine 0.70 0.028 L-Tyrosine 0.69 0.012 Gly-Met
0.61 0.011 D,L-.alpha.-Amino-N-Butyric Acid 0.60 0.012 Hyroxylamine
0.60 0.010 L-Methionine 0.56 0.003 Met-Ala 0.55 0.007
.alpha.-Amino-N-Valeric Acid 0.52 0.004 Adenine 0.22 0.000
TABLE-US-00025 TABLE 5 Carbon sources affecting specific production
of isoprene through the DXP pathway in E. coli during growth on
glucose. All carbon sources are normalized to the negative control
that was only fed glucose. Only compounds affecting the specific
isoprene production with statistical significance (p < 0.1) are
shown. Negative control is marked with grey. Isoprene production
normalized to P-value Compound negative control (T-test)
Phenylethylamine 1.74 0.014 Propionic Acid 1.67 0.010
D-Galacturonic Acid 1.60 0.056 Inosine 1.60 0.015 L-Galactonic
Acid-.gamma.-Lactone 1.55 0.059 D-Psicose 1.53 0.055 Glucuronamide
1.50 0.093 2-Aminoethanol 1.38 0.042 D-Cellobiose 1.38 0.044
Sucrose 1.37 0.080 Mucic Acid 1.35 0.095 L-Malic Acid 1.28 0.086
L-Phenylalanine 1.23 0.004 2,3-Butanediol 1.22 0.044 L-Ornithine
1.21 0.010 D-Gluconic Acid 1.17 0.035 D-Threonine 1.15 0.032
D-Lactic Acid Methyl Ester 1.15 0.011 Chondroitin Sulfate C 1.15
0.035 L-Arginine 1.15 0.099 Salicin 1.13 0.063 M-Inositol 1.13
0.033 D-Glucosaminic Acid 1.13 0.002 D-Mannose 1.11 0.036 Negative
Control 1.00 1.000 Turanose 0.94 0.042 .beta.-D-Allose 0.92 0.100
L-Isoleucine 0.90 0.040 Sedoheptulosan 0.89 0.049 D-Tagatose 0.87
0.090 L-Arabitol 0.85 0.090 D,L-Malic Acid 0.82 0.031 L-Arabinose
0.82 0.090 a-Methyl-D-Glucoside 0.82 0.068 Stachyose 0.82 0.033
D-Glucose-6-Phosphate 0.81 0.041 D-Ribose 0.74 0.007 D-Galactose
0.72 0.011 Lactitol 0.70 0.031 .beta.-Methyl-D-Galactoside 0.70
0.011 .beta.-Methyl-D-Xyloside 0.68 0.085
.alpha.-Methyl-D-Galactoside 0.62 0.062 2,3-Butanone 0.51 0.013
D-Melibiose 0.49 0.001 D-Raffinose 0.45 0.001 4-Hydroxy Benzoic
Acid 0.41 0.005 Sorbic Acid 0.40 0.052 Capric Acid 0.35 0.008
Dihydroxy Acetone 0.22 0.002 2-Deoxy-D-Ribose 0.20 0.002 2-Hydroxy
Benzoic Acid 0.18 0.000 Caproic Acid 0.18 0.001
TABLE-US-00026 TABLE 6 Carbon sources leading to a high specific
production of isoprene in E. coli that over-expresses enzymes from
the DXP pathway and isoprene synthase. The specific isoprene
productivity was normalized to .alpha.-D-glucose. The final optical
density (OD600) of the cultures is also shown in the table,
indicating the growth of E. coli on the specificcarbon sources. The
negative control was not fed any carbon source and is marked with
grey. Isoprene production normalized to Growth Compound
.alpha.-D-Glucose OD600 D-Galacturonic Acid 1.36 0.217 D-Trehalose
1.31 0.243 N-Acetyl-DGlucosamine 1.17 0.283 D-Mannitol 1.16 0.270
D-Fructose 1.09 0.250 D-Glucose-6-Phosphate 1.09 0.299
.alpha.-D-Glucose 1.00 0.279 D-Gluconic Acid 1.00 0.276 Methyl
Pyruvate 0.99 0.213 Pyruvic Acid 0.95 0.211 Inosine 0.93 0.191
L-Serine 0.92 0.213 D-Serine 0.90 0.221 Adenosine 0.88 0.187
L-Glutamic Acid 0.78 0.194 .alpha.-D-Lactose 0.75 0.198 Thymidine
0.66 0.202 D-Fructose-6-Phosphate 0.66 0.172 Mucic Acid 0.62 0.167
2-Deoxy Adenosine 0.57 0.160 Dulcitol 0.53 0.182
D-Glucose-1-Phosphate 0.49 0.167 m-Hydroxy Phenyl Acetic Acid 0.48
0.170 Propionic Acid 0.35 0.131 Sucrose 0.31 0.147 M-Tartaric Acid
0.24 0.144 Negative Control 0.17 0.145
Example 21
Increased Expression of Fpr Improves Isoprene Production
[0650] In this example, we demonstrate an increase in activity of
the GcpE and LytB enzymes of the DXP pathway by providing more of
an essential auxiliary factor, Fpr, which has been shown to
positively influence their in vitro and in vivo activities
(Seemann, M. et al. Agnew. Chem. Int. Ed., 41: 4337-4339 (2002);
Wolff, M. et al. FEBS Letters, 541: 115-120 (2003), which are
hereby incorporated by reference in their entireties). Fpr provides
the necessary electrons derived from NADPH via FldA for GcpE and
LytB to perform their catalytic functions (reviewed in report by L.
A. Furgerson, The mevalonate-independent Pathway to Isoprenoid
Compounds: Discovery, Elucidation, and Reaction Mechanisms,
published Feb. 13, 2006, which is hereby incorporated by reference
in its entirety).
[0651] The expression of fpr (encoding flavodoxin/ferredoxin
NADPH-oxidoreductase) is increased in an engineered, isoprene
producing strain of E. coli. Our previously tested higher DXP flux
strains produce only modest isoprene levels, and are observed to
accumulate significant levels of both cMEPP,
2-C-methyl-D-erythritol-2,4-cyclodiphosphate, and HMBPP,
(E)-4-hydroxy-3-methylbut-2-enyl diphosphate. The cMEPP and HMBPP
DXP intermediates are the substrates of GcpE and LytB,
respectively. The increased amount of Fpr may increase the activity
demonstrated by the DXP pathway enzymes GcpE and LytB resulting in
improved carbon flux to isoprene synthesis in the strain of
interest over that of the comparable BL21 (DE3) control strain
producing only endogenous levels of Fpr. The improved flux is
demonstrated by an increase in isoprene titer.
[0652] The flavodoxin/ferredoxin NADPH-oxidoreductase encoded by
fpr is intended to be expressed at increased levels from the E.
coli chromosome by incorporating a constitutive highly active GI
1.6-promoter in front of the fpr open-reading frame, while
replacing the endogenous promoter sequence. Alternatively, fpr can
be expressed ectopically from a multi-copy vector construct. For
either method, our goal is to express and accumulate Fpr at a level
surpassing that generated from the endogenous fldA locus. Our
preliminary qRT-PCR results suggest GI 1.6 fpr generates more
fpr-transcript than the endogenous locus, and will likely
accumulate more Fpr than the control as a result of the increased
level of fpr-message. This is confirmed by immuno-blot once we
receive the antibodies to Fpr.
[0653] Using a BL21(DE3) high DXP flux strain as the parental host
strain, the introduction of the up-regulated fpr locus is assessed
for the effects on isoprene production relative to the control
strains. In addition, metabolite studies on the DXP intermediates
provides insight into the beneficial affects of increased Fpr
levels on GcpE and LytB activities.
[0654] Initially, the following BL21 (DE3) test strain is
constructed and assessed for growth and the production of isoprene
relative to the control: BL21 (DE3) GI 1.6-dxs GI 1.6-fpr T7-MEARR
alba/pBBR1MCS-5. This strain is compared to the parental control
strain (BL21 (DE3) GI 1.6-dxs T7-MEARR alba/pBBR1MCS-5) for growth,
isoprene production, and DXP metabolite accumulation.
Growth
[0655] Strains are grown at 30.degree. C. in TM3 liquid media (13.6
g K.sub.2PO.sub.4, 13.6 g KH.sub.2PO.sub.4, 2.0 g
MgSO.sub.4*7H.sub.2O), 2.0 g citric acid monohydrate, 0.3 g ferric
ammonium citrate, 3.2 g (NH.sub.4).sub.2SO.sub.4, 0.2 g yeast
extract, 1.0 ml 1000.times. Modified Trace Metal Solution, adjusted
to pH 6.8 and q.s. to H.sub.2O, and filter sterilized) supplemented
to a final concentration with 0.1% yeast extract and 1.0% glucose
and including the appropriate antibiotics. Growth is monitored
periodically by recording each of the culture's optical density
measured at 600 nm using an Eppendorf Biophotometer spectrometer
(Eppendorf).
Isoprene Production
[0656] Isoprene production is analyzed using a headspace assay. For
the shake flask cultures, one ml of a culture is transferred from
shake flasks to 20 ml CTC headspace vials (Agilent vial cat#5188
2753; cap cat#5188 2759). The cap is screwed on tightly and the
vials incubated at the equivalent temperature with shaking at 250
rpm. After 30 minutes the vials are removed from the incubator and
analyzed. The analysis is performed using an Agilent 6890 GC/MS
system interfaced with a CTC Analytics (Switzerland) CombiPAL
autosampler operating in headspace mode. An Agilent HP-5MS GC/MS
column (30 m.times.0.25 mm; 0.25 .mu.m film thickness) is used for
separation of analytes. The sampler is set up to inject 500 .mu.L
of headspace gas. The GC/MS method utilized helium as the carrier
gas at a flow of 1 ml/minutes The injection port is held at
250.degree. C. with a split ratio of 50:1. The oven temperature is
held at 37.degree. C. for the 2 minute duration of the analysis.
The Agilent 5793N mass selective detector is run in single ion
monitoring (SIM) mode on m/z 67. The detector was switched off from
1.4 to 1.7 minutes to allow the elution of permanent gases. Under
these conditions isoprene (2-methyl-1,3-butadiene) is observed to
elute at 1.78 minutes. A calibration table is used to quantify the
absolute amount of isoprene and was found to be linear from 1
.mu.g/L to 200 .mu.g/L. The limit of detection is estimated to be
50 to 100 ng/L using this method. The specific productivity of each
strain is reported as ug/L OD Hr. Ratio of 1900 ul headspace:100 ul
broth in assay vials for 30 min. incubation results in the
following conversion of isopreneug/L of culture to specific
productivity: (isoprene/L determined by GC-MS).times.(38)/(OD 600
nm of the culture).
DXP Metabolite Accumulation
[0657] The DXP metabolites of the isoprene-producing parental and
test strains will be isolated and quantified as follows:
Metabolite Extraction
[0658] Cell metabolism is rapidly inactivated by withdrawing 3.5 mL
of the culture into a tube filled with 3.5 mL of dry ice-cold
methanol. Cell debris is pelleted by centrifugation and the
supernatant is loaded onto Strata-X-AW anion exchange column
(Phenomenex) containing 30 mg of sorbent. The pellet is
re-extracted twice, first with 3 mL of 50% MetOH containing 1 mM
NH.sub.4HCO.sub.3 buffer (pH=7.0) and then with 3 mL of 75% MetOH/1
mM NH.sub.4HCO.sub.3 buffer (pH=7.0). After each extraction, cell
debris is pelleted by centrifugation and the supernatants are
consecutively loaded onto the same anion exchange column. During
the extraction and centrifugation steps the samples are kept at
below +4.degree. C. Prior to metabolite elution, the anion exchange
columns are washed with water and methanol (1 mL of each) and the
analytes were eluted by adding 0.35 mL of concentrated
NH.sub.4OH/methanol (1:14, v/v) and then 0.35 mL of concentrated
NH.sub.4OH/water/methanol (1:2:12, v/v/v) mixtures. The eluant is
neutralized with 30 .mu.L of glacial acetic acid and cleared by
centrifugation in a microcentrifuge.
Metabolite Quantification
[0659] Metabolites are analyzed using a Thermo Scientific TSQ
Quantum Access mass spectrometer (Thermo Electron Corporation, San
Jose, Calif.). All system control, data acquisition, and mass
spectral data evaluation are performed using XCalibur and LCQuan
software (Thermo Electron Corp). For the LC-ESI-MS/MS method, a
chiral Nucleodex .beta.-OH 5 .mu.M HPLC column (100.times.2 mm,
Macherey-Nagel, Germany) equipped with a CC 8/4 Nucleodex beta-OH
guard cartridge is eluted with a mobile phase gradient shown in
Table 7 (flow rate of 0.4 mL/min). The sample injection volume was
10 .mu.L.
TABLE-US-00027 TABLE 7 HPLC gradient used to elute metabolites.
Mobile phase, % B Time, A (100 mM ammonium C min (water)
bicarbonate, pH = 8.0) (acetonitrile) 0.0 0.0 20.0 80.0 0.5 15.0
5.0 80.0 4.5 37.5 12.5 50.0 6.5 37.5 12.5 50.0 7.0 49.5 0.5 50.0
12.0 34.9 0.1 65.0 12.5 0.0 20.0 80.0 13.0 0.0 20.0 80.0
[0660] Mass detection is carried out using electrospray ionization
in the negative mode. The following m/z values for precursor ions
are selected to detect the metabolites of interest in SRM mode:
245.0 for IPP and DMAPP, 381.1 for FPP, 213.0 for DXP, 215.0 for
MEP, 260.0 for HDMAPP, and 277.0 for cMEPP. Concentrations of
metabolites are determined based on the integrated intensities of
peaks generated by PO.sub.3.sup.- product ion (m/z=79.0).
Calibration curves obtained by injection of corresponding standards
purchased from Echelon Biosciences Inc. Intracellular
concentrations of metabolites are calculated based on the
assumption that in 1 mL of the culture at OD=200 the integrated
volume of all cells is 50 .mu.L
Example 22
Improved Carbon Flux into the DXP Pathway Using a Heterologous
DXS
[0661] Living organisms synthesize isoprenoids via two distinct
pathways: the mevalonate (MVA) pathway and 2-C-methyl-D-erythritol
4-phosphate (MEP) pathway. MEP pathway starts from
1-deoxy-D-xylulose 5-phosphate (DXP), which is synthesized by
condensation of pyruvate and glyceraldehyde-3-phosphate. This
reaction is catalyzed by 1-deoxy-D-xylulose-5-phosphate synthase
(DXS). In some bacteria, including E. coli, DXP serves not only as
a precursor of isoprenoids but is also used for biosynthesis of two
important cofactors: thiamine (vitamin B1) and pyridoxol phosphate
(vitamin B6).
[0662] The rate of isoprenoid synthesis in E. coli is regulated at
the level of DXS. One of the mechanisms of this regulation may
involve feedback inhibition of DXS activity by metabolites
downstream the MEP pathway or/and intermediates of vitamin B1/B6
biosynthesis. Accordingly, the overall flux into the MEP pathway
may be increased in E. coli by expressing an enzyme from a
different organism that is not subject to inhibition by downstream
products. Heterologous DXS may also be superior to the native E.
coli DXS due lower K.sub.m or higher K.sub.cat values with respect
to pyruvate or glyceraldehyde-3-phosphate. Earlier studies have
shown that a single Y392F substitution in the DXS of E. coli
results in two-fold increase in the activity of the enzyme in
vitro, although catalytic properties of the modified enzyme have
not been studied in detail.
[0663] The choice of the sources of DXS for heterologous expression
in E. coli can be based on the following considerations (see Table
8). First, organisms which have genome coding for several dxs
isogenes can be selected. These organisms include plants (different
forms of DXS in plants are classified as DXS 1 and DXS2), and
bacteria (e.g. species of Streptomyces) having two or more dxs
isogenes. Second, bacteria in which isoprenoids are synthesized via
both the MEP (or DXP) pathway and the MVA pathway can be selected.
Third, bacteria, which synthesize isoprenoids via the MVA pathway
but contain a copy of the dxs gene in their genome specifically
needed to make the vitamin cofactors. The DXS sequence this group
of microorganisms is characterized by a significantly shorter loop
corresponding to the amino acids 203-242 of E. coli DXS sequence
(FIG. 74).
[0664] In one set of the experiment, DXS from a variety of
organisms (examples are listed in Table 8) is introduced into E.
coli cells over-expressing plant isoprene synthase and
isopentenyl-diphosphate delta-isomerase (IDI). (IDI activity in E.
coli is normally very low; therefore enhanced expression of this
enzyme is necessary to provide efficient conversion of
isopentenyl-diphosphate into dimethylallyl-diphosphate, the
substrate of isoprene synthase.). The resulting strains are tested
for isoprene production and accumulation of DXP pathway
intermediates, including but not limited to DXP, MEP, 4-(cytidine
5'-diphospho)-2-C-methyl-D-erythritol, 2-phospho-4-(cytidine
5'-diphospho)-2-C-methyl-D-erythritol, 2-C-methyl-D-erythritol
2,4-cyclodiphosphate and 1-hydroxy-2-methyl-2-butenyl
4-diphosphate, and compared to the control strain containing native
E. coli DXS expressed in the same context as in the tested mutants.
Increased concentrations of DXP intermediates and/or elevated rate
of isoprene evolution in mutants containing heterologous DXSs
indicated that the enzyme from the particular organism has higher
activity in E. coli and is not subject to feedback inhibition by
accumulated products.
[0665] In another set of experiments, a set of mutants
over-expressing either heterologous dxs genes or dxs from E. coli
(the control) are introduced into the background E. coli strain
containing plant isoprene synthase, IDI, and several enzymes of MVA
pathway allowing that strain to synthesize excessive amounts of
isoprenoids when grown in the media containing exogenous MVA. These
strains are tested for the accumulation of the intermediates
specific to the DXP pathway. As in the previous case, increased
concentrations of DXP intermediates compared to the control showed
that DXS from specific organisms have higher activity in E. coli
than the native enzyme and is not subject to feedback inhibition by
isopentenyl-diphosphate and/or downstream isoprenoid products. To
verify that a particular mutant have an improved rate of the
isoprene production specifically due to the modified DXS, isoprene
production rate is measured in cells grown on .sup.13C-uniformly
labeled glucose in the presence of non-labeled MVA. In this case,
.sup.13C composition of isoprene analyzed by mass spectrometry
unequivocally indicated that this compound is synthesized via the
DXP pathway from the labeled glucose, not from exogenous
non-labeled MVA.
[0666] In a third set of experiments, experiments are performed to
demonstrate that substitution of the tyrosine at position 392 of E.
coli DXS for phenylalanine results in higher flux rate into the DXP
pathway compared to the wild type enzyme. For this experiment the
wild-type and the mutated DXS are over-expressed in an E. coli
strain containing plant isoprene synthase and IDI. The two strains
are compared for isoprene production rate and accumulation of DXP
pathway intermediates. Increased concentrations of DXP
intermediates and/or elevated rate of isoprene evolution in the
strain bearing the superior properties of the engineered enzyme
demonstrated the superior attributes of the mutant enzyme.
TABLE-US-00028 TABLE 8 Examples of organisms have kinetic
properties of DXSs different from that of E. coli. Organism Reason
Myxococcus xanthus DK 1622 DXS is needed to Gramella forsetii
KT0803 synthesize vitamin Flavobacterium johnsoniae UW101
cofactor(s); isoprenoids Lactobacillus johnsonii NCC 533 are made
via the MVA Lactobacillus gasseri ATCC 33323, pathway Lactococcus
lactis subsp. lactis Il1403 Listeria monocytogenes EGD-e Both MVA
and DXP Lactobacillus plantarum pathways are present Streptomyces
griseolosporeus MF730-N6 in these organisms Streptomyces
hygroscopicus NRRL 3418 Organisms have Streptomyces spheroides
NCIMB 11891 multiple copies of DXS Streptomyces spheroides NCIMB
11891 Streptomyces griseolosporeus MF730-N6 Streptomyces coelicolor
Streptomyces griseolosporeus MF730-N6 DXS type1 and DXS type 2 from
higher plants
Example 23
The Identification of Combinations of Genes, Gene Expression or
Mutations that Increase Flux Through the DXP Pathway
[0667] Populations of cells with a high degree of genotypic
diversity are generated to identify combinations of genes, gene
expression or mutations that increase flux through the DXP pathway.
Three different methods are used in this example. First,
combinations of genes, either endogenous to E. coli or from
heterologous organisms, are assembled using the Multisite Gateway
(Invitrogen) procedure and introduced into the E. coli screening
strain. Second, libraries of genomic DNA, either from E. coli or
heterologous organisms, are generated and introduced in the E. coli
screening strain. Third, transposons that can result in either gene
disruption or activation due to an internal promoter that is
directed towards the inverted repeat of the transposable element
are introduced.
A. The Multisite Gateway (Invitrogen) Procedure for Generating
Synthetic Operons
[0668] Genes either endogenous to E. coli or from heterologous
organisms are assembled into synthetic operons that are
subsequently screened for increased flux through the DXP pathway
and resulting isoprene production. The Multisite Gateway
(Invitrogen) kit provides for a maximum of four discrete DNA
"elements" that can be assembled together into one operon. Four
genes are individually cloned into pENTR vectors, according to the
manufacturer's protocol. For example, the last two genes in the DXP
pathway, ispG and ispH are amplified by PCR with appropriate att
recombination sites (according to manufacturer's protocol) and
variable RBS (see Yarchuk et al., J. of Mol. Biol., 226(3):581-596
(1992), which is hereby incorporated by reference in its entirety)
to generate plasmid pools with varying expression levels of each
gene. The same procedure is applied to the electron carrier genes
fldA and fpr, and the four resulting plasmid pools are recombined
together onto Gateway destination vectors (pDEST-14 (Invitrogen),
pET54-DEST or pCOLA-2-DEST (Novagen)) according to the
manufacturer's protocol. The resulting plasmids harbor four gene
operons with varying expression levels of each ORF. The pooled
destination vectors are then introduced into E. coli strains by
selecting for antibiotic resistance markers (kanamycin or
ampicillin) and resulting pools are screened by GC-MS (described
below).
B. Generation of Genomic Libraries
[0669] Genomic DNA either endogenous to E. coli or from
heterologous organisms is cloned into the pSMART LCKan vector
(Lucigen) according to the manufacturer's recommended protocol (see
Lynch et al., Nat. Methods, 4(1):87-93 (2007), which is hereby
incorporated by reference in its entirety). DNA from E. coli BL21
and K12 strains, B. subtilis, Lb. plantarum, Lb. sakei, P. citrea,
S. coelicolor, S. spheroides, L. monocytogenes, A. tumefaciens, S.
meliloti, and C. jejuni is used to generate libraries. The genomic
DNA inserts of up to 20 kb in size are then introduced into E. coli
strains for screening. Positive transformants are selected for by
introduction of antibiotic resistance (kanamycin), pooled, and
screened by GC-MS.
C. Transposon Mutagenesis and Gene Activation
[0670] A transposon that can both inactive genes by disruption of
the ORF and also drive expression of proximal genes due to an
endogenous promoter in the transposable element is introduced into
E. coli for screening. The custom transposon is generated by
inserting either a constitutive or inducible promoter into the MCS
of the EZ-Tn5 transposon construction vectors (Epicentre). Examples
of internal promoters include PT7, Ptrc, Ptac, Pbad, Plac, PL
(phage lambda), the gi series, and Ptet. These promoters are cloned
into the transposable element, and the resulting custom transposon
is introduced into E. coli. Strains harboring transposon insertions
are identified by antibiotic resistance, pooled, and subjected to
screening by GC-MS.
E. coli Strains and Screening
[0671] Plasmid pools or transposons are introduced into different
E. coli strains for screening. Positive transformants are
identified by antibiotic resistance markers (typically Kan or Amp)
located on the plasmid or within the transposable element. Strains
include: A strain harboring a plasmid carrying dxs, dxr, idi, and
IspS (isoprene synthase) under control of the T7 promoter; a strain
harboring integrated and constitutively expressed dxs, dxr, and idi
with ispS also integrated or expressed from a plasmid; a strain
expressing the entire DXP operon under the control of the T7
Promoter; any strain harboring the current best conformation of DXP
pathway genes for isoprene production, yet still displays clear
accumulation of DXP pathway metabolites (e.g. HDMAPP). Individual
transformants are pooled (in groups of 100 to 1000 individuals per
pool) and screened via GC-MS in a 96-well glass block. The analysis
is performed (for the 2 mL and 96-well plate methods) using an
Agilent 6890 GC/MS system interfaced with a 5973 MS Leap CTC
CombiPAL autosampler operating in headspace mode. An Agilent HP-5
(5% Phenyl Methyl Siloxane (15 m.times.0.25 mm.times.0.25 uM))
column is used for separation of analytes. The sampler is set up to
inject 100 .mu.L of headspace gas. The GC/MS method utilizes helium
as the carrier gas at a flow of 1 ml/min. The injection port is
held at 250.degree. C. with a split ratio of 50:1. The oven
temperature is held at 37.degree. C. for the 2 min duration of the
analysis. The Agilent 5793N mass selective detector is run in
single ion monitoring (SIM) mode on mass 67. The detector is
switched off from 0.00 to 0.44 minutes to allow the elution of
permanent gases and on 0.44 mins to 0.60 mins. Under these
conditions isoprene (2-methyl-1,3-butadiene) is observed to elute
at 0.49 minutes. A calibration table is used to quantify the
absolute amount of isoprene and was found to be linear from 0
.mu.g/L to 5600 mg/L (using calibration gas). Positive pools are
then re-assayed to confirm any positive effect on isoprene
production. The individual plasmids or constructs in strains or
pools which display increased isoprene production are identified to
determine the precise nature of positive influence on DXP pathway
flux.
Genes of Organisms Examined
[0672] Genes including, but not limiting to, the following
organisms are examined: Arabidopsis thaliana, Zea mays,
Campylobacter jejuni, Sinorhizobium meliloti, Helicobacter pylori
Agrobacterium tumefaciens, Deinococcus radiodurans, Bacillus
subtilis, Pantoea citrea, Listeria monocytogenes, Lactobacillus
spp., and Streptomyces spp.
Materials
Multisite Gateway kit (Invitrogen)
[0673] Lucigen (Clonesmart Cloning Kits)--library construction
EZ-Tn5 System (EpiCentre)--gene disruption/activation
Plasmids
[0674] pET--PT7-driven full DXP pathway plasmid pET--PT7 driven
dxs, dxr. idi, ispS pET--best conformation of DXP pathway genes for
isoprene production pBBR--PT7 or Ptrc ispS pET-54-DEST, pCOLA-DEST
vectors (Novagen) pDEST14, pDEST15 (Invitrogen)
Example 24
Increased Isoprene Production in REMG39 by Overexpression of GcpE,
LytB PetF and PetH of T. elongatus BP-1 within CMP272
[0675] This example provides further demonstration of increased
isoprene production in REMG39 by overexpression of GcpE, LytB PetF
and PetH of T. elongatus BP-1 within CMP272, a BL21 derived
host.
[0676] As described and shown infra, increased expression of both
dxs and yeast idi allow increased flux through the endogenous DXP
pathway of E. coli. Previous work by the field (see, for example,
Chao et al., Biotechnol Prog., 18(2):394-400 (2002) and Zhang et
al., Protein Expression and Purification, 29(1): 132-139 (May
2003)) has lead to the conclusion that T7-based expression systems
are unstable and their behavior not entirely predictable when
subjected to 14-L fermentation conditions. The CMP271 and
subsequent CMP272 strain were constructed to: (1) replace our
current T7-governed plasmid-based expression of yeast idi with
expression originating from the chromosome; permitting the use of a
non-T7 based expression strain for DXP-mediated isoprene production
and/or (2) introduce the genomically encoded locus harboring the
genes for the lower MVA pathway enzymes and yeast IDI to provide
sufficient levels of yeast IDI for maximal flux to Isoprene
Synthase.
[0677] The CMP271 strain was made into an isoprene generating
strain by the addition of pDW33, harboring a P. alba isoprene
synthase allele, via electroporation, and subsequently yielding
strain CMP272.
[0678] The CMP272 strain serves as the baseline host in which
isoprene production has been successfully improved by the addition
of the T. elogatus IspG (GcpE) and IspH (LytB) encoding genes along
with their putative reducing shuttle system (PetF and PetH). The
construct harboring the T. elongatus genes,
Ptac-gcpE-lytB-petF-petH/pK184, has been described infra in the
example utilizing T. elongatus. The parental CMP272 and test strain
REMG39 were evaluate for growth, isoprene production, metabolite
profile, and product yield on carbon under 14-L fermentation
conditions described below. The results are depicted in FIG.
77.
A. Construction of Strains CMP271, CMP272, and REMG39
[0679] The GI 1.X-promoter insertions and subsequent loopout of the
antibiotic resistance markers described in this example were
carried out using the Red/ET system from Gene Bridges GmbH
according to the manufacturer's instructions. The strain BL21
(Novagene) was used. P1 lysate preparations and transductions were
performed as previously described (Thomason et al., 2007).
TABLE-US-00029 Primers MQ09-10F- (SEQ ID NO: 140) 5'
ggttaatcatttcactcttcaattatctataatgatgagtgatcag
aattacatgtgagaaattaattaaccctcacTaaagggcggccgcgaa MQ09-10R- (SEQ ID
NO: 141) 5' atattccaccagctatttgttagtgaataaaagtggttgaattatt
tgctcaggatgtggcatNgtcaagggctaatacgactcacta tagggctcgagg * for the
case of GI1.6 N = T in the primer sequence above. MQ09-11F- (SEQ ID
NO: 142) 5' gcccttgacNatgccacatcctgagcaaataattcaaccactttta
ttcactaacaaa tagctggtggaatata Tgactgccgacaacaatagtatgccc * for the
case of GI1.6 N = A in the primer sequence above. MQ09-11R- (SEQ ID
NO: 143) 5' gatgcgtccagtaaaataagcattacgttatgctcataaccccggc
aaatgtcggggt tttttatagcattctatgaatttg top Gb's CMP (SEQ ID NO: 144)
5' ACTGAAACGTTTTCATCGCTC MQ09-12R- (SEQ ID NO: 145) 5'
gatgcgtccagtaaaataagcattacgttatgctc galMR (SEQ ID NO: 146) 5'
gtcaggctggaatactcttcg galMF (SEQ ID NO: 147) 5'
gacgctttcgccaagtcagg
[0680] The strategy for inserting the GI1.X-yidi series into the E.
coli idi locus using the Gene Bridges GmbH methods is illustrated
in FIG. 77. The antibiotic resistance cassette GB-CMP containing
fragment (Frag A) was amplified by PCR using primer sets
MQ09-10F/MQ09-10R. The GI1.X-yidi containing fragment (Frag B) was
amplified by PCR using primer sets MQ09-11F/MQ09-11R. The
GB-CMP-GI1.X-yidi fragment was ultimately generated using the
primers MQ09-10F and MQ09-11R. The MQ09-10F and MQ09-11R primers
each contain at least 50 bases of homology to the E. coli idi locus
which allow recombination at the specific sites upon
electroporation of the PCR product in the presence of the pRed-ET
plasmid.
Amplification of the GB-CMP-GI1.X-yidi Fragment
PCR Reaction for GB-CmR (Frag A)
2 ul (100 ng GB-CmR)
10 ul HerculaseII Buffer
[0681] 0.5 ul dNTP's (100 mM) 1.25 ul primer (10 uM) MQ09-10F 1.25
ul primer (10 uM) MQ09-10R
2 ul DMSO
[0682] 32 ul diH2O +1 ul of HerculaseII Fusion from Stratagene
PCR Reaction for GB-CmR (Frag B)
2 ul (100 ng GB-CmR)
10 ul HerculaseII Buffer
[0683] 0.5 ul dNTP's (100 mM) 1.25 ul primer (10 uM) MQ09-11F 1.25
ul primer (10 uM) MQ09-11R
2 ul DMSO
[0684] 32 ul diH2O +1 ul of HerculaseII fusion from Stratagene
PCR Reaction for GB-CmR (Frag A+B)
1 ul (Frag A)
1 ul (Frag B)
10 ul HerculaseII Buffer
[0685] 0.5 ul dNTP's (100 mM) 1.25 ul primer (10 uM) MQ09-10F''
1.25 ul primer (10 uM) MQ09-11R
2 ul DMSO
[0686] 32 ul diH2O +1 ul of HerculaseII fusion from Stratagene
Cycle Parameter:
Frag A
[0687] (95.degree. C. 2 min., 95.degree. C. 20 sec., 55.degree. C.
20 sec., 72.degree. C. 1 min., 29.times., 72.degree. C. 3 min,
4.degree. C. until cool, use Eppendorf Mastercycler)
Frag B
[0688] (95.degree. C. 2 min., 95.degree. C. 20 sec., 55.degree. C.
20 sec., 72.degree. C. 35 sec., 29.times., 72.degree. C. 3 min,
4.degree. C. until cool, use Eppendorf Mastercycler)
Frag A & B
[0689] (95.degree. C. 2 min., 95.degree. C. 20 sec., 55.degree. C.
20 sec., 72.degree. C. 1.2 min., 29.times., 72.degree. C. 3 min,
4.degree. C. until cool, use Eppendorf Mastercycler)
[0690] The resulting PCR fragments Frag A, B, and A+B were
separated on a 1.2% E-gel (Invitrogen) for verification of
successful amplification, and purified using the QIAquick PCR
Purification kits according to manufacturer's instructions. The
purifed stocks of Frag A and Frag B were used in the Frag A+B PCR
reaction described above. The resulting purified stock of Frag A+B
is referred to as GB-CMP-GI1.X-yidi.
Amplification of the galM Locus of CMP263
[0691] One colony of CMP263 was stirred in 30 uL H.sub.2O and then
heated to 95.degree. C. for 5 min. The resulting solution was spun
down to pellet debris and 2 uL of the supernatant was used as the
template in the following PCR reaction:
2 ul colony in H.sub.2O (see above)
5 ul Herculase Buffer
[0692] 1 ul dNTP's (100 mM) 1 ul galMF primer (10 uM) 1 ul galMR
primer (10 uM)
39.5 ul H.sub.2O
[0693] +0.5 ul of Herculase Enhanced DNA polymerase from
Stratagene
Cycle Parameter:
[0694] 95.degree. C..times.2 min., [95.degree. C..times.30 sec.,
52.degree. C..times.30 sec., 72.degree. C..times.60 sec].times.30
cycles; 72.degree. C..times.7 min, 4.degree. C. until cool
(PCRExpress Thermocycler from ThermoHybaid).
[0695] The size of the resulting PCR fragment was determined on a
0.8% E-gel (Invitrogen), using DNA Molecular Weight X (Roche) as a
ladder; a corresponding PCR product was not obtained from BL21
cells, as expected for the negative control.
Integration of GB-CMP GI 1.X-yidi PCR Product into BL21/pRed-ET
Strain
[0696] The pRed-ET vector (Gene Bridges kit) was transformed into
BL21 by electroporation using the BIO RAD Gene Pulser system and a
transformation protocol suggested by the manufacturer (BIO RAD)
resulting in strain MD08-114 (BL21/pRed-ET). Approximately 400 ug
of the purified GB-CMP GI 1.X-yidi PCR fragment was electroporated
into MD08-114. The transformants were recovered in L Broth and then
plated on L agar containing chloramphenicol (5 ug/ml).
Chloramphenicol resistant colonies were analyzed by PCR for the
presence of the GB-CMP GI 1.X-yidi sequence at the desired locus
using the top Gb's CMP and MQ09-12R primers. The PCR fragments from
a number of transformants were sequenced using the MQ09-12R and top
GB's CMP primers (Quintara; Albany, Calif.) and the various
GI1.X-yidi strains of interest identified. One chloramphenicol
resistant clone harboring the GI1.6-yidi locus (BL21 FRT-CmR-FRT
GI1.6(A)-yidi) was chosen and designated MD09-211.
B. Strategy for Creating the CMP271 Strain
[0697] The GI1.6-dxs::kan locus of strain MCM625, described in
Example 9, was introduced into MD09-211 via P1-mediated
transduction and the resulting kanamycin and chloramphenicol
resistant strain named MD09-221. The antibiotic resistance markers
of strain MD09-221 were looped out using pCP20 from the pRed-ET kit
according to the manufacturer's instructions (GeneBridges).
Transformants of interest were verified by the loss of resistance
to chloramphenicol (5 ug/ml) and kanamycin (50 ug/ml); one
chloramphenicol and kanamycin sensitive clone was chosen and
designated MCM710. The FRT-Neo-FRT PL.2 mKKDyI locus (harboring an
additional copy of the yeast idigene) of strain MCM521, described
in US Appl. No. 61/289,959, was moved into MCM710 by P1-mediated
transduction. One kanamycin resistant clone was chosen and
designated MCM783. MCM783 was transduced with a P1 lysate of E.
coli K-12 MG1655, and selected on M9 medium (Na2HPO4 6 g/L, KH2PO4
3 g/L, NaCl 0.5 g/L, NH4Cl 0.5 g/L, 0.1 mM CaCl2, 2 mM MgSO4)+0.4%
w/v galactose. One galactose ultizing clone was chosen and
designated CMP263. The presence of the galM locus within the 17,257
bp of MG1655 that is not endogenous to BL21, but was now harbored
by CMP263, was verified by PCR using the primer set galMF/galMR;
this PCR reaction is described above. The kanmycin resistance
marker within strain CMP263 was looped out using Gene Bridges GmbH
methods. One kanamycin sensitive clone was chosen and designated
CMP271.
C. Strategy for Creating the CMP272 Strain
[0698] Electroporation of pDW33 into strain CMP271 was done using
the BIO RAD Gene Pulser system (0.1 cm cuvette cat.#165-2089) and a
transformation protocol suggested by the manufacturer (BIO RAD).
The vector construct harbors the PTrc-governed MEARR P. alba allele
encoding a truncated form of Isoprene Synthase. The template for
pDW33 construction, EWL230, has been described in US. Publ. No.
2009/0203102 and WO 2009/076676. A picture of the pDW33 vector map
is presented in FIG. 78.
Construction of pDW33
[0699] pDW33 was constructed in order to generate an isoprene
producing Escherichia coli strain harboring the truncated version
of P. alba isoprene synthase (the MEA variant) under control of the
Ptrc promoter.
Construction of Strain DW194:
[0700] The plasmid harboring truncated P. alba isoprene synthase
(IspS) was constructed by Quikchange PCR mutagenesis
(Stratagene--see Table below for primer sequences) upon the
template EWL230 (aka pTrc-P. alba). PCR reaction and cycling
parameters are described below. The PCR product was visualized by
gel electrophoresis (E-gel, Invitrogen), and then treated with 1
.mu.l DpnI restriction endonuclease (Roche) for three hours at
37.degree. C. Ten .mu.l of the PCR product was then de-salted using
a microdialysis membrane (MilliPore) and transformed into
electrocompetent E. coli strain MCM531 (previously described) using
standard molecular biology techniques. Cells were recovered in one
ml of LB medium for 1.5 hours at 30.degree. C., plated onto LB
solid agar plates containing 50 .mu.g/ml carbenicillin and 5 mM
mevalonic acid, and then incubated overnight at 37.degree. C. The
next day, positive colonies (of strain DW194, see below) were
selected for growth and plasmid purification (Qiagen), and
ultimately confirmed by DNA sequencing (Quintara) with the primers
listed below. The final plasmid, pDW33, carries the open reading
frame encoding the truncated version (MEA) of IspS.
TABLE-US-00030 Primers: QC EWL244 MEA F (SEQ ID NO: 148)
gaggaataaaccatggaagctcgtcgttct QC EWL244 MEA R (SEQ ID NO: 149)
agaacgacgagcttccatggtttattcctc EL-1006 (SEQ ID NO: 150)
gacagcttatcatcgactgcacg EL-1000 (SEQ ID NO: 151)
gcactgtctttccgtctgctgc A-rev (SEQ ID NO: 152) ctcgtacaggctcaggatag
A-rev-2 (SEQ ID NO: 153) ttacgtcccaacgctcaact QB1493 (SEQ ID NO:
154) cttcggcaacgcatggaaat MCM66 (aka pTrc Reverse) (SEQ ID NO: 155)
ccaggcaaattctgttttatcag
Strains:
TABLE-US-00031 [0701] Strain Background Plasmid Resistance Genotype
DW194 MCM531 pDW33 Carb BL21 (Novagen) PL.2mKKDyI, + pTrc-P. alba
(MEA)
QuikChange PCR Reaction:
[0702] 1 ul plasmid EWL230 (aka pTrc P. alba) 5 ul 10.times.
PfuUltra HF buffer 1 ul dNTPs (100 mM)
1 ul (50 uM) QC EWL244 MEA F
1 ul (50 uM) QC EWL244 MEA R
2 ul DMSO
[0703] 39 ul diH2O
1 ul PfuUltra HF Polymerase (Stratagene)
PCR Cycling Parameters:
1. 95.degree. C. 1 min.
2. 95.degree. C. 30 sec.
3. 55.degree. C. 1 min.
4. 68.degree. C. 6 min.
[0704] 5. Go to step 2-18 cycles
6. 4.degree. C.
TABLE-US-00032 [0705] Sequence of truncated P. alba IspS (MEA) (SEQ
ID NO: 156)
mearrsanyepnswdydyllssdtdesievykdkakkleaevrreinnekaefltllelidnvqrlglgyrfes-
dirgaldrfvs
sggfdavtktslhgtalsfrllrqhgfevsqeafsgfkdqngnflenlkedikailslyeasflalegenilde-
akvfaishlkelse
ekigkelaeqvnhalelplhrrtqrleavwsieayrkkedanqvllelaildynmiqsvyqrdlretsrwwrrv-
glatklhfar
drliesfywavgvafepqysdcrnsvakmfsfvtiiddiydvygtldelelftdaverwdvnaindlpdymklc-
flalyntin
eiaydnlkdkgenilpyltkawadlcnaflqeakwlynkstptfddyfgnawksssgplqlvfayfavvqnikk-
eeienlqk
yhdtisrpshifrlcndlasasaeiargetansvscymrtkgiseelatesvmnlidetwkkmnkeklggslfa-
kpfvetainl arqshctyhngdahtspdeltrkrvlsvitepilpfer Sequence of
pDW33: (SEQ ID NO: 157)
gtttgacagcttatcatcgactgcacggtgcaccaatgcttctggcgtcaggcagccatcggaagctgtggtat-
ggctgtgcagg
tcgtaaatcactgcataattcgtgtcgctcaaggcgcactcccgttctggataatgttttttgcgccgacatca-
taacggttctggca
aatattctgaaatgagctgttgacaattaatcatccggctcgtataatgtgtggaattgtgagcggataacaat-
ttcacacaggaaac
agcgccgctgagaaaaagcgaagcggcactgctctttaacaatttatcagacaatctgtgtgggcactcgaccg-
gaattatcgat
taactttattattaaaaattaaagaggtatatattaatgtatcgattaaataaggaggaataaaccatggaagc-
tcgtcgttctgcgaa
ctacgaacctaacagctgggactatgattacctgctgtcctccgacacggacgagtccatcgaagtatacaaag-
acaaagcgaa
aaagctggaagccgaagttcgtcgcgagattaataacgaaaaagcagaatttctgaccctgctggaactgattg-
acaacgtcca
gcgcctgggcctgggttaccgtttcgagtctgatatccgtggtgcgctggatcgcttcgtttcctccggcggct-
tcgatgcggtaa
ccaagacttccctgcacggtacggcactgtctttccgtctgctgcgtcaacacggttttgaggtttctcaggaa-
gcgttcagcggct
tcaaagaccaaaacggcaacttcctggagaacctgaaggaagatatcaaagctatcctgagcctgtacgaggcc-
agcttcctgg
ctctggaaggcgaaaacatcctggacgaggcgaaggttttcgcaatctctcatctgaaagaactgtctgaagaa-
aagatcggtaa
agagctggcagaacaggtgaaccatgcactggaactgccactgcatcgccgtactcagcgtctggaagcagtat-
ggtctatcga
ggcctaccgtaaaaaggaggacgcgaatcaggttctgctggagctggcaattctggattacaacatgatccagt-
ctgtataccag
cgtgatctgcgtgaaacgtcccgttggtggcgtcgtgtgggtctggcgaccaaactgcactttgctcgtgaccg-
cctgattgaga
gcttctactgggccgtgggtgtagcattcgaaccgcaatactccgactgccgtaactccgtcgcaaaaatgttt-
tctttcgtaaccat
tatcgacgatatctacgatgtatacggcaccctggacgaactggagctgtttactgatgcagttgagcgttggg-
acgtaaacgcc
atcaacgacctgccggattacatgaaactgtgctttctggctctgtataacactattaacgaaatcgcctacga-
caacctgaaagat
aaaggtgagaacatcctgccgtatctgaccaaagcctgggctgacctgtgcaacgctttcctgcaagaagccaa-
gtggctgtac
aacaaatctactccgacctttgacgactacttcggcaacgcatggaaatcctcttctggcccgctgcaactggt-
gttcgcttacttc
gctgtcgtgcagaacattaaaaaggaagagatcgaaaacctgcaaaaataccatgacaccatctctcgtccttc-
ccatatcttccg
tctgtgcaatgacctggctagcgcgtctgcggaaattgcgcgtggtgaaaccgcaaatagcgtttcttgttaca-
tgcgcactaaag
gtatctccgaagaactggctaccgaaagcgtgatgaatctgatcgatgaaacctggaaaaagatgaacaaggaa-
aaactgggt
ggtagcctgttcgcgaaaccgttcgtggaaaccgcgatcaacctggcacgtcaatctcactgcacttatcataa-
cggcgacgcg
catacctctccggatgagctgacccgcaaacgcgttctgtctgtaatcactgaaccgattctgccgtttgaacg-
ctaactgcagct
ggtaccatatgggaattcgaagctttctagaacaaaaactcatctcagaagaggatctgaatagcgccgtcgac-
catcatcatcat
catcattgagtttaaacggtctccagcttggctgttttggcggatgagagaagattttcagcctgatacagatt-
aaatcagaacgca
gaagcggtctgataaaacagaatttgcctggcggcagtagcgcggtggtcccacctgaccccatgccgaactca-
gaagtgaaa
cgccgtagcgccgatggtagtgtggggtctccccatgcgagagtagggaactgccaggcatcaaataaaacgaa-
aggctcag
tcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctcctgagtaggacaaatccgccggg-
agcggatttgaac
gttgcgaagcaacggcccggagggtggcgggcaggacgcccgccataaactgccaggcatcaaattaagcagaa-
ggccatc
ctgacggatggcctttttgcgtttctacaaactctttttgtttatttttctaaatacattcaaatatgtatccg-
ctcatgagacaataaccct
gataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttt-
tttgcggcattttgcct
tcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggtt-
acatcgaactgg
atctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagtt-
ctgctatgtggcgc
ggtattatcccgtgttgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttg-
agtactcaccag
tcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataac-
actgcggcca
acttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaact-
cgccttgatcgtt
gggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacg-
ttgcgca
aactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagtt-
gcaggaccactt
ctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtat-
cattgcagcact
ggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaa-
atagacaga
tcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagatt-
gatttaaaacttcattt
ttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgt-
tccactgagcgtcaga
ccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaa-
aaccaccgctacc
agcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcaga-
taccaaatactg
tccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgcta-
atcctgttaccagt
ggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagc-
ggtcgggct
gaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgag-
ctatgaga
aagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgca-
cgagg
gagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatt-
tttgtgatgctcgt
caggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggcctttt-
gctcacatgttc
tttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcag-
ccgaacgaccgag
cgcagcgagtcagtgagcgaggaagcggaagagcgcctgatgcggtattttctccttacgcatctgtgcggtat-
ttcacaccgc
atatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagtatacactccgctatcgctacgt-
gactgggtcatgg
ctgcgccccgacacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacaga-
caagctgtg
accgtctccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgaggcagcagatcaattc-
gcgcgcgaa
ggcgaagcggcatgcatttacgttgacaccatcgaatggtgcaaaacctttcgcggtatggcatgatagcgccc-
ggaagagagt
caattcagggtggtgaatgtgaaaccagtaacgttatacgatgtcgcagagtatgccggtgtctcttatcagac-
cgtttcccgcgtg
gtgaaccaggccagccacgtttctgcgaaaacgcgggaaaaagtggaagcggcgatggcggagctgaattacat-
tcccaacc
gcgtggcacaacaactggcgggcaaacagtcgttgctgattggcgttgccacctccagtctggccctgcacgcg-
ccgtcgcaa
attgtcgcggcgattaaatctcgcgccgatcaactgggtgccagcgtggtggtgtcgatggtagaacgaagcgg-
cgtcgaagc
ctgtaaagcggcggtgcacaatcttctcgcgcaacgcgtcagtgggctgatcattaactatccgctggatgacc-
aggatgccatt
gctgtggaagctgcctgcactaatgttccggcgttatttcttgatgtctctgaccagacacccatcaacagtat-
tattttctcccatga
agacggtacgcgactgggcgtggagcatctggtcgcattgggtcaccagcaaatcgcgctgttagcgggcccat-
taagttctgt
ctcggcgcgtctgcgtctggctggctggcataaatatctcactcgcaatcaaattcagccgatagcggaacggg-
aaggcgactg
gagtgccatgtccggttttcaacaaaccatgcaaatgctgaatgagggcatcgttcccactgcgatgctggttg-
ccaacgatcag
atggcgctgggcgcaatgcgcgccattaccgagtccgggctgcgcgttggtgcggatatctcggtagtgggata-
cgacgatac
cgaagacagctcatgttatatcccgccgtcaaccaccatcaaacaggattttcgcctgctggggcaaaccagcg-
tggaccgctt
gctgcaactctctcagggccaggcggtgaagggcaatcagctgttgcccgtctcactggtgaaaagaaaaacca-
ccctggcgc
ccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactg-
gaaagcggg cagtgagcgcaacgcaattaatgtgagttagcgcgaattgatctg
Transformation of pDW33 into CMP271
[0706] This step was done to build the isoprene-producing strain
CMP272 the pDW33 plasmid was transformed by electroporation into
CMP271. Transformants were recovered in L broth and plated on L
agar containing carbenicillin (50 ug/ml). The resulting strain was
designated as CMP272.
D. Strategy for Creating the REMG39 Strain
[0707] Electroporation of Ptac-gcpE-lytB-petF-petH/pK184 into
strain CMP272 was performed using the BIO RAD Gene Pulser system
(0.1 cm cuvette cat.#165-2089) and a transformation protocol
suggested by the manufacturer (BIO RAD). A plasmid preparation of
Ptac-gcpE-lytB-petF-petH/pK184 was provided by Gene Oracle, Inc.
Ptac-gcpE-lytB-petF-petH/pK184 has been described infra (see, e.g.,
Example 11).
Transformation of Ptac-gcpE-lytB-petF-petH/pK184 into CMP272
[0708] To build the REMG39 test strain,
Ptac-gcpE-lytB-petF-petH/pK184 was transformed by electroporation
into CMP272. Transformants were recovered in L broth and plated on
L agar containing carbenicillin (50 ug/ml) and kanamycin (50
ug/ml). The resulting strain was designated as REMG39.
E. Comparing CMP272 to REMG39 for Growth, Isoprene Production, DXP
Metabolite Profile, and Product Yield on Carbon During 14-L
Fermentation
[0709] The parental strain CMP272 was compared to the test strain
REMG39 under 14-L fermentation conditions. The benefit of the T.
elongatus IspG (GcpE) and IspH (LytB) activities on isoprene
production and overall flux through the otherwise endogenous DXP
pathway of E. coli is illustrated in FIG. 79 and FIG. 80A-B,
respectively. Expression of the T. elongatus genes improved
isoprene production approximately 2.7-fold over that of the
parental strain CMP272. Despite the higher levels of cMEPP observed
for the REM G39 strain during the initial 10 hour period, the
REMG39 strain accumulated reduced levels of the cMEPP intermediate
during the later portion of the fermentation compared to the
parental strain, an observation that is correlated with increased
specific productivity during post-exponential and maximal CER
growth (see FIG. 3B-D).
F. Large Scale Fermentation of Strain CMP272
[0710] Isoprene production from E. coli expressing genes from the
DXP pathway and isoprene synthase, grown in fed-batch culture at
the 15-L scale.
Medium Recipe (Per Liter Fermentation Medium):
[0711] K2HPO4 7.5 g, MgSO4*7H2O 2 g, citric acid monohydrate 2 g,
ferric ammonium citrate 0.3 g, yeast extract 5 g, 1000.times.
Modified Trace Metal Solution 1 ml. All of the components were
added together and dissolved in Di H2O. This solution was heat
sterilized (123.degree. C. for 20 minutes). The pH was adjusted to
7.0 with ammonium hydroxide (28%) and q.s. to volume. Glucose 10 g,
Mercury Vitamin Solution 8 mL, and antibiotics were added after
sterilization and pH adjustment.
1000.times. Modified Trace Metal Solution (Per Liter):
[0712] Citric Acids*H.sub.2O 40 g, MnSO4*H.sub.2O 30 g, NaCl 10 g,
FeSO4*7H2O 1 g, CoCl2*6H2O 1 g, ZnSO*7H2O 1 g, CuSO4*5H2O 100 mg,
H3BO3 100 mg, NaMoO4*2H2O 100 mg. Each component was dissolved one
at a time in Di H2O, pH was adjusted to 3.0 with HCl/NaOH, and then
the solution was q.s. to volume and filter sterilized with a 0.22
micron filter.
Mercury Vitamin Solution (Per Liter):
[0713] Thiamine hydrochloride 1.0 g, D-(+)-biotin 1.0 g, nicotinic
acid 1.0 g, D-pantothenic acid 4.8 g, pyridoxine hydrochloride 4.0
g. Each component was dissolved one at a time in Di H2O, pH was
adjusted to 3.0 with HCl/NaOH, and then the solution was q.s. to
volume and filter sterilized with 0.22 micron filter.
Feed Solution (Per Kilogram):
[0714] Glucose 0.57 kg, Di H.sub.2O 0.38 kg, K2HPO4 7.5 g, and 100%
Foamblast 10 g. All components were mixed together and autoclaved.
Mercury Vitamin Solution 6.7 mL was added after the solution had
cooled to 25.degree. C.
[0715] Fermentation was performed in a 15-L bioreactor with E. coli
BL21 cells overexpressing the first enzyme in the dxp pathway
(GI1.6-dxs), the last enzyme in the DXP pathway (GI1.6y-IDI), the
lower MVA pathway (PL.2-mKKDyI) and truncated isoprene synthase
from P. alba (pDW33) and containing a restored 17,257 bp
chromosomal galM-containing region derived from MG1655 (strain name
CMP272). This experiment was carried out to monitor isoprene
formation from glucose at the desired fermentation pH 7.0 and
temperature 34.degree. C. A frozen vial of the E. coli strain was
thawed and inoculated into tryptone-yeast extract medium for the
bioreactor. After the inoculum grew to optical density 1.0,
measured at 550 nm (OD.sub.550), 500 mL was used to inoculate a
15-L bioreactor and bring the initial tank volume to 5 L.
[0716] The feed solution was fed at an exponential rate until a top
feed rate of 4.8 g/min was reached. After this time, the glucose
feed was fed to meet metabolic demands at rates less than or equal
to 4.8 g/min. The total amount of glucose delivered to the
bioreactor during the 45 hr fermentation was 5.6 kg. Induction was
achieved by adding isopropyl-beta-D-1-thiogalactopyranoside (IPTG).
A single shot of IPTG was added to the tank to bring the
concentration to 200 uM when the cells were at an OD of 8.
[0717] The isoprene level in the off-gas from the bioreactors was
determined using a Hiden mass spectrometer. The isoprene titer
increased over the course of the fermentation to a maximum value of
0.97 g/L at 45 hr.
Equation for calculating Isoprene Titer: .intg.(Instantaneous
isoprene production rate, g/L/hr)dt from t=0 to 45 hrs [=] g/L
broth Equation for calculating Specific Productivity levels: (mg
isoprene.sub.t-mg isoprene.sub.to)/[(OD550.sub.t*L
broth.sub.t-OD550.sub.to*L broth.sub.to)/(2.7 OD*L/g
cell)]/(t-t.sub.0) [=] mg isoprene/g cell/hr
G. Large Scale Fermentation of Strain REMG39
[0718] Isoprene production from E. coli expressing genes from the
DXP pathway and isoprene synthase, grown in fed-batch culture at
the 15-L scale.
Medium Recipe (Per Liter Fermentation Medium):
[0719] K2HPO4 7.5 g, MgSO4*7H2O 2 g, citric acid monohydrate 2 g,
ferric ammonium citrate 0.3 g, yeast extract 0.5 g, 1000.times.
Modified Trace Metal Solution 1 ml. All of the components were
added together and dissolved in Di H2O. This solution was heat
sterilized (123.degree. C. for 20 minutes). The pH was adjusted to
7.0 with ammonium hydroxide (28%) and q.s. to volume. Glucose 10 g,
Mercury Vitamin Solution 8 mL, and antibiotics were added after
sterilization and pH adjustment.
1000.times. Modified Trace Metal Solution (Per Liter):
[0720] Citric Acids*H.sub.2O 40 g, MnSO4*H.sub.2O 30 g, NaCl 10 g,
FeSO4*7H2O 1 g, CoCl2*6H2O 1 g, ZnSO*7H2O 1 g, CuSO4*5H2O 100 mg,
H3BO3 100 mg, NaMoO4*2H2O 100 mg. Each component was dissolved one
at a time in Di H2O, pH was adjusted to 3.0 with HCl/NaOH, and then
the solution was q.s. to volume and filter sterilized with a 0.22
micron filter.
Mercury Vitamin Solution (Per Liter):
[0721] Thiamine hydrochloride 1.0 g, D-(+)-biotin 1.0 g, nicotinic
acid 1.0 g, D-pantothenic acid 4.8 g, pyridoxine hydrochloride 4.0
g. Each component was dissolved one at a time in Di H2O, pH was
adjusted to 3.0 with HCl/NaOH, and then the solution was q.s. to
volume and filter sterilized with 0.22 micron filter.
Feed Solution (Per Kilogram):
[0722] Glucose 0.57 kg, Di H.sub.2O 0.38 kg, K2HPO4 7.5 g, and 100%
Foamblast 10 g. All components were mixed together and autoclaved.
Macro Salt Solution 3.4 mL, 1000.times. Modified Trace Metal
Solution 0.8 ml, and Mercury Vitamin Solution 6.7 mL were added
after the solution had cooled to 25.degree. C.
Macro Salt Solution (Per Liter):
[0723] MgSO4*7H2O 296 g, citric acid monohydrate 296 g, ferric
ammonium citrate 49.6 g. All components were dissolved in water,
q.s. to volume and filter sterilized with 0.22 micron filter.
[0724] Fermentation was performed in a 15-L bioreactor with E. coli
BL21 cells overexpressing the first enzyme in the dxp pathway
(GI1.6-dxs), the last enzyme in the DXP pathway (GI1.6-yIDI), the
lower MVA pathway (PL.2-mKKDyI), various other genes from the DXP
pathway of T. elongates (Ptac-gcpE-lytB-petF-petH/pK184), and
truncated isoprene synthase from P. alba (pDW33) and containing a
restored 17,257 bp chromosomal galM-containing region derived from
MG1655 (strain name REMG39). This experiment was carried out to
monitor isoprene formation from glucose at the desired fermentation
pH 7.0 and temperature 34.degree. C. A frozen vial of the E. coli
strain was thawed and inoculated into tryptone-yeast extract medium
for the bioreactor. After the inoculum grew to optical density 1.0,
measured at 550 nm (OD.sub.550), 500 mL was used to inoculate a
15-L bioreactor and bring the initial tank volume to 5 L.
[0725] The feed solution was fed at an exponential rate until a top
feed rate of 4.8 g/min was reached. After this time, the glucose
feed was fed to meet metabolic demands at rates less than or equal
to 4.8 g/min. The total amount of glucose delivered to the
bioreactor during the 56 hr fermentation was 7.0 kg. Induction was
achieved by adding isopropyl-beta-D-1-thiogalactopyranoside (IPTG).
A single shot of IPTG was added to the tank to bring the
concentration to 300 uM when the cells were at an OD of 5. After a
run time of 36 h, whole broth, including cell mass, was drawn off
periodically to prevent overflow of the bioreactor.
[0726] The isoprene level in the off-gas from the bioreactors was
determined using a Hiden mass spectrometer. The isoprene titer
increased over the course of the fermentation to a maximum value of
2.7 g/L at 56 hr.
Equation for calculating Isoprene Titer: .intg.(Instantaneous
isoprene production rate, g/L/hr)dt from t=0 to 56 hrs [=] g/L
broth Equation for calculating Specific Productivity levels: (mg
isoprene.sub.t-mg isoprene.sub.to)/[(OD550.sub.t*L
broth.sub.t-OD550.sub.to*L broth.sub.to)/(2.7 OD*L/g
cell)]/(t-t.sub.0) [=] mg isoprene/g cell/hr
Example 25
DXP Metabolite Determination
A. Metabolite Extraction: Processing 14-L Fermentor Samples.
[0727] Cell metabolism was rapidly inactivated by withdrawing
several milliliters of the fermentor culture into a pre-weighed
tube filled with 9.0 mL of dry ice-cold methanol. The resulting
sample was weighed again to calculate the amount of withdrawn cell
culture and then put to -80.degree. C. for storage until further
analysis. In order to extract metabolites, 500 .mu.L of
methanol-quenched fermentation sample was spun down by
centrifugation for 4 min at 4500.times.g, at -9.degree. C. The
pellet was then re-extracted twice, first with 350 .mu.L of 85%
methanol buffered with 5 mM ammonium acetate in water (pH=7.0) and
then with 350 .mu.L of 50% methanol in the ammonium acetate buffer.
After each extraction, cell debris was pelleted by centrifugation
and all three supernatants were pooled together for further
analysis.
Metabolite Quantitation
[0728] Extracted metabolites were analyzed by LC-ESI-MS/MS on a
Quantum triple quadrupole mass spectrometer (Thermo Electron
Corporation, San Jose, Calif.). The system control, data
acquisition, and mass spectral data evaluation were performed using
XCalibur and LCQuan software (Thermo Electron Corp). LC separation
was done on a Synergi 45 .mu.M Hydro-RP HPLC column (150.times.2
mm, Phenomenex, USA) at a flow rate of 0.4 mL/min and the column
temperature of 40.degree. C. The LC gradient was t=0 min, 12% B;
t=5 min, 12% B; t=9 min, 23% B; t=20 min, 99% B; t=23 min, 99% B;
t=24 min, 12% B; t=29 min, 12% B, where solvent A was 10 mM
tributylamine/15 mM acetic acid in water and solvent B was
LCMS-grade methanol. The sample injection volume was 10 .mu.L.
[0729] Mass detection was carried out using electrospray ionization
in the negative mode. The following m/z values for precursor ions
were selected to detect the metabolites of interest in SRM mode:
213.0 for DXP, 215.0 for MEP, 245.0 for IPP and DMAPP, 260.0 for
HDMAPP, and 277.0 for cMEPP, 381.1 for FPP, 520.1 for CDP-ME, 600.0
for CDP-MEP. Concentrations of metabolites were determined based on
integrated intensities of peaks generated by PO.sub.3.sup.- product
ion (m/z=79.0) using calibration curves obtained by injection of
corresponding standards (Echelon Biosciences Inc). The
concentration of CDP-MEP was expressed in arbitrary units because
of the unavailability of commercial standard. Intracellular
concentrations of metabolites were calculated based on a standard
assumption that in 1 mL of the culture at OD=200 the integrated
volume of all cells is 50 .mu.L.
Example 26
Result of Increased Activity of IspG
[0730] This example demonstrates that increased activity of IspG
can be detrimental to isoprene production when it occurs in the
absence of increased FldA expression.
[0731] Data obtained using 14-L REMG39 indicates that despite the
increased production of isoprene in REMG39, the strain is still
limited for IspG activity; this is suggested by the approx. 19 mM
cMEPP level the REMG39 strain maintains across the majority of the
fermentation (see FIG. 80B). One way to improve IspG activity is to
increase its expression, as was observed for strain REM E7.sub.--12
(FIG. 85B). However, increasing IspG activity in the test strain
REM E7.sub.--12 compared to the parental strain CMP272 proved to be
detrimental to isoprene production (FIG. 9A). An alternative method
to increase the IspG activity generated from the CMP272 strain
background is to increase fldA expression (test strain REM
C9.sub.--12; FIG. 85B). The largest benefit determined at small
scale that increased both the increased IspG activity and
endogenous IspH activity as well as improved isoprene production
from the CMP272 background was to co-overexpress fldA and ispG
(test strain REM D6.sub.--12; FIG. 85).
A. Construction of test strains REM C9.sub.--12, REM D6.sub.--12,
and REM E7.sub.--12
[0732] The construction of GI1.6 fldA/pCL, GI1.6 fldA-ispG/pCL, and
GI1.6 ispG/pCL were done using standard molecular biology
techniques (Sambrook et al., 1989). The pCL1920 (pCL) cloning
vector has been described in publications, see, e.g., Lerner, C. G.
et al., Nucleic Acids Research, Vol. 18: 4631(1990). FIG. 82-84
depict the resulting plasmid constructs. The CMP272 strain was used
for the transformations described below.
[0733] Chromosomal DNA from strain REM I6.sub.--4 was used as a PCR
template for the generation of the PCR fragment harboring GI1.6
fldA, which was used to create GI1.6 fldA/pCL. Generation of strain
REM I6.sub.--4 is described below. The DNA ultimately derived from
the DXP operon pET24a plasmid (see, e.g., Example 11) was used as
the PCR template for both the generation of the PCR fragments
harboring ispG and GI1.6 ispG, which were used to create GI1.6
fldA-ispG/pCL and GI1.6 ispG/pCL, respectively. The DXP operon
pET24a plasmid and GI1.6 gcpE-lytB-yidi pCR Blunt II TOPO vector
PCR templates utilized have been described previously (see, e.g.,
Example 11).
B. The Generation of REM I6 4, the Precursor to GI1.6 fldA/pCL
[0734] The GI 1.X-promoter insertions and subsequent loopout of the
antibiotic resistance markers described in this example were
carried out using the Red/ET system from Gene Bridges GmbH
according to the manufacturer's instructions. The strain BL21(DE3)
(Invitrogen) was used. The BIO RAD Gene Pulser system (0.1 cm
cuvette cat.#165-2089) and a transformation protocol suggested by
the manufacturer (BIO RAD) was used for the electroporations
described.
TABLE-US-00033 Primers fldA confirm-F (SEQ ID NO: 158) 5'
tgattccgcaagactgcctgt fldA confirm-R (SEQ ID NO: 159) 5'
ttcggtattaccggtgtcgct fldA cmpGI1.X-F (SEQ ID NO: 160) 5'
ctatgattgc ctttatccgt gggcaatttt ccacccccat
aattaaccctcactaaagggcggccgc fldA cmpGI1.X-R (SEQ ID NO: 161) 5'
aagatgccagtgatagccatgagtgaaataacctcttgaa
ggttacctccgggaaacgcggttgatttgtttagtggttgaattatttg
ctcaggatgtggcatngtcaagggcgtgacggctcgc taatacgactcactatagggctcgag *
for the case of GI1.6 fldA N = T in the primer sequence above. top
Gb's CMP (SEQ ID NO: 144) 5' actgaaacgttttcatcgctc bottom Pgb2 (SEQ
ID NO: 163) 5' ggtttagttcctcaccttgtc
[0735] The GI1.X promoters introduced upstream of the endogenous
fldA coding region using the Gene Bridges GmbH methods are
illustrated in FIG. 81. The antibiotic resistance cassette GB-CMP
was amplified by PCR using primer sets fldA cmpGI1.X-F/fldA
cmpGI1.X-R. The primers contain 40 bases of homology to the region
immediately 5' to the fldA coding region to allow recombination at
the specific locus upon electroporation of the PCR product in the
presence of the pRed-ET plasmid. The FRT "scar" sequences remaining
after Flipase-mediated excision of the antibiotic markers are also
depicted in the figure.
Amplification of the GB-CmpR-fldA Fragment
[0736] To amplify the GB-CmpR cassette for inserting the GI
1.X-promoters immediately upstream of the fldA locus the following
PCR reaction was set up:
1 ul template (100 ng GB-CmpR)
10 ul HerculaseII Buffer
[0737] 0.5 ul dNTP's (100 mM) 1.25 ul primer (10 uM) fldA
cmpGI1.X-F 1.25 ul primer (10 uM) fldA cmpGI1.X-R 35 ul diH2O +1 ul
of HerculaseII fusion from Stratagene
Cycle Parameter:
[0738] 95.degree. C..times.2 min., (95.degree. C..times.30 sec.,
63.degree. C..times.30 sec., 72.degree. C..times.2 min.).times.29
cycles; 72.degree. C..times.5 min., 4.degree. C. until cool
(Biometra T3000 Combi Thermocycler)
[0739] The resulting PCR fragments were separated on a 0.8% E-gel
(Invitrogen) for verification of successful amplification, and
purified using the QIAquick PCR Purification kits (Qiagen)
according to manufacturer's instructions. The resulting stock was
GB-CmpR-GI 1.X-fldA fragment.
Integration of GB-CmpR-GI 1.X fldA PCR Product into
BL21(DE3)/pRed-ET Strain
[0740] The pRed-ET vector (Gene Bridges kit) was transformed into
BL21(DE3) by electroporation resulting in strain DW30
(BL21(DE3)/pRed-ET). The purified GB-CmpR-GI 1.X-fldA PCR fragment
was electroporated into DW30. The transformants were recovered in L
Broth and then plated on L agar containing chloramphenicol (10
ug/ml). Chloramphenicol resistant colonies were analyzed by PCR for
the presence of the GB-CmpR cassette and the GI 1.X-promoters using
primers fldA confirm-F, fldA confirm-R, top GB's CMP, and bottom
Pgb2. The PCR fragments from a number of transformants were
sequenced using the fldA confirm-R and top GB's CMP primers
(Sequetech; Mountain View, Calif.) and the various GI 1.X fldA
strains of interest identified. The chloramphenicol resistant
strain, BL21(DE3) CMP::GI1.6fldA, was designated REM
I6.sub.--4.
C. Strategy for Creating REM C9.sub.--12
[0741] Electroporation of GI1.6 fldA/pCL into CMP272 was performed
using the BIO RAD Gene Pulser system (0.1 cm cuvette cat.#165-2089)
and a transformation protocol suggested by the manufacturer (BIO
RAD). Cells of the strain REM I6.sub.--4 encoding GI1.6 fldA were
used as the PCR template for vector construction.
TABLE-US-00034 Primers Sequences 5' SalI GI1.X-5' (SEQ ID NO: 164)
cgag gtcgac gcgagccgtcacgcccttgac 3' NruI/SacII fldA stop-5' (SEQ
ID NO: 165) gctc tcgcga gagc ccgcgg tcaggcattgagaatttcgtcgag M13
(-20) 5' (SEQ ID NOS: 63, 69 and 98) M13 reverse 5' (SEQ ID NO: 99)
CAGGAAACAGCTATGAC
Amplification of the GI1.6 fldA Fragment
[0742] To amplify the GI1.6 fldA fragment for inserting the GI1.6
fldA fragment into pCL the following PCR reaction was set up:
1 ul template (approx. 1 ul volume of I6.sub.--4 cells)
10 ul HerculaseII Buffer
[0743] 0.5 ul dNTP's (100 mM) 1.25 ul primer (10 uM) 5' SalI GI1.X
1.25 ul primer (10 uM) 3' NruI/SacII fldA stop 35 ul diH2O +1 ul of
HerculaseII fusion from Stratagene
Cycle Parameter:
[0744] 95.degree. C..times.2 min., [95.degree. C..times.30 sec.,
60.degree. C..times.30 sec., 72.degree. C..times.3 min.].times.29
cycles; 72.degree. C..times.5 min., 4.degree. C. until cool
(Biometra T3000 Combi Thermocycler)
[0745] The resulting PCR fragment was separated on a 1.2% E-gel
(Invitrogen) for verification of successful amplification, and
purified using the QIAquick PCR Purification kits according to
manufacturer's instructions. The resulting stock was GI 1.6-fldA
fragment.
Cloning of the GI1.6 fldA Fragment into pCL
[0746] Approximately 600 ng of the GI1.6 fldA fragment was digested
with SalI (Roche) according to the manufacturer's specifications
and approx. 200 ng of the pCL plasmid was digested with SalI and
SmaI (Roche) according to the manufacturer's specifications. The
digests were subsequently combined and cleaned using the Qiagen
QiaQuick Gel Extraction Kit. Approximately one half of the cleaned
cut DNA was ligated using T4 DNA Ligase from New England Biolabs
according to the manufacturer's suggested protocol. Chemically
competent TOP10 cells (Invitrogen) were transformed with the
ligation reaction using a standard heat-shock protocol (Sambrook et
al., 1989), recovered in L broth for 1 hour at 37.degree. C. and
then plated on L agar containing spectinomycin (50 ug/ml) and
5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-GAL at 40
ug/ml; Sigma). White, spectinomycin resistant colonies were
selected, grown overnight in L broth containing spectinomycin (50
ug/ml), and harvested for subsequent plasmid preparation. Plasmid
constructs were isolated using Qiagen Qiaprep Spin Miniprep Kit.
Plasmid preparations of interest were sequenced (Sequetech;
Mountain View, Calif.) using primers M13 (-20) and M13 Reverse, and
the correct GI1.6 fldA/pCL clone identified, which has been
designated as strain REM A1.sub.--11 (TOP10 w/GI1.6 fldA/pCL; 5'
Sal I-3' SacII/NruI uncut (blunt 3') end PCR fragment into 5'
SalI-3'Sma I of pCL). A picture of the GI1.6 fldA/pCL vector map is
presented in FIG. 82.
Transformation of GI1.6 fldA/pCL into CMP271
[0747] To build the isoprene producing test strain REM C9.sub.--12,
the GI1.6 fldA/pCL plasmid was transformed by electroporation into
CMP272. Transformants were recovered in L broth and plated on L
agar containing spectinomycin (50 ug/ml) and carbenicillin (50
ug/ml). The resulting strain was designated REM C9.sub.--12.
Strategy for Creating REM D6.sub.--12
[0748] Electroporation of GI1.6 fldA-ispG/pCL into CMP272 was
performed using the BIO RAD Gene Pulser system (0.1 cm cuvette
cat.#165-2089) and a transformation protocol suggested by the
manufacturer (BIO RAD). The DXP operon pET24a plasmid was used as
the PCR template for vector construction.
Primers Sequences
TABLE-US-00035 [0749] Primers Sequences 5' SacII Ec ispG w/ rbs-5'
(SEQ ID NO: 166) tcca ccgcgg gctc gaa ggag atatacc atg cat aac cag
gct cca att caa 3' NruI Ec ispG stop-5' (SEQ ID NO: 167) gctc
tcgcga tta ttt ttc aac ctg ctg aac gtc M13For-5' (SEQ ID NO: 168)
gttgtaaaacgacggccagt 5' BamHI Ec ispG w/ rbs-5' (SEQ ID NO: 169)
tacg ggatcc atttga ggag taagcc atg cat aac cag gct cca att caa 3'
SacI Ec ispG w/ stop-5' (SEQ ID NO: 170) gctg gagctc cac tta ttt
ttc aac ctg ctg aac gtc pRA42-5' (SEQ ID NO: 171)
gatgatcaacatgacgcatggc pRA43-5' (SEQ ID NO: 172)
cattccgatccgtattggcg
Amplification of they' SacII-ispG-3' NruI Fragment
[0750] To amplify the ispG fragment for inserting into GI1.6
fldA/pCL the following PCR reaction was set up:
1 ul template (approx. 1 ul volume of I6.sub.--4 cells)
10 ul HerculaseII Buffer
[0751] 0.5 ul dNTP's (100 mM) 1.25 ul primer (10 uM) 5' SacII Ec
ispG w/rbs 1.25 ul primer (10 uM) 3' NruI Ec ispG stop 35 ul diH2O
+1 ul of HerculaseII fusion from Stratagene
Cycle Parameter:
[0752] 95.degree. C..times.2 min., [95.degree. C..times.30 sec.,
60.degree. C..times.30 sec., 72.degree. C..times.2 min.].times.29
cycles; 72.degree. C..times.5 min., 4.degree. C. until cool
(Biometra T3000 Combi Thermocycler)
[0753] The resulting PCR fragment was separated on a 1.2% E-gel
(Invitrogen) for verification of successful amplification, and
purified using the QIAquick PCR Purification kits according to
manufacturer's instructions. The resulting stock was 5'
SacII-ispG-3' NruI fragment.
Cloning of the GI1.6 fldA Fragment into pCL
[0754] Approximately 600 ng of the 5' SacII-ispG-3' NruI fragment
was digested with Sac II (New England BioLabs) according to the
manufacturer's specifications and approx. 200 ng of the GI1.6
fldA/CL plasmid was digested with SacII and NruI (New England
BioLabs) according to the manufacturer's specifications. The
digests were subsequently combined and cleaned using the Qiagen
QiaQuick Gel Extraction Kit. Approximately one half of the cleaned
cut DNA was ligated using T4 DNA Ligase (New England Biolabs)
according to the manufacturer's suggested protocol. Chemically
competent TOP10 cells (Invitrogen) were transformed with the
ligation reaction using a standard heat-shock protocol (Sambrook et
al., 1989), recovered in L broth for 1 hour at 37.degree. C. and
then plated on L agar containing spectinomycin (50 ug/ml). Some
spectinomycin resistant colonies were selected, grown overnight in
L broth containing spectinomycin (50 ug/ml), and harvested for
subsequent plasmid preparation. Plasmid constructs were isolated
using Qiagen Qiaprep Spin Miniprep Kit. Plasmid preparations of
interest were sequenced (Sequetech; Mountain View, Calif.) using
primers 5' SacII Ec ispG, 3' NruI Ec ispG stop, M13For, 5' BamHI Ec
ispG w/rbs, 3' SacII Ec ispG w/stop, pRA42, and pRA43 and the
correct GI1.6fldA-ispG/pCL clone identified, which has been
designated as strain REM D9.sub.--11 (TOP10 w/GI1.6 fldA-ispG/pCL;
5' Sac II-3' NruI uncut (blunt 3' end) PCR fragment into 5'
SacII-3'NruI of pCL). A picture of the GI1.6 fldA-ispG/pCL vector
map is presented in FIG. 83.
Transformation of GI1.6 fldA-ispG/pCL into CMP271
[0755] To build the isoprene producing test strain REM D6.sub.--12,
the GI1.6 fldA-ispG/pCL plasmid was transformed by electroporation
into CMP272. Transformants were recovered in L broth and plated on
L agar containing spectinomycin (50 ug/ml) and carbenicillin (50
ug/ml). The resulting strain was designated REM D6.sub.--12.
E. Strategy for Creating REM E7 12
[0756] Electroporation of GI1.6 ispG/pCL into CMP272 was performed
using the BIO RAD Gene Pulser system (0.1 cm cuvette cat.#165-2089)
and a transformation protocol suggested by the manufacturer (BIO
RAD). The GI1.6 gcpE-lytB-yidi pCR Blunt II TOPO vector was used as
the PCR template for vector construction.
TABLE-US-00036 Primers Sequences 5' SalI GI1.X-5' (SEQ ID NO: 164)
cgag gtcgac gcgagccgtcacgcccttgac 3' SacI Ec ispG w/ stop-5' (SEQ
ID NO: 170) gctg gagctc cac tta ttt ttc aac ctg ctg aac gtc M13For
5' (SEQ ID NO: 168) gttgtaaaacgacggccagt M13Rev 5' (SEQ ID NO: 173)
tcacacaggaaacagctatga
Amplification of the GI1.6 ispG Fragment
[0757] To amplify the GI1.6 ispG fragment for inserting into pCL
the following PCR reaction was set up:
1 ul template (approx. 1 ul volume of I6.sub.--4 cells)
10 ul HerculaseII Buffer
[0758] 0.5 ul dNTP's (100 mM) 1.25 ul primer (10 uM) 5' SalI GI1.X
1.25 ul primer (10 uM) 3' SacI Ec ispG w/stop 35 ul diH2O +1 ul of
HerculaseII fusion from Stratagene
Cycle Parameter:
[0759] 95.degree. C..times.2 min., [95.degree. C..times.30 sec.,
60.degree. C..times.30 sec., 72.degree. C..times.2 min.].times.29
cycles; 72.degree. C..times.5 min., 4.degree. C. until cool
(Biometra T3000 Combi Thermocycler)
[0760] The resulting PCR fragment was separated on a 1.2% E-gel
(Invitrogen) for verification of successful amplification, and
purified using the QIAquick PCR Purification kits according to
manufacturer's instructions. The resulting stock was GI1.6 ispG
fragment.
Cloning of the G11.6 ispG Fragment into pCL
[0761] Approximately 600 ng of the GI1.6 ispG fragment and 200 ng
of the pCL vector were digested with SalI and SacI (Roche)
according to the manufacturer's specifications. The digests were
subsequently combined and cleaned using the Qiagen QiaQuick Gel
Extraction Kit. Approximately one half of the cleaned cut DNA was
ligated using T4 DNA Ligase (New England Biolabs) according to the
manufacturer's suggested protocol. Chemically competent TOP10 cells
(Invitrogen) were transformed with the ligation reaction using a
standard heat-shock protocol (Sambrook et al., 1989), recovered in
L broth for 1 hour at 37.degree. C. and then plated on L agar
containing spectinomycin (50 ug/ml) and
5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-GAL at 40
ug/ml; Sigma). White spectinomycin resistant colonies were
selected, grown overnight in L broth containing spectinomycin (50
ug/ml), and harvested for subsequent plasmid preparation. Plasmid
constructs were isolated using Qiagen Qiaprep Spin Miniprep Kit.
Plasmid preparations of interest were sequenced (Sequetech;
Mountain View, Calif.) using primers 3' SacI Ec ispG w/stop,
M13For, and M13 Rev and the correct GI1.6 ispG/pCL clone
identified, which has been designated as strain REM H5.sub.--11
(TOP10 w/GI1.6 ispG/pCL; 5' SalI-3' SacI PCR fragment into 5'
SalI-3'SacI of pCL). A picture of the GI1.6 ispG/pCL vector map is
presented in FIG. 84.
Transformation of GI1.6 ispG/pCL into CMP271
[0762] To build the isoprene producing test strain REM E7.sub.--12,
the GI1.6 ispG/pCL plasmid was transformed by electroporation into
CMP272. Transformants were recovered in L broth and plated on L
agar containing spectinomycin (50 ug/ml) and carbenicillin (50
ug/ml). The resulting strain was designated REM E7.sub.--12.
F. Analysis of Test Strains REM C9.sub.--12, REM D6.sub.--12, and
REM E7.sub.--12 and the Parental Strain CMP272 for Growth, Isoprene
Production, and DXP Metabolite Accumulation.
[0763] The parental strain CMP272 was compared against the test
strains (REM C9.sub.--12, REM D6.sub.--12, and REM E7.sub.--12) in
a shake flask assay as well as in a DXP metabolite determination
study FIG. 85A and FIG. 85B, respectively. The detriment,
approximately 20% decrease in isoprene production, of expressing
ispG alone within the CMP272 background (strain REM E7.sub.--12) is
shown in FIG. 85A. The increased benefit on isoprene production in
small scale of co-expressing fldA along with ispG in comparison to
expressing either fldA or ispG alone from the CMP272 host is also
depicted in FIG. 85A. A 1.4-fold improvement in isoprene production
was observed for the REM D6.sub.--12 strain relative to the
parental control strain CMP272. The benefit of increasing the level
of fldA expression on endogenous levels of E. coli IspG and IspH
activity in strain REM C9.sub.--12 as well as improving the
activity of IspH within the ispG-overexpressing strain REM
D6.sub.--12 is indicated by the metabolite profile described in
FIG. 85B. More specifically, the additional FldA in strain REM
C9.sub.--12 decreased the levels of both the IspG and IspH
substrates, cMEPP and HDMAPP, respectively, relative to the
parental strain CMP272 (cMEPP, 17% decrease; HDMAPP, 16% decrease);
while the additional FldA within the co (fldA and
ispG)-overexpression strain REM D6.sub.--12 compared to the REM
E7.sub.--12 strain overexpressing ispG alone was seen to decrease
HDMAPP roughly 4.3-fold.
Growth
[0764] Strains CMP272, REM C9.sub.--12, REM D6.sub.--12, and REM
E7.sub.--12 were grown as 2-5 ml cultures at 30.degree. C. in TM3
liquid media (13.6 g K.sub.2PO.sub.4, 13.6 g KH.sub.2PO.sub.4, 2.0
g MgSO.sub.4*7H.sub.2O), 2.0 g citric acid monohydrate, 0.3 g
ferric ammonium citrate, 3.2 g (NH.sub.4).sub.2SO.sub.4, 0.2 g
yeast extract, 1.0 ml 1000.times. Modified Trace Metal Solution,
adjusted to pH 6.8 and q.s. to H.sub.2O, and filter sterilized)
supplemented to a final concentration with 0.1% yeast extract and
1.0% glucose and including spectinomycin (50 ug/ml) and
carbenicillin (50 ug/ml). Induction of LacI-regulated gene
expression was achieved by adding
isopropyl-beta-D-1-thiogalactopyranoside (IPTG) to a concentration
of 600 uM. Growth was monitored periodically by recording each of
the culture's optical density measured at 600 nm using an Eppendorf
Biophotometer spectrometer (Eppendorf).
Isoprene Production
[0765] Isoprene production was anlayzed using a headspace assay.
For the shake flask cultures, 200 ul of a culture was transferred
from shake flasks to 2 ml CTC headspace vials (SUN-SRI 2 mL HS
vials, VWR#66020-950, and caps, VWR#66008-170). The cap was screwed
on tightly and the vials incubated at the equivalent temperature
with shaking at 250 rpm. After 30 minutes the vials were removed
from the incubator and analyzed. The analysis was performed using
an Agilent 6890 GC/MS system interfaced with a CTC Analytics
(Switzerland) CombiPAL autosampler operating in headspace mode. An
Agilent HP-5MS GC/MS column (15 m.times.0.25 mm; 0.25 .mu.m film
thickness) was used for separation of analytes. The sampler was set
up to inject 100 .mu.L of headspace gas. The GC/MS method utilized
helium as the carrier gas at a flow of 1 ml/minutes The injection
port was held at 250.degree. C. with a split ratio of 50:1. The
oven temperature was held at 37.degree. C. for 0.6 minute, the
duration of the analysis. The Agilent 5793N mass selective detector
was run in single ion monitoring (SIM) mode on m/z 67. The detector
was switched off from 0 to 0.42 minutes to allow the elution of
permanent gases. Under these conditions isoprene
(2-methyl-1,3-butadiene) was observed to elute at approx. 0.49
minutes. A calibration table was used to quantify the absolute
amount of isoprene and was found to be linear from 1 .mu.g/L to
5000 .mu.g/L. The limit of detection was estimated to be 50 to 100
ng/L using this method. The specific productivity of each strain is
reported as ug/L OD Hr. Note, ratio of 1900 ul headspace:100 ul
broth in assay vials for 30 min. incubation results in the
following conversion of isoprene ug/L of culture to specific
productivity: (isoprene/L determined by GC-MS).times.(38)/(OD 600
nm of the culture).
DXP Metabolite Accumulation
[0766] The DXP metabolites of the isoprene-producing parental and
test strains, CMP272 and REM C9.sub.--12, REM D6.sub.--12, and REM
E7.sub.--12, respectively, that are described above and depicted in
FIG. 85B were isolated and quantified as follows:
Metabolite Extraction: Processing Samples from Small-Scale
Experiments.
[0767] To measure accumulation of metabolites in small-scale
experiments 0.4 to 1.5 mL of cell culture was centrifuged for 3 min
at 7500.times.g, at -9.degree. C. Immediately after centrifugation
the supernatant was aspirated to a clean tube for analysis of
excreted metabolites and 100 .mu.L of dry ice-cold methanol was
added to pelleted cells. The resulting samples were then stored at
-80.degree. C. until further processing.
[0768] To determine concentrations of excreted metabolites, 500
.mu.L of methanol was added to 300 .mu.L of the supernatant and the
resulting mixture was centrifuged for 10 min at 20000.times.g at
4.degree. C. to remove insoluble material before the LCMS
analysis.
[0769] For metabolites extraction from the pellet (further referred
as intracellular metabolites), 10 .mu.L of water was added to
methanol-containing samples, the pellet was resuspended in the
resulting methanol/water mix and cell debris were spun down by
4-min centrifugation at 4500.times.g. The pellet was re-extracted
two more times, first with 100 .mu.L of 75% methanol buffered with
1 mM ammonium acetate in water (pH=8.0), then with 90 .mu.L of 50%
methanol in the ammonium acetate buffer. After each extraction,
cell debris was pelleted by centrifugation and the supernatants
from all three extractions were combined and analyzed by LCMS.
During the extraction procedure, samples were kept on ice or in a
refrigerated centrifuge whenever possible to minimize metabolites
degradation.
Metabolite Quantitation
[0770] Extracted metabolites were analyzed by LC-ESI-MS/MS on a
Quantum triple quadrupole mass spectrometer (Thermo Electron
Corporation, San Jose, Calif.). The system control, data
acquisition, and mass spectral data evaluation were performed using
XCalibur and LCQuan software (Thermo Electron Corp). LC separation
was done on a Synergi 45 .mu.M Hydro-RP HPLC column (150.times.2
mm, Phenomenex, USA) at a flow rate of 0.4 mL/min and the column
temperature of 40.degree. C. The LC gradient was t=0 min, 12% B;
t=5 min, 12% B; t=9 min, 23% B; t=20 min, 99% B; t=23 min, 99% B;
t=24 min, 12% B; t=29 min, 12% B, where solvent A was 10 mM
tributylamine/15 mM acetic acid in water and solvent B was
LCMS-grade methanol. The sample injection volume was 10 .mu.L.
[0771] Mass detection was carried out using electrospray ionization
in the negative mode. The following m/z values for precursor ions
were selected to detect the metabolites of interest in SRM mode:
213.0 for DXP, 215.0 for MEP, 245.0 for IPP and DMAPP, 260.0 for
HDMAPP, and 277.0 for cMEPP, 381.1 for FPP, 520.1 for CDP-ME, 600.0
for CDP-MEP. Concentrations of metabolites were determined based on
integrated intensities of peaks generated by PO.sub.3.sup.- product
ion (m/z=79.0) using calibration curves obtained by injection of
corresponding standards (Echelon Biosciences Inc). The
concentration of CDP-MEP was expressed in arbitrary units because
of the unavailability of commercial standard. Intracellular
concentrations of metabolites were calculated based on a standard
assumption that in 1 mL of the culture at OD=200 the integrated
volume of all cells is 50 .mu.L.
Example 27
Effects of Increased Activity of IspG
[0772] This example demonstrates that increased activity of IspG
can be detrimental to isoprene production as a result of
insufficient IspH activity within strain REM G4.sub.--11.
[0773] As described in the example above, increased expression of
fldA alone or in combination with ispG within the CMP272 strain
background improved isoprene production (FIG. 85). These learnings
were applied to strain REMG39, as the overall goal was to improve
IspG activity within this (benchmark) strain background. To
reiterate, the REMG39 strain exhibited characteristics perceived to
reflect a bottleneck at the point of IspG activity in flux through
the DXP pathway toward isoprene production (see 14-L REMG39
example). In FIG. 86A, the benefit of increasing IspG activity
within the REM G4.sub.--11 strain at small scale is made apparent
(35% increase in isoprene production over the parental control;)
however, as shown in FIGS. 79 and 80, this benefit did not
translate to the large scale fermentation. Results of the large
scale fermentation presented in FIG. 80 indicate that increased
IspH activity is required by the REM G4.sub.--11 strain; this is
suggested by the high (>15 mM) HDMAPP levels observed during
exponential phase growth of REM G4.sub.--1 (FIG. 80C).
A. Construction of Test Strains REM G2.sub.--11 and REM
G4.sub.--11
[0774] To further improve the IspG activity generated by the REMG39
strain background, the vector constructs GI1.6 fldA/pCL and GI1.6
fldA-ispG/pCL were introduced into the strain, subsequently
generating the test strains REM G2.sub.--11 and REM G4.sub.--11,
respectively.
B. Strategy for Creating REM G2.sub.--11 and REM G4.sub.--11
[0775] Electroporation of GI1.6 fldA/pCL and GI1.6 fldA-ispG/pCL
into REMG39 was performed using the BIO RAD Gene Pulser system and
a transformation protocol suggested by the manufacturer (BIO RAD).
Plasmid preparations of GI1.6 fldA/pCL, generated from strain REM
A1.sub.--11, and GI1.6 fldA-ispG/pCL, generated from strain REM
D9.sub.--11, were used; these strains and constructs are described
above.
Transformation of GI1.6 fldA/pCL and GI1.6 fldA-ispG/pCL into
CMP271
[0776] To build the isoprene producing test strains REM G2.sub.--11
and REM G4.sub.--11, the GI1.6 fldA/pCL and GI1.6 fldA-ispG/pCL
plasmids were transformed, separately, by electroporation into
REMG39. Transformants were recovered in L broth and plated on L
agar containing spectinomycin (50 ug/ml), kanamycin (50 ug/ml), and
carbenicillin (50 ug/ml). The resulting strains were designated REM
G2.sub.--11 and REM G4.sub.--11, respectively.
C. Analysis of Test Strains REM G2.sub.--11, REM G4.sub.--11, and
the Parental Strain REMG39 for Growth, Isoprene Production, and DXP
Metabolite Accumulation.
[0777] The parental strain REMG39 was compared against the test
strains (REM G2.sub.--11, REM and REM G4.sub.--11) in a shake flask
assay as well as in a DXP metabolite determination study. The
increase in isoprene production provided by the presence of GI1.6
fldA/pCL and GI1.6 fldA-ispG/pCL within the REMG39 background is
depicted in FIG. 86A. The test strain REM G4.sub.--11 produced
approximately 1.35-fold more isoprene than the parental control
strain REMG39 at the 3.5 hour time point, where REM G2.sub.--11
generated approximately 1.25-fold more isoprene than the parental
control at the 3.5 hour time point. As seen in FIG. 86B, both of
the test strains, REM G2.sub.--11 and REM G4.sub.--11, were found
to accumulate less of the IspG substrate, cMEPP, than the parental
strain REMG39 at the 3.5 hour time point (REMG2.sub.--11 had
approx. 66% of the parental control cMEPP level; and REM
G4.sub.--11 had approx. 9% of the parental control cMEPP level).
The REM G4.sub.--11 strain did however accumulate a 5.4-fold higher
level of HDMAPP, the substrate of IspH, than both the parental
control and test strain REM G2.sub.--11 (FIG. 86B).
Growth
[0778] Strains REMG39, REM G2.sub.--11, and REM G4.sub.--11 were
grown at 30.degree. C. as 2-5 ml cultures in TM3 liquid media (13.6
g K.sub.2PO.sub.4, 13.6 g KH.sub.2PO.sub.4, 2.0 g
MgSO.sub.4*7H.sub.2O), 2.0 g citric acid monohydrate, 0.3 g ferric
ammonium citrate, 3.2 g (NH.sub.4).sub.2SO.sub.4, 0.2 g yeast
extract, 1.0 ml 1000.times. Modified Trace Metal Solution, adjusted
to pH 6.8 and q.s. to H.sub.2O, and filter sterilized) supplemented
to a final concentration with 0.1% yeast extract and 1.0% glucose
and including spectinomycin (50 ug/ml) and carbenicillin (50
ug/ml). Induction of LacI-regulated gene expression was achieved by
adding isopropyl-beta-D-1-thiogalactopyranoside (IPTG) to a
concentration of 400 uM. Growth was monitored periodically by
recording each of the culture's optical density measured at 600 nm
using an Eppendorf Biophotometer spectrometer (Eppendorf).
Isoprene Production
[0779] Isoprene production was anlayzed using a headspace assay.
For the shake flask cultures, 200 ul of a culture was transferred
from shake flasks to 2 ml CTC headspace vials (SUN-SRI 2 mL HS
vials, VWR#66020-950, and caps, VWR#66008-170). The cap was screwed
on tightly and the vials incubated at the equivalent temperature
with shaking at 250 rpm. After 30 minutes the vials were removed
from the incubator and analyzed. The analysis was performed using
an Agilent 6890 GC/MS system interfaced with a CTC Analytics
(Switzerland) CombiPAL autosampler operating in headspace mode. An
Agilent HP-5MS GC/MS column (15 m.times.0.25 mm; 0.25 .mu.m film
thickness) was used for separation of analytes. The sampler was set
up to inject 100 .mu.L of headspace gas. The GC/MS method utilized
helium as the carrier gas at a flow of 1 ml/minutes The injection
port was held at 250.degree. C. with a split ratio of 50:1. The
oven temperature was held at 37.degree. C. for 0.6 minute, the
duration of the analysis. The Agilent 5793N mass selective detector
was run in single ion monitoring (SIM) mode on m/z 67. The detector
was switched off from 0 to 0.42 minutes to allow the elution of
permanent gases. Under these conditions isoprene
(2-methyl-1,3-butadiene) was observed to elute at approx. 0.49
minutes. A calibration table was used to quantify the absolute
amount of isoprene and was found to be linear from 1 .mu.g/L to
5000 .mu.g/L. The limit of detection was estimated to be 50 to 100
ng/L using this method. The specific productivity of each strain is
reported as ug/L OD Hr. Note, ratio of 1900 ul headspace:100 ul
broth in assay vials for 30 min. incubation results in the
following conversion of isoprene ug/L of culture to specific
productivity: (isoprene/L determined by GC-MS).times.(38)/(OD 600
nm of the culture).
DXP Metabolite Accumulation
[0780] The DXP metabolites of the isoprene-producing parental and
test strains, REMG39 and REM G2.sub.--11 and REM G4.sub.--11,
respectively, that are described above and depicted in FIG. 10B
were isolated and quantified as follows:
Metabolite Extraction: Processing Samples from Small-Scale
Experiments.
[0781] To measure accumulation of metabolites in small-scale
experiments 0.4 to 1.5 mL of cell culture was centrifuged for 3 min
at 7500.times.g, at -9.degree. C. Immediately after centrifugation
the supernatant was aspirated to a clean tube for analysis of
excreted metabolites and 100 .mu.L of dry ice-cold methanol was
added to pelleted cells. The resulting samples were then stored at
-80.degree. C. until further processing.
[0782] To determine concentrations of excreted metabolites, 500
.mu.L of methanol was added to 300 .mu.L of the supernatant and the
resulting mixture was centrifuged for 10 min at 20000.times.g at
4.degree. C. to remove insoluble material before the LCMS
analysis.
[0783] For metabolites extraction from the pellet (further referred
as intracellular metabolites), 10 .mu.L of water was added to
methanol-containing samples, the pellet was resuspended in the
resulting methanol/water mix and cell debris were spun down by
4-min centrifugation at 4500.times.g. The pellet was re-extracted
two more times, first with 100 .mu.L of 75% methanol buffered with
1 mM ammonium acetate in water (pH=8.0), then with 90 .mu.L of 50%
methanol in the ammonium acetate buffer. After each extraction,
cell debris was pelleted by centrifugation and the supernatants
from all three extractions were combined and analyzed by LCMS.
During the extraction procedure, samples were kept on ice or in a
refrigerated centrifuge whenever possible to minimize metabolites
degradation.
Metabolite Quantitation
[0784] Extracted metabolites were analyzed by LC-ESI-MS/MS on a
Quantum triple quadrupole mass spectrometer (Thermo Electron
Corporation, San Jose, Calif.). The system control, data
acquisition, and mass spectral data evaluation were performed using
XCalibur and LCQuan software (Thermo Electron Corp). LC separation
was done on a Synergi 45 .mu.M Hydro-RP HPLC column (150.times.2
mm, Phenomenex, USA) at a flow rate of 0.4 mL/min and the column
temperature of 40.degree. C. The LC gradient was t=0 min, 12% B;
t=5 min, 12% B; t=9 min, 23% B; t=20 min, 99% B; t=23 min, 99% B;
t=24 min, 12% B; t=29 min, 12% B, where solvent A was 10 mM
tributylamine/15 mM acetic acid in water and solvent B was
LCMS-grade methanol. The sample injection volume was 10 .mu.L.
[0785] Mass detection was carried out using electrospray ionization
in the negative mode. The following m/z values for precursor ions
were selected to detect the metabolites of interest in SRM mode:
213.0 for DXP, 215.0 for MEP, 245.0 for IPP and DMAPP, 260.0 for
HDMAPP, and 277.0 for cMEPP, 381.1 for FPP, 520.1 for CDP-ME, 600.0
for CDP-MEP. Concentrations of metabolites were determined based on
integrated intensities of peaks generated by PO.sub.3.sup.- product
ion (m/z=79.0) using calibration curves obtained by injection of
corresponding standards (Echelon Biosciences Inc). The
concentration of CDP-MEP was expressed in arbitrary units because
of the unavailability of commercial standard. Intracellular
concentrations of metabolites were calculated based on a standard
assumption that in 1 mL of the culture at OD=200 the integrated
volume of all cells is 50 .mu.L.
D. Analysis of Test Strain REM G4.sub.--11 for Growth, Isoprene
Production, and DXP Metabolite Accumulation at Large Scale.
[0786] The increased HDMAPP present in the REM G4.sub.--11 cells
was higher than the parental control strain; however, the averaged
0.63 mM HDMAPP intracellular concentration measured in the REM
G4.sub.--11 cells was significantly less than the >10 mM
intracellular HDMAPP level that has been correlated with poor cell
growth and reduced isoprene production (see FIG. 10B). However,
surprisingly strain REM G4.sub.--11 performed less well and
produced roughly 3-fold less isoprene than the parental control at
the 14-L fermentor scale (FIG. 3). The moderate accumulation of
HDMAPP observed to occur in the REM G4.sub.--11 cells at small
scale was found to be exaggerated under large scale fermentation
conditions, reaching intracellular HDMAPP levels >20 mM (FIG.
4C). The decrease in cMEPP and corresponding increase in HDMAPP
observed for the REM G4.sub.--11 strain relative to the parental
control strain REMG39 strongly suggests that: [0787] 1) IspG
activity has been improved within the REM G4.sub.--11 strain.
[0788] 2) a bottleneck in DXP flux now occurs at the point of IspH
activity in the REM G4.sub.--11 strain.
E. Large Scale Fermentation of Strain REM G4.sub.--11
[0789] The large scale fermentation of the parental strain REMG39
is described above. Isoprene production from E. coli expressing
genes from the DXP pathway and isoprene synthase, grown in
fed-batch culture at the 15-L scale.
Medium Recipe (Per Liter Fermentation Medium):
[0790] K2HPO4 7.5 g, MgSO4*7H2O 2 g, citric acid monohydrate 2 g,
ferric ammonium citrate 0.3 g, yeast extract 0.5 g, 1000.times.
Modified Trace Metal Solution 1 ml. All of the components were
added together and dissolved in Di H2O. This solution was heat
sterilized (123.degree. C. for 20 minutes). The pH was adjusted to
7.0 with ammonium hydroxide (28%) and q.s. to volume. Glucose 10 g,
Mercury Vitamin Solution 8 mL, and antibiotics were added after
sterilization and pH adjustment.
1000.times. Modified Trace Metal Solution (Per Liter):
[0791] Citric Acids*H.sub.2O 40 g, MnSO4*H.sub.2O 30 g, NaCl 10 g,
FeSO4*7H2O 1 g, CoCl2*6H2O 1 g, ZnSO*7H2O 1 g, CuSO4*5H2O 100 mg,
H3BO3 100 mg, NaMoO4*2H2O 100 mg. Each component was dissolved one
at a time in Di H2O, pH was adjusted to 3.0 with HCl/NaOH, and then
the solution was q.s. to volume and filter sterilized with a 0.22
micron filter.
Mercury Vitamin Solution (Per Liter):
[0792] Thiamine hydrochloride 1.0 g, D-(+)-biotin 1.0 g, nicotinic
acid 1.0 g, D-pantothenic acid 4.8 g, pyridoxine hydrochloride 4.0
g. Each component was dissolved one at a time in Di H2O, pH was
adjusted to 3.0 with HCl/NaOH, and then the solution was q.s. to
volume and filter sterilized with 0.22 micron filter.
Feed Solution (Per Kilogram):
[0793] Glucose 0.57 kg, Di H.sub.2O 0.38 kg, K2HPO4 7.5 g, and 100%
Foamblast 10 g. All components were mixed together and autoclaved.
Macro Salt Solution 3.4 mL, 1000.times. Modified Trace Metal
Solution 0.8 ml, and Mercury Vitamin Solution 6.7 mL were added
after the solution had cooled to 25.degree. C.
Macro Salt Solution (Per Liter):
[0794] MgSO4*7H2O 296 g, citric acid monohydrate 296 g, ferric
ammonium citrate 49.6 g. All components were dissolved in water,
q.s. to volume and filter sterilized with 0.22 micron filter.
[0795] Fermentation was performed in a 15-L bioreactor with E. coli
BL21 cells overexpressing the first enzyme in the dxp pathway
(GI1.6-dxs), the last enzyme in the DXP pathway (GI1.6-yIDI), the
lower MVA pathway (PL.2-mKKDyI), various other genes from the DXP
pathway of T. elongates (Ptac-gcpE-lytB-petF-petH/pK184), the E.
coli ispG and fldA genes (GI1.6 fldA-ispG/pCL), and truncated
isoprene synthase from P. alba (pDW33) and containing a restored
17,257 bp chromosomal galM-containing region derived from MG1655
(strain name REM G4.sub.--11). This experiment was carried out to
monitor isoprene formation from glucose at the desired fermentation
pH 7.0 and temperature 34.degree. C. A frozen vial of the E. coli
strain was thawed and inoculated into tryptone-yeast extract medium
for the bioreactor. After the inoculum grew to optical density 1.0,
measured at 550 nm (OD.sub.550), 500 mL was used to inoculate a
15-L bioreactor and bring the initial tank volume to 5 L.
[0796] The feed solution was fed at an exponential rate until a top
feed rate of 4.9 g/min was reached. After this time the glucose
feed was fed to meet metabolic demands a rates less than or equal
to 4.9 g/min. The total amount of glucose delivered to the
bioreactor during the 44 hr fermentation was 3.0 kg. Induction was
achieved by adding isopropyl-beta-D-1-thiogalactopyranoside (IPTG).
The inital IPTG concentration when the tank was first inoculated
was 50 uM. Shots of 50 uM were added over the next five hours to
bring the IPTG concentration to 350 uM when the cells were at an
OD.sub.550 of 10.
[0797] The isoprene level in the off-gas from the bioreactors was
determined using a Hiden mass spectrometer. The isoprene titer
increased over the course of the fermentation to a maximum value of
0.98 g/L at 44 hours.
Equation for calculating Isoprene Titer: .intg.(Instantaneous
isoprene production rate, g/L/hr)dt from t=0 to 44 hrs [=] g/L
broth Equation for calculating Specific Productivity levels: (mg
isoprene.sub.t-mg isoprene.sub.to)/[(OD550.sub.t*L
broth.sub.t-OD550.sub.to*L broth.sub.to)/(2.7 OD*L/g
cell)]/(t-t.sub.0) [=] mg isoprene/g cell/hr
Example 28
DXP Metabolite Determination
A. Metabolite Extraction: Processing 14-L Fermentor Samples.
[0798] Cell metabolism was rapidly inactivated by withdrawing
several milliliters of the fermentor culture into a pre-weighted
tube filled with 9.0 mL of dry ice-cold methanol. The resulting
sample was weighted again to calculate the amount of withdrawn cell
culture and then put to -80.degree. C. for storage until further
analysis. In order to extract metabolites, 500 .mu.L of
methanol-quenched fermentation sample was spun down by
centrifugation for 4 min at 4500.times.g, at -9.degree. C. The
pellet was then re-extracted twice, first with 350 .mu.L of 85%
methanol buffered with 5 mM ammonium acetate in water (pH=7.0) and
then with 350 .mu.L of 50% methanol in the ammonium acetate buffer.
After each extraction, cell debris was pelleted by centrifugation
and all three supernatants were pooled together for further
analysis.
B. Metabolite Quantitation
[0799] Extracted metabolites were analyzed by LC-ESI-MS/MS on a
Quantum triple quadrupole mass spectrometer (Thermo Electron
Corporation, San Jose, Calif.). The system control, data
acquisition, and mass spectral data evaluation were performed using
XCalibur and LCQuan software (Thermo Electron Corp). LC separation
was done on a Synergi 45 .mu.M Hydro-RP HPLC column (150.times.2
mm, Phenomenex, USA) at a flow rate of 0.4 mL/min and the column
temperature of 40.degree. C. The LC gradient was t=0 min, 12% B;
t=5 min, 12% B; t=9 min, 23% B; t=20 min, 99% B; t=23 min, 99% B;
t=24 min, 12% B; t=29 min, 12% B, where solvent A was 10 mM
tributylamine/15 mM acetic acid in water and solvent B was
LCMS-grade methanol. The sample injection volume was 10 .mu.L.
[0800] Mass detection was carried out using electrospray ionization
in the negative mode. The following m/z values for precursor ions
were selected to detect the metabolites of interest in SRM mode:
213.0 for DXP, 215.0 for MEP, 245.0 for IPP and DMAPP, 260.0 for
HDMAPP, and 277.0 for cMEPP, 381.1 for FPP, 520.1 for CDP-ME, 600.0
for CDP-MEP. Concentrations of metabolites were determined based on
integrated intensities of peaks generated by PO.sub.3.sup.- product
ion (m/z=79.0) using calibration curves obtained by injection of
corresponding standards (Echelon Biosciences Inc). The
concentration of CDP-MEP was expressed in arbitrary units because
of the unavailability of commercial standard. Intracellular
concentrations of metabolites were calculated based on a standard
assumption that in 1 mL of the culture at OD=200 the integrated
volume of all cells is 50 .mu.L.
Example 29
Increased Isoprene Production by Expression of the IspH Enzyme and
Coincident Demonstration of Maintained Accumulation of Higher DXP
Metabolite Levels
[0801] This example demonstrates increased isoprene production by
expression of the IspH enzyme from Anabaena sp. PCC7120 in strain
REM H8.sub.--12 and coincident demonstration of maintained
accumulation of higher DXP metabolite levels in the REM H8.sub.--12
strain exhibiting increased IspG activity.
[0802] Data in the above example(s) generated with test strain REM
G4.sub.--11 indicates that increased IspG activity within an
enhanced DXP fluxing strain needs to be balanced by sufficient IspH
activity in order to avoid high levels of HDMAPP accumulation
during 14-L fermentation. Intracellular levels of HDMAPP, the
substrate for IspH, in excess of 10 mM have been correlated in both
small scale and large scale experiments with poor cell growth,
reduced flux through the DXP pathway, and subsequently reduced
isoprene generation from isoprene production strains. Therefore,
increased IspH activity within an enhanced DXP pathway strain (REM
I7.sub.--11; described below) was achieved by over-expressing the
ispH allele of Anabaena sp. PCC7120, generating test strain REM
H8.sub.--12. Demonstrated in FIG. 89 is the small scale benefit
increased IspH activity, provided by expression of the IpsH of
Anabaena sp. PCC7120, has on isoprene production by test strain REM
H8.sub.--12. At 14-L scale, the test strain REM H8.sub.--12
produced the highest (2.6 g/L) isoprene titer recorded for a strain
exhibiting the enhanced IspG activity provided by GI1.6
fldA-ispG/pCL (FIG. 90A). Furthermore, unlike the REM G4.sub.--11
strain at the 14-L scale, strain REM H8.sub.--12 is able to
maintain flux through the DXP pathway, as indicated by the
maintained accumulation of the MEPP and cMEPP intermediates
(compare FIG. 80C to FIG. 90C).
A. Construction of Test Strain REM H8.sub.--12, and the Parental
Strain REM I7.sub.--11.
[0803] REM I7.sub.--11 and REM H8.sub.--12 are derivatives of
WW119. This strain was constructed by electoporation of Strain
WW103 with plasmid pDW33 (see Example 30 for construction of
WW119). WW119 exhibits improved DXP-flux, but generates similar
isoprene levels to that of the previous parental strain CMP272;
this is potentially due to a bottleneck in flux at the point of
IspG. WW119 harbors two improvements over the CMP272 strain. These
beneficial modifications include increased dxs expression and
increased dxr expression and are described infra. REM I7.sub.--11
was generated by introducing GI1.6 fldA-ispG/pCL into WW119 and REM
H8.sub.--12 was made by moving Ptac Anabaena ispH aspA term/pEWL454
into REM I7.sub.--11; both plasmids were incorporated into their
corresponding host strain via electroporation transformation
methods.
TABLE-US-00037 Primers 5' AseI F-pgl pET-15b (SEQ ID NO: 174) 5'
cagtct ATTAAT atgAAGCAAACAGTTTATATC 3' BamHI R-pgl pET-15b (SEQ ID
NO: 175) 5' TAGCAGCC GGATCCTTAGTGTGCGTTAACCACCAC EL-1098: (SEQ ID
NO: 139) 5' TAACTTTAAGGAGGTATACATATGGAGCTCACGCGTGCGGCCGC
CTCGAGCTGCAGTACAAATAAAAAAGGCACGTCAG EL-1099: (SEQ ID NO: 138) 5'
GGATCCGTAATCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATT
CCACACATTATACGAGCCGATGATTAATTGTCAACAGAATTCC TTTCCAGTCGGGAAACCTGTCG
EL-1100: (SEQ ID NO: 137) 5' CGTCGTTTTACAACGTCGTG EL-1101: (SEQ ID
NO: 136) 5' GAACTCCAAGACGAGGCAGC EL-1102: (SEQ ID NO: 135) 5'
GTGATATTGCTGAAGAGCTTGG EL-1103: (SEQ ID NO: 134) 5'
GGACTCAAGACGATAGTTACC EL-1104: (SEQ ID NO: 133) 5'
CACGACAGGTTTCCCGACTGG EL-1150 (SEQ ID NO: 132) 5'
GAGCGCCCAATACGCAAACC Neo.21 (SEQ ID NO: 131) 5'
GGCGATAGAAGGCGATGC
Amplification of the pgl Locus of REM I1.sub.--9
[0804] To verify/amplify the pgl locus of REM I1.sub.--9 the
following PCR reaction was set up:
1 ul template (approx. 1 ul volume of I1.sub.--9 cells)
10 ul HerculaseII Buffer
[0805] 0.5 ul dNTP's (100 mM) 1.25 ul primer (10 uM) 5' Asel F-pgl
pET-15b 1.25 ul primer (10 uM) 3' BamHI R-pgl pET-15b 35 ul diH2O
+1 ul of HerculaseII fusion from Stratagene
Cycle Parameter:
[0806] 95.degree. C..times.2 min., [95.degree. C..times.30 sec.,
55.degree. C..times.30 sec., 72.degree. C..times.2 min.].times.29
cycles; 72.degree. C..times.5 min., 4.degree. C. until cool
(Biometra T3000 Combi Thermocycler)
[0807] The resulting PCR fragment was separated on a 1.2% E-gel
(Invitrogen) for verification of successful amplification. A pgl+
verified clone was selected as REM I1.sub.--9
Amplification of the pEWL454 Fragment
[0808] To generate pEWL454 the following PCR reaction was set
up:
1 ul template (approx. 1 ul volume of pK184 w/aspA term vector
(Gene Oracle, Inc.)) 5 ul 10.times.Pfu Ultra II Fusion DNA
polymerase 2.5 ul dNTP's (10 mM) 1.0 primer (10 uM) EL-1098 1.0
primer (10 uM) EL-1099 39.5 ul diH2O +1 ul of Pfu Ultra II Fusion
DNA polymerase from Stratagene
Cycle Parameter:
[0809] 95.degree. C..times.2 min., [95.degree. C..times.30 sec.,
60.degree. C..times.30 sec., 72.degree. C..times.52 sec.].times.29
cycles; 72.degree. C..times.3 min., 4.degree. C. until cool (MJ
Research PTC-200 Peltier Thermal Cycler)
[0810] The resulting PCR fragment was separated on a 1.2% E-gel
(Invitrogen) for verification of successful amplification.
[0811] Strain REM I1.sub.--9 description
[0812] The strain REM I1.sub.--9 was used to clone the Anabaena sp.
PCC7120 ispH allele, which had been codon optimized for expression
in E. coli (provided by Gene Oracle, Inc.). Surprisingly Gene
Oracle, Inc. was unable to provide an E. coli strain harboring the
desired clone. Therefore, strain REM I1.sub.--9 was used as a host
to obtain the Ptac Anabaena ispH aspA term/pEWL454 clone of
interest using a survival based strategy.
[0813] Strain REM I1.sub.--9 is derived from MD09-220
(BL21(DE3)PL.2 mKKDyI::FRT-.DELTA.ispH::FRT) and has been described
previously. The FRT-neo-FRT-GI1.6-dxs locus of strain MCM625 was
transduced into the genome of MD09-220 via standard P1 lystate/P1
transduction protocol (Thomason et al., 2007) and the resulting
kanamycin resistant strain named REM C5.sub.--9. Using Gene
Bridge's GmbH methods the antibiotic marker was looped out,
generating strain REM H5.sub.--9. Subsequently, the pgl and galP
region of MG1655 was transduced into strain REM H5.sub.--9 using
standard P1 lystate/P1 transduction protocol (Thomason et al.,
2007), and the cells selected for growth on M9 agar (Na2HPO4 6 g/L,
KH2PO4 3 g/L, NaCl 0.5 g/L, NH4Cl 0.5 g/L, 0.1 mM CaCl2, 2 mM
MgSO4, 1.5% agar) containing 0.4% w/v galactose and 500 uM
mevalonic acid. The presence of the pgl locus in the
galactose-utlizing, mevalonic acid-dependent, kanamycin sensitive
cells was verified by PCR (see above) and one clone selected as REM
I1.sub.--9 (BL21(DE3) PL.2 mKKDy/::FRT-.DELTA.ispH::FRT pgl
FRT::GI1.6-dxs).
Cloning of the Anabaena sp. PCC7120 ispH Allele into pEWL454
[0814] Approximately 90 ng of a precut 5' BamHI-3' PstI purified
DNA fragment harboring the Anabaena sp. PCC7120 ispH allele codon
optimized for expression in E. coli (provided by Gene Oracle, Inc.)
was ligated to precut 5' BamHI-3' PstI purified DNA vector backbone
pEWL454 (provided by Gene Oracle, Inc.), harboring the tac promoter
and aspA terminator sequences separated by a multiple cloning site
(MCS) within a pK184 (Jobling and Holmes, 1990) derived plasmid,
using T4 DNA Ligase (New England Biolabs) according to the
manufacturer's suggested protocol. The aspA terminator sequences
present in pEWL454 were synthesized by Gene Oracle, Inc. Using the
PCR method outlined above, the lac promoter sequence present in
pK184 was removed and the tac promoter and MCS harbored within
pEWL454 was inserted using the oligos detailed above (Integrated
DNA Technologies). The resulting PCR fragment was ligated using T4
DNA Ligase from New England Biolabs according to the manufacturer's
suggested protocol. Chemically competent TOP10 cells (Invitrogen)
were transformed with the ligation reaction using a standard
heat-shock protocol (Sambrook et al., 1989), recovered in L broth
for 1 hour at 37.degree. C. and then plated on L agar containing
kanamycin (50 ug/ml). A kanamycin resistant clone was selected,
grown overnight in L broth containing kanamycin (50 ug/ml), and
harvested for subsequent plasmid preparation. Plasmid constructs
were isolated using Qiagen Qiaprep Spin Miniprep Kit. Plasmid
preparations of interest were sequenced (Quintara; Albany, Calif.)
using primers EL-1100, EL-1101, EL-1102, EL-1103, and EL-1104 and
the correct pEWL454 clone identified, which has been designated as
strain EWL454 (TOP10 w/pEWL454; pK184-derived cloning vector
harboring Ptac-RBS-NdeI-SacI-MluI-NotI-XhoI-PstI-aspA terminator).
A picture illustrating pEWL454 is shown in FIG. 87.
[0815] Water-washed REM I1.sub.--9 cells were transformed with the
ligation reaction via electroporation using the BIO RAD Gene Pulser
system (0.1 cm cuvette cat.#165-2089) and a transformation protocol
suggested by the manufacturer (BIO RAD). The cells were recovered
in L broth plus 500 uM mevalonic acid (available commercially, for
example, Sigma-Aldrich) for 1 hour at 37.degree. C. and then plated
on L agar containing kanamycin (50 ug/ml). Kanamycin resistant
colonies that grew in the absence of mevalonic acid were selected,
grown overnight in L broth containing kanamycin (50 ug/ml), and
harvested for subsequent plasmid preparation; the presence of the
Anabaena ispH allele relieved the cell's dependence on mevalonic
acid for growth. Plasmid constructs were isolated using Qiagen
Qiaprep Spin Miniprep Kit. Plasmid preparations of interest were
sequenced (Sequetech; Mountain View, Calif.) using primers EL-1105
and Neo.21 and the correct Ptac Anabaena ispH aspA term/pEWL454
clone identified, which has been designated as strain REM
F5.sub.--12 (REM I1.sub.--9 w/Ptac Anabaena ispH aspA term/pEWL454;
5' BamHI-3' PstI synthetic fragment into 5' BamHI-3' PstI of
pEWL454). A picture of the resulting Ptac Anabaena ispH aspA
term/pEWL454 construct is shown in FIG. 88.
B. Strategy for Creating REM I7.sub.--11 and REM H8.sub.--12
[0816] REM I7.sub.--11 was constructed by transformation of GI1.6
fldA-ispG/pCL into WW119. The transformation was performed by
electroporation using a BIO RAD Gene Pulser system (0.1 cm cuvette
cat.#165-2089) and a transformation protocol suggested by the
manufacturer (BIO RAD). A plasmid preparation of GI1.6
fldA-ispG/pCL, generated from strain REM D9.sub.--11, was used;
this strain and corresponding plasmid construct are described
infra.
[0817] REM H8.sub.--12 was constructed by transformation of Ptac
Anabaena ispH aspA term/pEWL454. The transformation was performed
by electroporation using a BIO RAD Gene Pulser system (0.1 cm
cuvette cat.#165-2089) and a transformation protocol suggested by
the manufacturer (BIO RAD). A plasmid preparation of Ptac Anabaena
ispH aspA term/pEWL454 was made from strain REM F5.sub.--12.
Transformation of GI1.6 fldA-ispG/pCL into WW119 and Ptac Anabaena
ispH aspA Term/pEWL454 into REM I7.sub.--11
[0818] To build the isoprene producing parental strain, REM
I7.sub.--11, from which the test strain REM H8.sub.--12 is derived,
the GI1.6 fldA-ispG/pCL plasmid was transformed by electroporation
into WW119. Transformants were recovered in L broth and plated on L
agar containing spectinomycin (50 ug/ml) and carbenicillin (50
ug/ml). The resulting strain was designated REM I7.sub.--11.
[0819] REM I7.sub.--11 was then transformed by electroporation with
Ptac Anabaena ispH aspA term/pEWL454. Transformants were recovered
in L broth and plated on L agar containing spectinomycin (50
ug/ml), kanamycin (50 ug/ml), and carbenicillin (50 ug/ml). The
resulting strain was designated REM H8.sub.--12.
C. Analysis of Test Strain REM H8.sub.--12 and the Parental Strain
REM I7.sub.--11 for Growth, Isoprene Production, and DXP Metabolite
Accumulation at Small Scale.
[0820] The parental strain REM I7.sub.--11 was compared against the
test strain REM H8.sub.--12 in a shake flask assay as well as in a
DXP metabolite determination study. The increased benefit on
isoprene production of the REM H8.sub.--12 strain harboring the
Ptac Anabaena ispH aspA term/pEWL454 construct over the parental
control strain REM 17.sub.--11 is depicted in FIG. 89. The
increased IspH activity present in the REM H8.sub.--12 strain
compared to the parent strain REM I7.sub.--11 is reflected by the
averaged 10-fold decrease in HDMAPP across the 3 hour and 3.75 hour
time points (FIG. 89). This elevated IspH activity provided by
expression of the Anabaena sp. PCC7120 ispH allele permitted a 2.1
to 3.2-fold increase in isoprene production from the REM
H8.sub.--12 test strain over the parental control (FIG. 89). The
REM H8.sub.--12 test strain also grew moderately better (approx.
20% faster) than the parental strain REM I7.sub.--11.
Growth
[0821] Strains REM I7.sub.--11 and REM H8.sub.--12 were grown at
30.degree. C. in 2-5 ml cultures of TM3 liquid media (13.6 g
K.sub.2PO.sub.4, 13.6 g KH.sub.2PO.sub.4, 2.0 g
MgSO.sub.4*7H.sub.2O), 2.0 g citric acid monohydrate, 0.3 g ferric
ammonium citrate, 3.2 g (NH.sub.4).sub.2SO.sub.4, 0.2 g yeast
extract, 1.0 ml 1000.times. Modified Trace Metal Solution, adjusted
to pH 6.8 and q.s. to H.sub.2O, and filter sterilized) supplemented
to a final concentration with 0.1% yeast extract and 1.0% glucose
and including spectinomycin (50 ug/ml) and carbenicillin (50
ug/ml).). Induction of LacI-regulated gene expression was achieved
by adding isopropyl-beta-D-1-thiogalactopyranoside (IPTG) to a
concentration of 500 uM. Growth was monitored periodically by
recording each of the culture's optical density measured at 600 nm
using an Eppendorf Biophotometer spectrometer (Eppendorf).
Isoprene Production
[0822] Isoprene production was analyzed using a headspace assay.
For the shake flask cultures, 200 ul of a culture was transferred
from shake flasks to 2 ml CTC headspace vials (SUN-SRI 2 mL HS
vials, VWR#66020-950, and caps, VWR#66008-170). The cap was screwed
on tightly and the vials incubated at the equivalent temperature
with shaking at 250 rpm. After 30 minutes the vials were removed
from the incubator and analyzed. The analysis was performed using
an Agilent 6890 GC/MS system interfaced with a CTC Analytics
(Switzerland) CombiPAL autosampler operating in headspace mode. An
Agilent HP-5MS GC/MS column (15 m.times.0.25 mm; 0.25 .mu.m film
thickness) was used for separation of analytes. The sampler was set
up to inject 100 .mu.L of headspace gas. The GC/MS method utilized
helium as the carrier gas at a flow of 1 ml/minutes The injection
port was held at 250.degree. C. with a split ratio of 50:1. The
oven temperature was held at 37.degree. C. for 0.6 minute, the
duration of the analysis. The Agilent 5793N mass selective detector
was run in single ion monitoring (SIM) mode on m/z 67. The detector
was switched off from 0 to 0.42 minutes to allow the elution of
permanent gases. Under these conditions isoprene
(2-methyl-1,3-butadiene) was observed to elute at approx. 0.49
minutes. A calibration table was used to quantify the absolute
amount of isoprene and was found to be linear from 1 .mu.g/L to
5000 .mu.g/L. The limit of detection was estimated to be 50 to 100
ng/L using this method. The specific productivity of each strain is
reported as ug/L OD Hr. Note, ratio of 1900 ul headspace:100 ul
broth in assay vials for 30 min. incubation results in the
following conversion of isoprene ug/L of culture to specific
productivity: (isoprene/L determined by GC-MS).times.(38)/(OD 600
nm of the culture).
DXP Metabolite Accumulation
[0823] The DXP metabolites of the isoprene-producing parental
strain REM I7.sub.--11 and test strain REM H8.sub.--12 that are
described above and depicted in FIG. 89 were isolated and
quantified as follows:
Metabolite Extraction: Processing Samples from Small-Scale
Experiments.
[0824] To measure accumulation of metabolites in small-scale
experiments 0.4 to 1.5 mL of cell culture was centrifuged for 3 min
at 7500.times.g, at -9.degree. C. Immediately after centrifugation
the supernatant was aspirated to a clean tube for analysis of
excreted metabolites and 100 .mu.L of dry ice-cold methanol was
added to pelleted cells. The resulting samples were then stored at
-80.degree. C. until further processing.
[0825] To determine concentrations of excreted metabolites, 500
.mu.L of methanol was added to 300 .mu.L of the supernatant and the
resulting mixture was centrifuged for 10 min at 20000.times.g at
4.degree. C. to remove insoluble material before the LCMS
analysis.
[0826] For metabolites extraction from the pellet (further referred
as intracellular metabolites), 10 .mu.L of water was added to
methanol-containing samples, the pellet was resuspended in the
resulting methanol/water mix and cell debris were spun down by
4-min centrifugation at 4500.times.g. The pellet was re-extracted
two more times, first with 100 .mu.L of 75% methanol buffered with
1 mM ammonium acetate in water (pH=8.0), then with 90 .mu.L of 50%
methanol in the ammonium acetate buffer. After each extraction,
cell debris was pelleted by centrifugation and the supernatants
from all three extractions were combined and analyzed by LCMS.
During the extraction procedure, samples were kept on ice or in a
refrigerated centrifuge whenever possible to minimize metabolites
degradation.
Metabolite Quantitation
[0827] Extracted metabolites were analyzed by LC-ESI-MS/MS on a
Quantum triple quadrupole mass spectrometer (Thermo Electron
Corporation, San Jose, Calif.). The system control, data
acquisition, and mass spectral data evaluation were performed using
XCalibur and LCQuan software (Thermo Electron Corp). LC separation
was done on a Synergi 45 .mu.M Hydro-RP HPLC column (150.times.2
mm, Phenomenex, USA) at a flow rate of 0.4 mL/min and the column
temperature of 40.degree. C. The LC gradient was t=0 min, 12% B;
t=5 min, 12% B; t=9 min, 23% B; t=20 min, 99% B; t=23 min, 99% B;
t=24 min, 12% B; t=29 min, 12% B, where solvent A was 10 mM
tributylamine/15 mM acetic acid in water and solvent B was
LCMS-grade methanol. The sample injection volume was 10 .mu.L.
[0828] Mass detection was carried out using electrospray ionization
in the negative mode. The following m/z values for precursor ions
were selected to detect the metabolites of interest in SRM mode:
213.0 for DXP, 215.0 for MEP, 245.0 for IPP and DMAPP, 260.0 for
HDMAPP, and 277.0 for cMEPP, 381.1 for FPP, 520.1 for CDP-ME, 600.0
for CDP-MEP. Concentrations of metabolites were determined based on
integrated intensities of peaks generated by PO.sub.3.sup.- product
ion (m/z=79.0) using calibration curves obtained by injection of
corresponding standards (Echelon Biosciences Inc). The
concentration of CDP-MEP was expressed in arbitrary units because
of the unavailability of commercial standard. Intracellular
concentrations of metabolites were calculated based on a standard
assumption that in 1 mL of the culture at OD=200 the integrated
volume of all cells is 50 .mu.L.
D. Analysis of Test Strain REM H8.sub.--12 for Growth, Isoprene
Production, and DXP Metabolite Accumulation at 14-L Fermentation
Scale.
[0829] REM_H8.sub.--12 produced 2.6 g/L isoprene in 14-L
fermentation (FIG. 14A). In addition to increased isoprene, the REM
H8.sub.--12 test strain maintained roughly 2-fold higher levels of
the MEP metabolite (product of DXR) and greater than 15-fold higher
levels of cMEPP (substrate for IspG) across the entire 14-L
fermentation than previously observed for the GI1.6 fldA-ispG/pCL
containing strain REM G4.sub.--11 (compare FIG. 80C to FIG.
90C).
E. Large Scale Fermentation of Strain REM H8.sub.--12
[0830] Isoprene production from E. coli expressing genes from the
DXP pathway and isoprene synthase, grown in fed-batch culture at
the 15-L scale.
1000.times. Modified Trace Metal Solution (Per Liter):
[0831] Citric Acids*H.sub.2O 40 g, MnSO4*H.sub.2O 30 g, NaCl 10 g,
FeSO4*7H2O 1 g, CoCl2*6H2O 1 g, ZnSO*7H2O 1 g, CuSO4*5H2O 100 mg,
H3BO3 100 mg, NaMoO4*2H2O 100 mg. Each component was dissolved one
at a time in Di H2O, pH was adjusted to 3.0 with HCl/NaOH, and then
the solution was q.s. to volume and filter sterilized with a 0.22
micron filter.
Mercury Vitamin Solution (Per Liter):
[0832] Thiamine hydrochloride 1.0 g, D-(+)-biotin 1.0 g, nicotinic
acid 1.0 g, D-pantothenic acid 4.8 g, pyridoxine hydrochloride 4.0
g. Each component was dissolved one at a time in Di H2O, pH was
adjusted to 3.0 with HCl/NaOH, and then the solution was q.s. to
volume and filter sterilized with 0.22 micron filter.
Feed Solution (Per Kilogram):
[0833] Glucose 0.57 kg, Di H.sub.2O 0.38 kg, K2HPO4 7.5 g, and 100%
Foamblast 10 g. All components were mixed together and autoclaved.
Macro Salt Solution 3.4 mL, 1000.times. Modified Trace Metal
Solution 0.8 ml, and Mercury Vitamin Solution 6.7 mL were added
after the solution had cooled to 25.degree. C.
Macro Salt Solution (Per Liter):
[0834] MgSO4*7H2O 296 g, citric acid monohydrate 296 g, ferric
ammonium citrate 49.6 g. All components were dissolved in water,
q.s. to volume and filter sterilized with 0.22 micron filter.
[0835] Fermentation was performed in a 15-L bioreactor with E. coli
BL21 cells overexpressing the first enzyme in the dxp pathway
(GI1.6-dxs), the last enzyme in the DXP pathway (GI1.6-yIDI), the
lower MVA pathway (PL.2-mKKDyI), various other genes from the DXP
pathway of T. elongates (Ptac-gcpE-lytB-petF-petH/pK184), the E.
coli ispG and fldA genes (GI1.6 fldA-ispG/pCL), and truncated
isoprene synthase from P. alba (pDW33) and containing a restored
17,257 bp chromosomal galM-containing region derived from MG1655
(strain name REM H8.sub.--12). This experiment was carried out to
monitor isoprene formation from glucose at the desired fermentation
pH 7.0 and temperature 34.degree. C. A frozen vial of the E. coli
strain was thawed and inoculated into tryptone-yeast extract medium
for the bioreactor. After the inoculum grew to optical density 1.0,
measured at 550 nm (OD.sub.550), 500 mL was used to inoculate a
15-L bioreactor and bring the initial tank volume to 5 L.
[0836] The feed solution was fed at an exponential rate until a top
feed rate of 5.8 g/min was reached. After this time, the glucose
feed was fed to meet metabolic demands at rates less than or equal
to 5.8 g/min. The total amount of glucose delivered to the
bioreactor during the 44 hr fermentation was 4.4 kg. Induction was
achieved by adding isopropyl-beta-D-1-thiogalactopyranoside (IPTG).
A single shot of IPTG was added to the tank to bring the
concentration to 300 uM when the cells were at an OD.sub.550 of
7.
[0837] The isoprene level in the off-gas from the bioreactors was
determined using a Hiden mass spectrometer. The isoprene titer
increased over the course of the fermentation to a maximum value of
2.6 g/L at 44 hr.
Equation for calculating Isoprene Titer: .intg.(Instantaneous
isoprene production rate, g/L/hr)dt from t=0 to 84 hrs [=] g/L
broth
F. DXP Metabolite Determination
Metabolite Extraction: Processing 14-L Fermentor Samples.
[0838] Cell metabolism was rapidly inactivated by withdrawing
several milliliters of the fermentor culture into a pre-weighted
tube filled with 9.0 mL of dry ice-cold methanol. The resulting
sample was weighted again to calculate the amount of withdrawn cell
culture and then put to -80.degree. C. for storage until further
analysis. In order to extract metabolites, 500 .mu.L of
methanol-quenched fermentation sample was spun down by
centrifugation for 4 min at 4500.times.g, at -9.degree. C. The
pellet was then re-extracted twice, first with 350 .mu.L of 85%
methanol buffered with 5 mM ammonium acetate in water (pH=7.0) and
then with 350 .mu.L of 50% methanol in the ammonium acetate buffer.
After each extraction, cell debris was pelleted by centrifugation
and all three supernatants were pooled together for further
analysis.
Metabolite Quantitation
[0839] Extracted metabolites were analyzed by LC-ESI-MS/MS on a
Quantum triple quadrupole mass spectrometer (Thermo Electron
Corporation, San Jose, Calif.). The system control, data
acquisition, and mass spectral data evaluation were performed using
XCalibur and LCQuan software (Thermo Electron Corp). LC separation
was done on a Synergi 45 .mu.M Hydro-RP HPLC column (150.times.2
mm, Phenomenex, USA) at a flow rate of 0.4 mL/min and the column
temperature of 40.degree. C. The LC gradient was t=0 min, 12% B;
t=5 min, 12% B; t=9 min, 23% B; t=20 min, 99% B; t=23 min, 99% B;
t=24 min, 12% B; t=29 min, 12% B, where solvent A was 10 mM
tributylamine/15 mM acetic acid in water and solvent B was
LCMS-grade methanol. The sample injection volume was 10 .mu.L.
[0840] Mass detection was carried out using electrospray ionization
in the negative mode. The following m/z values for precursor ions
were selected to detect the metabolites of interest in SRM mode:
213.0 for DXP, 215.0 for MEP, 245.0 for IPP and DMAPP, 260.0 for
HDMAPP, and 277.0 for cMEPP, 381.1 for FPP, 520.1 for CDP-ME, 600.0
for CDP-MEP. Concentrations of metabolites were determined based on
integrated intensities of peaks generated by PO.sub.3.sup.- product
ion (m/z=79.0) using calibration curves obtained by injection of
corresponding standards (Echelon Biosciences Inc). The
concentration of CDP-MEP was expressed in arbitrary units because
of the unavailability of commercial standard. Intracellular
concentrations of metabolites were calculated based on a standard
assumption that in 1 mL of the culture at OD=200 the integrated
volume of all cells is 50 .mu.L.
Example 30
Discovery of Apparent Biochemical Feedback Inhibition of Dxr and
Alleviation of Negative Effects Thereof
[0841] We made the surprising observation that in a DXP strain
production of isoprene was shut off while cells were still in a
vigorous growth phase. In addition these cells also
accumulate1-deoxyxylulose-5-phosphate, the substrate for Dxr.
Without being bound by theory, one possible hypothesis to explain
this observation is that the pathway is subject to regulation
either at the genetic level or at the biochemical level. Jawaid et.
al., PLoS One, 4(12):e8288 (2009) reported that a fraction of Dxr
protein from Francisella tularensis was phosphorylated at ser177
when overexpressed in E. coli. This phosphorylation was presumed to
inactivate the protein based on the observation that the mutations
S177D and S177E led to inactive protein. We subsequently showed
that purified Dxr from E. coli is inactivated when incubated with
dimethylallyl diphosphate (DMAPP) or
1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate (HMBPP). Further, an
E. coli strain with a genetically modified deoxyxylulose phosphate
(DXP) pathway was shown to accumulate DMAPP and/or HMBPP to levels
higher than that observed in wild type. Without being bound by
theory, based on the result of in vitro inactivation of Dxr and in
vivo metabolite accumulation observed in the engineered DXP pathway
strain, we postulate that the shut down of the pathway and the
accumulation of 1-deoxyxylulose-5-phosphate is due to the in vivo
inactivation of Dxr in the engineered strain. We discovered that
shut down of the pathway in engineered strains is prevented by
rebalancing pathway enzymes and maintaining levels of HDMAPP and
DMAPP at concentrations below 1 to 2 mM DMAPP and 1 to 2 mM HDMAPP.
These observations are exemplified in FIG. 90. FIG. 90A shows the
isoprene production for strain REM H8.sub.--12, a strain with an
improved DXP pathway as judged by sustained isoprene production and
reaching a titer of 2.6 g/L, compared to REMG4.sub.--11 a less well
balanced DXP pathway strain. Growth for REM H8.sub.--12 is shown in
panel B of FIG. 90, while the growth of REMG4.sub.--11 is shown in
FIG. 79C (grey triangles). Corresponding metabolite levels for REM
H8.sub.--12 are shown in FIG. 90C. By 8 hours the HDMAPP levels are
below 1 to 2 mM and isoprene production is maintained for a period
of 30 hours or more (FIG. 90A open squares). In comparison FIG. 80C
shows the metabolite levels for REM G4.sub.--11. The HDMAPP levels
are significantly above 1 to 2 mM for a period of 10-12 hours and
isoprene production is maintained only for about 10 to 15 hours, 15
to 20 hours short of expectation (FIG. 90A open circles). The final
titer of this strain was 0.98 g/L.
A. Methods
Strains Description
[0842] REM I7.sub.--11--This strain arose from the modification of
CMP271 detailed infra. CMP271 was transduced with P1 lysate MCM754,
obtained as described below, harboring a modified PL.6 promoter
(DNA seq.#1) replacing the native promoter in front of the dxs
gene.
FRT-neo-FRT PL.x(Trimmed) Integrated at dxs.gb DNA Seq.#1 Sequence
Includes Upstream FRT to and Including ATG of dxs
TABLE-US-00038 (SEQ ID NO: 130)
cgcgaagttcctattctctagaaagtataggaacttcattctaccgggtaggggaggcgcttttcccaaggcag-
tctggagcatgc
gctttagcagccccgctgggcacttggcgctacacaagtggcctctggcctcgcacacattccacatccaccgg-
taggcgccaa
ccggctccgttctttggtggccccttcgcgccaccttccactcctcccctagtcaggaagttcccccccgcccc-
gcagctcgcgt
cgtgcaggacgtgacaaatggaagtagcacgtctcactagtctcgtgcagatggacagcaccgctgagcaatgg-
aagcgggt
aggcctttggggcagcggccaatagcagctttgctccttcgctttctgggctcagaggctgggaaggggtgggt-
ccgggggcg
ggctcaggggcgggctcaggggcggggcgggcgcccgaaggtcctccggaggcccggcattctgcacgcttcaa-
aagcgc
acgtctgccgcgctgttctcctcttcctcatctccgggcctttcgacctgcagcagcacgtgttgacaattaat-
catcggcatagtat
atcggcatagtataatacgacaaggtgaggaactaaaccatgggatcggccattgaacaagatggattgcacgc-
aggttctccg
gccgcttgggtggagaggctattcggctatgactgggcacaacagacgatcggctgctctgatgccgccgtgtt-
ccggctgtca
gcgcaggggcgcccggttctttttgtcaagaccgacctgtccggtgccctgaatgaactgcaggacgaggcagc-
gcggctatc
gtggctggccacgacgggcgttccttgcgcagctgtgctcgacgttgtcactgaagcgggaagggactggctgc-
tattgggcg
aagtgccggggcaggatctcctgtcatctcaccttgctcctgccgagaaagtatccatcatggctgatgcaatg-
cggcggctgca
tacgcttgatccggctacctgcccattcgaccaccaagcgaaacatcgcatcgagcgagcacgtactcggatgg-
aagccggtct
tgtcgatcaggatgatctggacgaagagcatcaggggctcgcgccagccgaactgttcgccaggctcaaggcgc-
gcatgccc
gacggcgaggatctcgtcgtgacccatggcgatgcctgcttgccgaatatcatggtggaaaatggccgcttttc-
tggattcatcga
ctgtggccggctgggtgtggcggaccgctatcaggacatagcgttggctacccgtgatattgctgaagagcttg-
gcggcgaatg
ggctgaccgcttcctcgtgctttacggtatcgccgctcccgattcgcagcgcatcgccttctatcgccttcttg-
acgagttcttctga
gcgggactctggggttcgaataaagaccgaccaagcgacgtctgagagctccctggcgaattcggtaccaataa-
aagagcttta
ttttcatgatctgtgtgttggtttttgtgtgcggcgcggaagttcctattctctagaaagtataggaacttcct-
cgagccctatagtgag
tcgtattaagataaccatctgcggtgataaattatctctggcggtgttgacntaaataccactggcggtgatac-
tgagcacatcagc aggacgcactgcaaaggaggtaaaaaaacatg
[0843] Looping out the associated antibiotic marker according to
Gene Bridges instructions yielded strain WW102. This strain was
additionally transduced with P1 lysate MCM755 harboring a promoter
named gi1.6 (DNA seq.#2).
FRT-neo-FRT-gi1.x-d.times.r Region BL21.gb DNA seq#2; Sequence
Includes Upstream FRT to and Including ATG of dxr
TABLE-US-00039 (SEQ ID NO: 129)
actaaagggcggccgcgaagttcctattctctagaaagtataggaacttc
attctaccgggtaggggaggcgcttttcccaaggcagtctggagcatgcg
ctttagcagccccgctgggcacttggcgctacacaagtggcctctggcct
cgcacacattccacatccaccggtaggcgccaaccggctccgttctttgg
tggccccttcgcgccaccttccactcctcccctagtcaggaagttccccc
ccgccccgcagctcgcgtcgtgcaggacgtgacaaatggaagtagcacgt
ctcactagtctcgtgcagatggacagcaccgctgagcaatggaagegggt
aggcctttggggcageggccaatagcagetttgctecttcgctttctggg
ctcagaggctgggaaggggtgggtccgggggcgggctcaggggcgggctc
aggggcggggcgggcgcccgaaggtcctccggaggcccggcattctgcac
gcttcaaaagcgcacgtctgccgcgctgttctcctcttcctcatctccgg
gcctttcgacctgcagcagcacgtgttgacaattaatcatcggcatagta
tatcggcatagtataatacgacaaggtgaggaactaaaccatgggatcgg
ccattgaacaagatggattgcacgcaggttctccggccgcttgggtggag
aggctattcggctatgactgggcacaacagacgatcggctgctctgatgc
cgccgtgttccggctgtcagcgcaggggcgcccggttctttttgtcaaga
ccgacctgtccggtgccctgaatgaactgcaggacgaggcagcgcggcta
tcgtggctggccacgacgggcgttccttgcgcagctgtgctcgacgttgt
cactgaagcgggaagggactggctgctattgggcgaagtgccggggcagg
atctcctgtcatctcaccttgctcctgccgagaaagtatccatcatggct
gatgcaatgcggcggctgcatacgcttgatccggctacctgcccattcga
ccaccaagcgaaacatcgcatcgagcgagcacgtactcggatggaagccg
gtcttgtcgatcaggatgatctggacgaagagcatcaggggctcgcgcca
gccgaactgttcgccaggctcaaggcgcgcatgcccgacggcgaggatct
cgtcgtgacccatggcgatgcctgcttgccgaatatcatggtggaaaatg
gccgcttttctggattcatcgactgtggccggctgggtgtggcggaccgc
tatcaggacatagcgttggctacccgtgatattgctgaagagcttggcgg
cgaatgggctgaccgcttcctcgtgctttacggtatcgccgctcccgatt
cgcagcgcatcgccttctatcgccttcttgacgagttcttctgagcggga
ctctggggttcgaataaagaccgaccaagcgacgtctgagagctccctgg
cgaattcggtaccaataaaagagctttattttcatgatctgtgtgttggt
ttttgtgtgcggcgcggaagttcctattctctagaaagtataggaacttc
ctcgagccctatagtgagtcgtattagccettgacnatgccacatcctga
gcaaataattcaaccacttttattcactaacaaatagctggtggaatata tg
[0844] This promoter was targeted to replace the native promoter of
the dxr gene. Looping out the antibiotic marker according to Gene
Bridges instructions yielded strain WW103. Strain WW103 was
transformed by electroporation with plasmid pDW33 (Example 24 Part
C) providing ispS, the isoprene synthase expression cassette and
the resultant strain is designated WW119.
B. Detailed Strain Construction Protocols
Construction of Strain CMP271
Construction of Strain
[0845] Construction of P1 lysates MCM754 and MCM755 are detailed
below:
TABLE-US-00040 Primers (provided by Integrated DNA Technologies;
Coralville, Iowa USA) MCM320 (SEQ ID NO: 128)
5'-tcgatacctcggcactggaagcgctagcggactacatcatccagcgt
aataaataaacaataagtattaataggcccctgaattaaccctcactaaa gggcgg MCM321
(SEQ ID NO: 127) 5'-tgttcgggattatggcgcaccacgtccagcgtgctgcaaccaatcga
gccggtcgagcccagaatggtgagttgcttcatatattccaccagctatt
tgttagtgaataaaagtggttgaattatttgctcaggatgtggcatNgtc
aagggctaatacgactcactatagggctcg MCM337 (SEQ ID NO: 126)
5'-acaaaaacgccgctcagtagatccttgcggatcggctggcggcgttt
tgctttttattctgtctcaactctggatgtttcaattaaccctcactaaa gggcgg MCM347
(SEQ ID NO: 125) 5'-aacagtcgtaactcctgggtggagtcgaccagtgccagggtcgggta
tttggcaatatcaaaactcatgtttttttacctcctttgcagtgcgtcct
gctgatgtgctcagtatcaccgccagtggtatttaNgtcaacaccgcca
gagataatttatcaccgcagatggttatcttaatacgactcactataggg ctcg MCM327 (SEQ
ID NO: 59) 5'-ttgtagacatagtgcagcgcca MCM330 (SEQ ID NO: 124)
5'-ccctgttgctgtagcatcgttt GB-DW (SEQ ID NO: 60)
5'-aaagaccgaccaagcgacgtctga
C. Creation of Amplicon for Promoter Integration
[0846] PL.6(trim)-dxs
[0847] PCR reactions were carried out in quadruplicate using the
Herculase II Fusion Kit (Stratagene).
35 uL ddH.sub.2O 10 uL 5.times. buffer 1.25 uL 10 uM primer MCM320,
(gel purified) 1.25 uL 10 uM primer MCM347, (gel purified) 0.5 uL
dNTPs 1 uL polymerase 1 uL FRT-PGK-gb2-neo-FRT template DNA,
GeneBridges Cat. No. K006
[0848] Reactions were cycled as follows:
95 C.times.2 min followed by (95 C.times.15 sec; 55 C.times.15 sec;
72 C.times.1 min).times.30 cycles 72 C.times.3 min 30 sec 4 C until
cold. gi1.6-dxr
[0849] Four PCR reactions were carried out in using the Herculase
II Fusion Kit (Stratagene). Reactions varied by the presence or
absence of 2 uL DMSO and an annealing temperature of 55 C or 60
C.
35 uL ddH.sub.2O 10 uL 5.times. buffer 1.25 uL 10 uM primer MCM321,
IDT (gel purified) 1.25 uL 10 uM primer MCM337, IDT (gel purified)
0.5 uL dNTPs 1 uL polymerase 1 uL FRT-PGK-gb2-neo-FRT template DNA,
GeneBridges Cat. No. K006
+/-2 uL DMSO
[0850] Reactions were cycled as follows:
95 C.times.2 min followed by (95 C.times.20 sec; 55 C or 60
C.times.20 sec; 72 C.times.1 min).times.30 cycles 72 C.times.3 min;
4 C until
[0851] For each amplicon, four reactions were pooled and purified
using a QIAquick PCR Purification kit (Qiagen) PCR column, eluting
in 30 uL EB.
D. Integration of Amplicon onto Chromosome
[0852] Strain MCM327 (BL21) carrying pRedET-carb (GeneBridges) was
grown in L broth (LB) containing carbenicillin (50 ug/ml) at 30 C
overnight and then diluted 1:100 into fresh LB+carb50 and cultured
at 30 C for 2 hr. 130 uL of 10% arabinose was added and cells
cultured at 37 C for approximately 2 hours. Cells were prepared for
electroporation by washing 3.times. in one half culture volume iced
ddH.sub.2O and resuspended in one tenth culture volume of the same.
100 uL of cell suspension was combined with 3 uL DNA amplicon in a
2 mm electroporation cuvette, electroporated at 25 uFD, 200 ohms,
2.5 kV, (Gene Pulser MXcell; BioRad) and immediately quenched with
500 uL LB. Cells were recovered shaking at 37 C for 1-3 hrs and
then transformants selected overnight on L agar (LA) plates
containing kanamycin (10 ug/ml) at 37 C.
[0853] Single colonies arising from transformations with each DNA
amplicon were patched to LA+kan50 and grown overnight at 37 C.
Clones were inoculated into 5 mL LB+kan10, grown to an
OD.sub.600.about.1 and then frozen by mixing 1 mL 50% glycerol and
0.5 mL culture, placing on dry ice until solid, and then storing at
-80 C. These manipulations resulted in strain MCM754 [PL.6(trim)
dxs] and strain MCM755 (gi1.6 dxr).
[0854] The integrated promoters were amplified for sequencing by
colony PCR using the Herculase II Fusion kit (Stratagene).
35 uL ddH.sub.2O 10 uL 5.times. buffer 1.25 uL 10 uM primers GB-DW
1.25 uL 10 uM primer MCM327 (dxs) or MCM330 (dxr) 0.5 uL dNTPs 1 uL
polymerase Colony scraping
[0855] Reactions were cycled as follows:
95 C for 2 min; (95 C for 20 sec; 55 C for 20 sec; 72 C for 30
sec).times.30 cycles; 72 C 3 min; 4 C until cold
[0856] PCR products were sequenced (Quintara Biosciences) following
treatment by ExoSAP.
TABLE-US-00041 P1 lysate MCM754, containing PL.6-dxs, was sequenced
with primers GB-DW and MCM327 (SEQ ID NO: 123)
5'-Aaagaccgaccaagcgacgtctgagagctccctggcgaattcggtac
caataaaagagctttattttcatgatctgtgtgttggtttttgtgtgcgg
cgcggaagttcctattctctagaaagtataggaacttcctcgagccctat
agtgagtcgtattaagataaccatctgcggtgataaattatctctggcgg
tgttgacataaataccactggcggtgatactgagcacatcagcaggacgc
actgcaaaggaggtaaaaaaacatgagttttgatattgccaaatacccga
ccctggcactggtcgactccacccaggagttacgactgtt P1 lysate MCM755,
containing gi1.6-dxr, sequenced with primers GB-DW and MCM330 (SEQ
ID NO: 122) 5'-aaagaccgaccaagcgacgtctgagagctccctggcgaattcggtac
caataaaagagctttattttcatgatctgtgtgttggtttttgtgtgcgg
cgcggaagttcctattctctagaaagtataggaacttcctcgagccctat
agtgagtcgtattagcccttgacaatgccacatcctgagcaaataattca
accacttttattcactaacaaatagctggtggaatatatgaagcaactca
ccattctgggctcgaccggctcgattggttgcagcacgctggacgtggtg
cgccataatcccgaacacttccgcgtagttgcgctggtggcaggcaaaaa
tgtcactcgcatggtagaacagtgcctggaattctctccccgctatgccg
taatggacgatgaagcgagtgcgaaacttcttaaaacgatgctacagcaa caggg
E. Preparation of P1 Lysates from Strains MCM754 and MCM755.
[0857] 100 uL of respective overnight cultures (LB+kan 10) were
diluted into 10 mL LB+0.2% glucose+5 mM CaCl2, and grown with
shaking at 250 rpm, 37 C. After 30 min., 100 uL of a generic P1
lysate from MG1655 was added and the culture returned to the shaker
for .about.3 hours. The lysed culture was transferred to a 15 mL
tube, 200 uL chloroform added, and it was vortexed for 30 sec. The
sample was centrifuged at 4500 g for 10 min and then the aqueous
supernatant transferred to a fresh 15 mL tube. 200 uL chloroform
was added and the lysate stored at 4 C.
F. Cloning and Purification of the Enzyme.
[0858] Dxr from E. coli was cloned and purified by methods well
known to those of skill in the art. The gene was inserted into the
pET15b vector as described by the vendor to include a N-terminal
His tag sequence (Invitrogen, Carlsbad, Calif.). A BL21(.lamda.DE3)
E. coli culture harboring the plasmid and expressing the protein
was harvested, the cell pellet lysed in a French pressure cell and
protein was purified using a Ni-NTA column following the protocol
recommended by the manufacturer (GE Healthcare, Pittsburgh,
Pa.).
G. Dxr Inactivation by Incubation with DMAPP and HDMAPP.
[0859] The purified protein, 5 uM, was incubated at several
concentration of DMAPP or HMBPP (Echelon Bioscience, Salt Lake
City, Utah) in buffer consisting of 100 mM Tris, 100 mM NaCl pH 8,
5 mM MgCl.sub.2, 0.2 mM NADPH, 0.2 mM DXP, and 250 nM DXR. D at
37.degree. C. for two hour in a total volume of 50 uL. Dxr activity
was measured periodically according to standard assay, see, e.g.,
Koppisch et al, Biochemistry, 41:236-43 (2002) with a 20-fold
dilution of the inactivation reaction mixture. Control incubations
and assays of the enzyme were conducted under similar conditions in
the absence of DMAPP or HMBPP in the inactivation reaction. Where
appropriate additional control activity assays were conducted in
the presence of a 20-fold diluted concentration of inactivators
(DMAPP or HMBPP). A larger aliquot of enzyme (about 400 ug) was
inactivated similarly with DMAPP for analysis by mass spectroscopy
to verify the anticipated amino acid residue modification. As shown
in FIG. 92 enzyme activity declined during the inactivation
incubation and yielding an inactivation half-life of 0.72
hours.
Example 31
Co-Expression of DXP and MVA Pathways for the Production of
Isoprene in E. coli
[0860] Comparison of the energetics and carbon utilization
efficiency for the DXP pathway and the MVA pathway reveal that the
DXP pathway is more efficient in carbon utilization but less
efficient in redox balance than the MVA pathway. When glucose is
the carbon source stoichiometric yield on carbon of the DXP pathway
is about 85% (grams of isoprene produced per grams of glucose
utilized). The energy balance of the DXP pathway is less efficient
when compared to the MVA pathway. For DXP glucose to isoprene
suffers a shortage of 3 moles of NAD(P)H per mole of isoprene
formed and is minus 2 moles of ATP. For the similar comparison of
glucose to isoprene via MVA this pathway produces an excess of 4
moles of NAD(P)H; ATP is balanced, however, the carbon utilization
efficiency is only about 55%. Without being bound by theory, a more
balanced and more efficient production host can be made by
combining the two pathways in a single host to optimize redox
chemistry and efficiency of carbon utilization.
[0861] In this example, we provide evidence consistent with that
combination of the two pathways in a single host can be established
in practice. Combination of the two pathways should lead to an
improved process. A series of cultures comprising two strains, REM
H8.sub.--12 and REM I7.sub.--11, described above, were set up in a
48-deep-well plate (cat# P-5ML-48-C-S Axygen Scientific,
California, USA) with each well providing a 2 mL culture. The
media, named TM3, is described below. The two strains were grown
overnight at 30 degrees Celsius at 250 rpm in TM3 medium
supplemented with 1% glucose and 0.1% yeast extract. In the
morning, the two strains were inoculated into the 48-deep well
block in replicate. The TM3 medium was supplemented with 1%
[U-.sup.13C]-glucose and 0.1% yeast extract. The cultures were
shaken at 30 degrees C. at 600 rpm (Shel-Lab Inc. Model SI6R
Refrigerated Shaking Incubator; Oregon, USA). Culture OD was
determined after two hours and then at timed intervals out to 4.25
hours. The cultures were induced at two hours of growth by the
addition of 400 uM IPTG. After one hour of induction the cultures
of each strain also received from 0 to 8 mM (R)-mevalonic acid
[cat#; Sigma M4667]. At timed intervals a 100 uL aliquot of each
culture was transferred to a 98-deep well glass block (cat#3600600
Zinsser; North America) which was immediately sealed with an
impermeable adhesive aluminum film and incubated for 30 minutes
with shaking at 450 rmp on an Eppendorf thermomixer (Eppendorf;
North America.). The cultures were killed by heating at 70 degrees
C. for 7 minutes on a second Eppendorf thermomixer. The glass block
was transferred to an Agilent 6890 GC attached to an Agilent 5973
MS and outfitted with a LEAP CTC CombiPAL autosampler for head
space analysis. The column was an Agilent HP-5 (5% Phenyl Methyl
Siloxane (15 m.times.0.25 mm.times.0.25 um)). A 100 uL gas volume
was injected on the column. Other conditions were as follows. Oven
Temperature: 37 C (held isothermal for 0.6 mins); Carrier Gas:
Helium (flow -1 mL/min), split ratio of 50:1 at 250.degree. C. on
the injection port; Single Ion Monitoring mode (SIM) on mass 67 or
73; Detector off: 0.00 min-0.42 mins; Dectector on: 0.42 mins-0.60
mins; elution time for Isoprene (2-methyl-1,3 butadiene) was
.about.0.49 min for a total analysis time of 0.6 mins. Calibration
of the instrument was performed by methods well known to those of
skill in the art.
[0862] Isoprene head space measurements were normalized by culture
OD.sub.600 to yield a measure of specific isoprene production in
units of ug/L/H/OD. All reactions were followed for 4 hours. FIGS.
92A and B show the results for this experiment. Isoprene is
simultaneously produced from [U-.sup.13C]-glucose (FIG. 92 panel B)
as well as from mevalonic acid (FIG. 92 panel A). The data indicate
that the isoprene produced from [U-.sup.13C]-glucose by the two
strains is independent of isoprene produced by mevalonate. Panel B
of FIG. 92 further shows that the specific productivity of isoprene
from [U-.sup.13C]-glucose is the same for both strains at
mevalonate concentrations ranging from 0 to 8 mM. These
measurements were made at m/z of 73 indicative of
[U-.sup.13C]-glucose utilization. At the same time, the isoprene
specific productivity increased with increasing mevalonic acid
concentration over the same concentration range. This measurement
was made at m/z of 67 indicative of mevalonate (all .sup.12C)
utilization. The overall conclusion of this experiment is that
isoprene produced by the DXP pathway is not affected by isoprene
produced from mevalonic acid by the lower MVA pathway.
TM3 (Per Liter Fermentation Medium):
[0863] K.sub.2HPO.sub.4 13.6 g, KH.sub.2PO.sub.4 13.6 g,
MgSO.sub.4*7H.sub.2O 2 g, citric acid monohydrate 2 g, ferric
ammonium citrate 0.3 g, yeast extract 1.0 g, 1000.times. Modified
Trace Metal stock solution 1 ml. All of the components were added
together and dissolved in Di H2O. The pH is adjusted to 6.8 with
NH.sub.4OH and the solution is filter sterilized over a 0.22 micron
membrane. Antibiotics were added post-sterile as needed.
U-.sup.13C-Glucose and [R]-mevalonic acid were added post sterile
as indicated.
1000.times. Modified Trace Metal Stock Solution (Per Liter):
[0864] Citric Acids*H.sub.2O 40 g, MnSO.sub.4*H.sub.2O 30 g, NaCl
10 g, FeSO.sub.4*7H.sub.2O 1 g, CoCl.sub.2*6H.sub.2O 1 g,
ZnSO.sub.4*7H.sub.2O 1 g, CuSO.sub.4*5H.sub.2O 100 mg,
H.sub.3BO.sub.3 100 mg, NaMoO.sub.4*2H.sub.2O 100 mg. Each
component was dissolved one at a time in Di H.sub.2O, pH was
adjusted to 3.0 with HCl/NaOH, and then the solution was q.s. to
volume and filter sterilized with a 0.22 micron filter.
Example 32
Demonstration of Isoprene Generated by Strain REM A2.sub.--17 Via
Dual Isoprenoid Biosynthetic Pathways
[0865] Described here is the construction of an isoprene producing
E. coli strain that harbors both an exogenous MVA isoprenoid
biosynthetic pathway and an enhanced DXP biosynthetic pathway. Data
presented here indicates that isoprene produced by strain REM
A2.sub.--17 is derived from both types of isoprenoid biosynthetic
pathways simultaneously. For this particular example, roughly 3:2
to 1:1 MVA-flux:DXP-flux contributions to isoprene production were
observed; see FIG. 96-102.
Construction of Strain REM A2.sub.--17
[0866] The genomic insertions described in this example were
carried out using the Red/ET system from Gene Bridges GmbH
according to the manufacturer's instructions. The strain BL21
(Novagene) was used. P1 lysate preparations and transductions were
performed as previously described (Thomason et al., 2007). The
pBBR1MCS-5 vector has been described (Kovach et al., 1994) as have
vector constructs MCM82, pMCM296, pDW34, pDW33, GI1.6
fldA-ispG/pCL, and Ptac Anabaena ispH aspA term/pEWL454 (see, e.g.,
Example 29 above and WO 2009/076676). MCM82 contains the pCL
PtrcUpperPathway encoding E. faecalis mvaE and mvaS). The Trc
promoter, Trp promoter and aspA terminator sequences were obtained
from the information provided by NCBI www.ncbi.nlm.nih.gov and
EcoCyc www.ecocyc.org.
Construction of pDW15 (Ptrc-Upper MVA Pathway on pBBR1MCS-5)
[0867] To insert the upper MVA pathway onto the pBBR1MCS-5 vector,
the entire expression cassette containing Ptrc, mvaE, mvaS, and the
rrn terminator was amplified by PCR from MCM82 using the primers
Upper5'XhoI and Upper3'XbaI. See below for PCR primer sequences
(Table 9), reaction and cycling parameters. The approximately 4.2
kb PCR product was confirmed by gel electrophoresis (E-Gel,
Invitrogen) and then purified using QiaQuick purification columns
(Qiagen) according to the manufacturers recommended protocol.
Purified PCR product and the pBBR1MCS-5 vector were then treated
with XbaI and XhoI restriction endonucleases overnight at
37.degree. C. See below for reaction conditions. The next day,
reactions were heated to 65.degree. C. to deactivate restriction
enzymes prior to ligation. Ligation reactions (see below for
conditions) were carried out at 4.degree. C. overnight.
Approximately 5 .mu.l of the ligation reactions were transformed
into chemically competent E. coli TOP10 cells (Invitrogen)
according to the manufacturer's recommended protocol, recovered at
37.degree. C. in LB for 1 hour, and then plated onto LB plates
containing X-gal and Gentamicin at 10 .mu.g/ml. Colonies displaying
no .beta.-galactosidase activity were selected for further analysis
by PCR using primers M13 Reverse and MCM163 to confirm the presence
of the insert. The plasmid from one of these colonies was purified
(Qiagen) and completely sequenced (Quintara Biosciences, see Table
9 for primer sequences) to verify that it contained the complete
upper MVA pathway expression cassette in the correct orientation.
The sequence and map of pDW15 is listed below and in FIG. 93,
respectively.
PCR Reaction and Cycling Parameters:
[0868] 1 .mu.l MCM82 (approx. 30 ng) [0869] 10 .mu.l 5.times.
Herculase Buffer (Stratagene) [0870] 0.5 .mu.l dNTPs (100 mM)
[0871] 1 .mu.l Upper5'XhoI (20 uM) [0872] 1 .mu.l Upper3'XbaI (20
uM) [0873] 35.5 .mu.l diH2O [0874] 1 .mu.l Herculase DNA Polymerase
(Stratagene)
[0875] 1. 95.degree. C. 4 min.
[0876] 2. 95.degree. C. 20 min, 52.degree. C. 20 sec., 72.degree.
C. 4 min., 5.times.
[0877] 3. 95.degree. C. 20 min, 55.degree. C. 20 sec., 72.degree.
C. 4 min., 25.times.
[0878] 4. 72.degree. C. 10 min,
[0879] 5. 4.degree. C. until cool
DNA Digestion:
[0880] 6 .mu.l diH2O [0881] 2 .mu.l 10.times. Buffer H (Roche)
[0882] 10 .mu.l DNA (pBBR1MCS-5 or PCR insert) [0883] 1 .mu.l XhoI
(Roche) [0884] 1 ulXbaI (Roche)
[0885] 1. 37.degree. C. overnight
[0886] 2. 65.degree. C. 20 min (heat kill)
Ligation:
[0887] 2 .mu.l diH2O [0888] 1 .mu.l 10.times. ligase buffer (NEB)
[0889] 1 .mu.l T4 DNA ligase (NEB) [0890] 2 .mu.l vector
(pBBR1MCS-5) [0891] 4 .mu.l insert (upper MVA expression
cassette)
[0892] 1. 4.degree. C. overnight
[0893] 2. microdialyze (Millipore) and transform into competent E.
coli (Invitrogen)
TABLE-US-00042 TABLE 9 PCR and Sequencing Primers Upper5'XhoI
atgctcgagctgttgacaattaatcatccggctc (SEQ ID NO: 121) Upper3'XbaI
cgatctagaaaggcccagtctttcgactgagcc (SEQ ID NO: 120) MCM163 CF07-58
atgaaaacagtagttattattgatgc (SEQ ID NO: 119) CF07-59
cttaaatcatttaaaatagc (SEQ ID NO: 118) CF07-82
atgacaattgggattgataaaattag (SEQ ID NO: 117) CF07-86
gaaatagccccattagaagtatc (SEQ ID NO: 116) CF07-87
ttgccaatcatatgattgaaaatc (SEQ ID NO: 115) CF07-88
gctatgcttcattagatccttatcg (SEQ ID NO: 114) CF07-89
gaaacctacatccaatcttttgccc (SEQ ID NO: 113)
TABLE-US-00043 Sequence of pDW15 (SEQ ID NO: 176)
accttcgggagcgcctgaagcccgttctggacgccctggggccgttgaatcgggatatgcaggccaaggccgcc-
gcgatcat
caaggccgtgggcgaaaagctgctgacggaacagcgggaagtccagcgccagaaacaggcccagcgccagcagg-
aacgc
gggcgcgcacatttccccgaaaagtgccacctggcggcgttgtgacaatttaccgaacaactccgcggccggga-
agccgatct
cggcttgaacgaattgttaggtggcggtacttgggtcgatatcaaagtgcatcacttcttcccgtatgcccaac-
tttgtatagagag
ccactgcgggatcgtcaccgtaatctgcttgcacgtagatcacataagcaccaagcgcgttggcctcatgcttg-
aggagattgat
gagcgcggtggcaatgccctgcctccggtgctcgccggagactgcgagatcatagatatagatctcactacgcg-
gctgctcaa
acctgggcagaacgtaagccgcgagagcgccaacaaccgcttcttggtcgaaggcagcaagcgcgatgaatgtc-
ttactacg
gagcaagttcccgaggtaatcggagtccggctgatgttgggagtaggtggctacgtctccgaactcacgaccga-
aaagatcaa
gagcagcccgcatggatttgacttggtcagggccgagcctacatgtgcgaatgatgcccatacttgagccacct-
aactttgtttta
gggcgactgccctgctgcgtaacatcgttgctgctgcgtaacatcgttgctgctccataacatcaaacatcgac-
ccacggcgtaa
cgcgcttgctgcttggatgcccgaggcatagactgtacaaaaaaacagtcataacaagccatgaaaaccgccac-
tgcgccgtta
ccaccgctgcgttcggtcaaggttctggaccagttgcgtgagcgcatacgctacttgcattacagtttacgaac-
cgaacaggctta
tgtcaactgggttcgtgccttcatccgtttccacggtgtgcgtccatgggcaaatattatacgcaaggcgacaa-
ggtgctgatgcc
gctggcgattcaggttcatcatgccgtttgtgatggcttccatgtcggcagaatgcttaatgaattacaacagt-
ttttatgcatgcgcc
caatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactgg-
aaagcgggc
agtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggc-
tcgtatgttgtgtg
gaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgccaagcgcgcaattaaccct-
cactaaaggg
aacaaaagctgggtaccgggccccccctcgagctgttgacaattaatcatccggctcgtataatgtgtggaatt-
gtgagcggata
acaatttcacacaggaaacagcgccgctgagaaaaagcgaagcggcactgctctttaacaatttatcagacaat-
ctgtgtgggca
ctcgaccggaattatcgattaactttattattaaaaattaaagaggtatatattaatgtatcgattaaataagg-
aggaataaaccatgg
atccgagctcaggaggtaaaaaaacatgaaaacagtagttattattgatgcattacgaacaccaattggaaaat-
ataaaggcagct
taagtcaagtaagtgccgtagacttaggaacacatgttacaacacaacttttaaaaagacattccactatttct-
gaagaaattgatca
agtaatctttggaaatgttttacaagctggaaatggccaaaatcccgcacgacaaatagcaataaacagcggtt-
tgtctcatgaaat
tcccgcaatgacggttaatgaggtctgcggatcaggaatgaaggccgttattttggcgaaacaattgattcaat-
taggagaagcg
gaagttttaattgctggcgggattgagaatatgtcccaagcacctaaattacaacgttttaattacgaaacaga-
aagctacgatgcg
cctttttctagtatgatgtatgatggattaacggatgcctttagtggtcaggcaatgggcttaactgctgaaaa-
tgtggccgaaaagt
atcatgtaactagagaagagcaagatcaattttctgtacattcacaattaaaagcagctcaagcacaagcagaa-
gggatattcgct
gacgaaatagccccattagaagtatcaggaacgcttgtggagaaagatgaagggattcgccctaattcgagcgt-
tgagaagcta
ggaacgcttaaaacagtttttaaagaagacggtactgtaacagcagggaatgcatcaaccattaatgatggggc-
ttctgctttgatt
attgcttcacaagaatatgccgaagcacacggtcttccttatttagctattattcgagacagtgtggaagtcgg-
tattgatccagcct
atatgggaatttcgccgattaaagccattcaaaaactgttagcgcgcaatcaacttactacggaagaaattgat-
ctgtatgaaatca
acgaagcatttgcagcaacttcaatcgtggtccaaagagaactggctttaccagaggaaaaggtcaacatttat-
ggtggcggtatt
tcattaggtcatgcgattggtgccacaggtgctcgtttattaacgagtttaagttatcaattaaatcaaaaaga-
aaagaaatatggag
tggcttctttatgtatcggcggtggcttaggactcgctatgctactagagagacctcagcaaaaaaaaaacagc-
cgattttatcaaa
tgagtcctgaggaacgcctggcttctcttcttaatgaaggccagatttctgctgatacaaaaaaagaatttgaa-
aatacggctttatc
ttcgcagattgccaatcatatgattgaaaatcaaatcagtgaaacagaagtgccgatgggcgttggcttacatt-
taacagtggacg
aaactgattatttggtaccaatggcgacagaagagccctcagttattgcggctttgagtaatggtgcaaaaata-
gcacaaggattta
aaacagtgaatcaacaacgcttaatgcgtggacaaatcgttttttacgatgttgcagatcccgagtcattgatt-
gataaactacaagt
aagagaagcggaagtttttcaacaagcagagttaagttatccatctatcgttaaacggggcggcggcttaagag-
atttgcaatatc
gtacttttgatgaatcatttgtatctgtcgactttttagtagatgttaaggatgcaatgggggcaaatatcgtt-
aacgctatgttggaag
gtgtggccgagttgttccgtgaatggtttgcggagcaaaagattttattcagtattttaagtaattatgccacg-
gagtcggttgttacg
atgaaaacggctattccagtttcacgtttaagtaaggggagcaatggccgggaaattgctgaaaaaattgtttt-
agcttcacgctat
gcttcattagatccttatcgggcagtcacgcataacaaaggaatcatgaatggcattgaagctgtagttttagc-
tacaggaaatgat
acacgcgctgttagcgcttcttgtcatgcttttgcggtgaaggaaggtcgctaccaaggcttgactagttggac-
gctggatggcga
acaactaattggtgaaatttcagttccgcttgctttagccacggttggcggtgccacaaaagtcttacctaaat-
ctcaagcagctgct
gatttgttagcagtgacggatgcaaaagaactaagtcgagtagtagcggctgttggtttggcacaaaatttagc-
ggcgttacggg
ccttagtctctgaaggaattcaaaaaggacacatggctctacaagcacgttctttagcgatgacggtcggagct-
actggtaaaga
agttgaggcagtcgctcaacaattaaaacgtcaaaaaacgatgaaccaagaccgagccatggctattttaaatg-
atttaagaaaa
caataaaggaggtaaaaaaacatgacaattgggattgataaaattagtttttttgtgcccccttattatattga-
tatgacggcactggc
tgaagccagaaatgtagaccctggaaaatttcatattggtattgggcaagaccaaatggcggtgaacccaatca-
gccaagatatt
gtgacatttgcagccaatgccgcagaagcgatcttgaccaaagaagataaagaggccattgatatggtgattgt-
cgggactgagt
ccagtatcgatgagtcaaaagcggccgcagttgtcttacatcgtttaatggggattcaacctttcgctcgctct-
ttcgaaatcaagg
aagcttgttacggagcaacagcaggcttacagttagctaagaatcacgtagccttacatccagataaaaaagtc-
ttggtcgtagcg
gcagatattgcaaaatatggcttaaattctggcggtgagcctacacaaggagctggggcggttgcaatgttagt-
tgctagtgaacc
gcgcattttggctttaaaagaggataatgtgatgctgacgcaagatatctatgacttttggcgtccaacaggcc-
acccgtatcctat
ggtcgatggtcctttgtcaaacgaaacctacatccaatcttttgcccaagtctgggatgaacataaaaaacgaa-
ccggtcttgatttt
gcagattatgatgctttagcgttccatattccttacacaaaaatgggcaaaaaagccttattagcaaaaatctc-
cgaccaaactgaa
gcagaacaggaacgaattttagcccgttatgaagaaagtatcgtctatagtcgtcgcgtaggaaacttgtatac-
gggttcactttat
ctgggactcatttcccttttagaaaatgcaacgactttaaccgcaggcaatcaaattggtttattcagttatgg-
ttctggtgctgtcgct
gaatttttcactggtgaattagtagctggttatcaaaatcatttacaaaaagaaactcatttagcactgctgga-
taatcggacagaact
ttctatcgctgaatatgaagccatgtttgcagaaactttagacacagacattgatcaaacgttagaagatgaat-
taaaatatagtattt
ctgctattaataataccgttcgttcttatcgaaactaaagatctgcagctggtaccatatgggaattcgaagct-
tgggcccgaacaa
aaactcatctcagaagaggatctgaatagcgccgtcgaccatcatcatcatcatcattgagtttaaacggtctc-
cagcttggctgttt
tggcggatgagagaagattttcagcctgatacagattaaatcagaacgcagaagcggtctgataaaacagaatt-
tgcctggcgg
cagtagcgcggtggtcccacctgaccccatgccgaactcagaagtgaaacgccgtagcgccgatggtagtgtgg-
ggtctcccc
atgcgagagtagggaactgccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttctagagcg-
gccgccac
cgcggtggagctccaattcgccctatagtgagtcgtattacgcgcgctcactggccgtcgttttacaacgtcgt-
gactgggaaaa
ccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggccc-
gcaccgatcg
cccttcccaacagttgcgcagcctgaatggcgaatggaaattgtaagcgttaatattttgttaaaattcgcgtt-
aaatttttgttaaatc
agctcattttttaaccaataggccgactgcgatgagtggcagggcggggcgtaatttttttaaggcagttattg-
gtgcccttaaacg
cctggtgctacgcctgaataagtgataataagcggatgaatggcagaaattcgaaagcaaattcgacccggtcg-
tcggttcagg
gcagggtcgttaaatagccgcttatgtctattgctggtttaccggtttattgactaccggaagcagtgtgaccg-
tgtgcttctcaaat
gcctgaggccagtttgctcaggctctccccgtggaggtaataattgacgatatgatcatttattctgcctccca-
gagcctgataaaa
acggtgaatccgttagcgaggtgccgccggcttccattcaggtcgaggtggcccggctccatgcaccgcgacgc-
aacgcggg
gaggcagacaaggtatagggcggcgaggcggctacagccgatagtctggaacagcgcacttacgggttgctgcg-
caaccca
agtgctaccggcgcggcagcgtgacccgtgtcggcggctccaacggctcgccatcgtccagaaaacacggctca-
tcgggcat
cggcaggcgctgctgcccgcgccgttcccattcctccgtttcggtcaaggctggcaggtctggttccatgcccg-
gaatgccggg
ctggctgggcggctcctcgccggggccggtcggtagttgctgctcgcccggatacagggtcgggatgcggcgca-
ggtcgcc
atgccccaacagcgattcgtcctggtcgtcgtgatcaaccaccacggcggcactgaacaccgacaggcgcaact-
ggtcgcgg
ggctggccccacgccacgcggtcattgaccacgtaggccgacacggtgccggggccgttgagcttcacgacgga-
gatccag
cgctcggccaccaagtccttgactgcgtattggaccgtccgcaaagaacgtccgatgagcttggaaagtgtctt-
ctggctgacca
ccacggcgttctggtggcccatctgcgccacgaggtgatgcagcagcattgccgccgtgggtttcctcgcaata-
agcccggcc
cacgcctcatgcgctttgcgttccgtttgcacccagtgaccgggcttgttcttggcttgaatgccgatttctct-
ggactgcgtggcca
tgcttatctccatgcggtagggtgccgcacggttgcggcaccatgcgcaatcagctgcaacttttcggcagcgc-
gacaacaatta
tgcgttgcgtaaaagtggcagtcaattacagattttctttaacctacgcaatgagctattgcggggggtgccgc-
aatgagctgttgc
gtaccccccttttttaagttgttgatttttaagtctttcgcatttcgccctatatctagttctttggtgcccaa-
agaagggcacccctgcg
gggttcccccacgccttcggcgcggctccccctccggcaaaaagtggcccctccggggcttgttgatcgactgc-
gcggccttc
ggccttgcccaaggtggcgctgcccccttggaacccccgcactcgccgccgtgaggctcggggggcaggcgggc-
gggcttc
gccttcgactgcccccactcgcataggcttgggtcgttccaggcgcgtcaaggccaagccgctgcgcggtcgct-
gcgcgagc
cttgacccgccttccacttggtgtccaaccggcaagcgaagcgcgcaggccgcaggccggaggcttttccccag-
agaaaatta
aaaaaattgatggggcaaggccgcaggccgcgcagttggagccggtgggtatgtggtcgaaggctgggtagccg-
gtgggca
atccctgtggtcaagctcgtgggcaggcgcagcctgtccatcagcttgtccagcagggttgtccacgggccgag-
cgaagcgag
ccagccggtggccgctcgcggccatcgtccacatatccacgggctggcaagggagcgcagcgaccgcgcagggc-
gaagcc
cggagagcaagcccgtagggcgccgcagccgccgtaggcggtcacgactttgcgaagcaaagtctagtgagtat-
actcaagc
attgagtggcccgccggaggcaccgccttgcgctgcccccgtcgagccggttggacaccaaaagggaggggcag-
gcatgg
cggcatacgcgatcatgcgatgcaagaagctggcgaaaatgggcaacgtggcggccagtctcaagcacgcctac-
cgcgagc
gcgagacgcccaacgctgacgccagcaggacgccagagaacgagcactgggcggccagcagcaccgatgaagcg-
atgg
gccgactgcgcgagttgctgccagagaagcggcgcaaggacgctgtgttggcggtcgagtacgtcatgacggcc-
agcccgg
aatggtggaagtcggccagccaagaacagcaggcggcgttcttcgagaaggcgcacaagtggctggcggacaag-
tacggg
gcggatcgcatcgtgacggccagcatccaccgtgacgaaaccagcccgcacatgaccgcgttcgtggtgccgct-
gacgcag
gacggcaggctgtcggccaaggagttcatcggcaacaaagcgcagatgacccgcgaccagaccacgtttgcggc-
cgctgtg
gccgatctagggctgcaacggggcatcgagggcagcaaggcacgtcacacgcgcattcaggcgttctacgaggc-
cctggag
cggccaccagtgggccacgtcaccatcagcccgcaagcggtcgagccacgcgcctatgcaccgcagggattggc-
cgaaaa
gctgggaatctcaaagcgcgttgagacgccggaagccgtggccgaccggctgacaaaagcggttcggcaggggt-
atgagcc
tgccctacaggccgccgcaggagcgcgtgagatgcgcaagaaggccgatcaagcccaagagacggcccgag
Construction of PTrp mMVK/pDW15
TABLE-US-00044 Primers * primers were modified with 5'
phosphorylation *5' phos Ptrp 5' mMVK (SEQ ID NO: 177)
5'-TGGCAAATATTCTGAAATGAGCTGTTGACAATT
AATCATCGAACTAGTTAACTAGTACGCAAGTTCACGTAAAAAGGGTATCG
ACATGGTATCCTGTTCTGCGCCGGGTAAGA *3' phos aspA term 3' mMVK (SEQ ID
NO: 178) 5'-CAAGAAAAAAGGCACGTCATCTGACGTGCCTT TTTTATTTGT
ATTAATCTACTTTCAGACCTTGCTCGGTCGG 5' mMVK seq prim (SEQ ID NO: 179)
5'-GATACGTATGTTTCTACCTTC 3' mMVK seq prim (SEQ ID NO: 180)
5'-GAAGGTAGAAACATACGTATC EL1003 (SEQ ID NO: 181)
5'-GATAGTAACGGCTGCGCTGCTACC MCM 177 (SEQ ID NO: 182)
5'-GGGCCCGTTTAAACTTTAACTAGACTTTAATCTACTTTCAGACCTT GC
Amplification of the PTrp mMVK Fragment PCR Reaction for PTrp mMVK
0.5 ul vector template pDW34
10 ul HerculaseII Buffer
[0894] 0.5 ul dNTP's (100 mM) 1.25 ul primer (10 uM) 5' phos Ptrp
5' mMVK 1.25 ul primer (10 uM) 3' phos aspA term 3' mMVK 36 ul
diH2O +0.5 ul of HerculaseII fusion from Stratagene
Cycle Parameter:
[0895] 95.degree. C..times.2 min., [95.degree. C..times.30 sec.,
60.degree. C..times.30 sec., 72.degree. C..times.2 min.].times.29
cycles; 72.degree. C..times.5 min., 4.degree. C. until cool
(Biometra T3000 Combi Thermocycler)
[0896] The resulting PCR fragment was separated on a 0.8% E-gel
(Invitrogen) for verification of successful amplification, and
purified using the QIAquick PCR Purification kits (Qiagen)
according to manufacturer's instructions. The resulting purified
stock is referred to as PTrp mMVK; note the primers used contained
5' phosphorylated ends.
Cloning of the PTrp mMVK fragment into pDW15
[0897] Approximately 500 ng of the pDW15 plasmid was digested with
SfoI (New England Biolabs) according to the manufacturer's
specifications. The SfoI cut vector was then dephosphorylated using
rAPpid Alkaline Phosphatase (Roche) according to the manufacturer's
suggested protocol. The digested/dephosphorylated DNA was cleaned
using the Qiagen QiaQuick Gel Extraction Kit prior to ligation. A
portion of the PTrp mMVK fragment (5' ends phosphorylated) was
ligated to the cleaned/SfoI cut/dephosphorylated pDW15 plasmid
using T4 DNA Ligase from New England Biolabs according to the
manufacturer's suggested protocol. Chemically competent TOP10 cells
(Invitrogen) were transformed with the ligation reaction using a
standard heat-shock protocol (Sambrook et al., 1989), recovered in
L broth for 1 hour at 37.degree. C. and then plated on L agar
containing gentamicin (10 ug/ml). Gentamicin resistant colonies
were selected, grown overnight in L broth containing gentamicin (10
ug/ml), and harvested for subsequent plasmid preparation. Plasmid
constructs were isolated using Qiagen Qiaprep Spin Miniprep Kit.
Plasmid preparations of interest were sequenced (Sequetech;
Mountain View, Calif.) using primers 5' mMVK seq prim, 3' mMVK seq
prim, EL1003, and MCM 177, and the correct PTrp mMVK/pDW15 clone
identified; the resulting clone of interest has been designated as
strain REM H9.sub.--14 (TOP10 w/PTrp mMVK/pDW15; SfoI site
destroyed with PTrp mMVK inserted in the orientation as the Ptrc
mvaE-mvaS operon present in the construct; see FIG. 94).
TABLE-US-00045 Sequence of PTrp mMVK/pDW15 (SEQ ID NO: 183)
accttcgggagcgcctgaagcccgttctggacgccctggggccgttgaatcgggatatgcaggccaaggccgcc-
gcgatcat
caaggccgtgggcgaaaagctgctgacggaacagcgggaagtccagcgccagaaacaggcccagcgccagcagg-
aacgc
gggcgcgcacatttccccgaaaagtgccacctggcggcgttgtgacaatttaccgaacaactccgcggccggga-
agccgatct
cggcttgaacgaattgttaggtggcggtacttgggtcgatatcaaagtgcatcacttcttcccgtatgcccaac-
tttgtatagagag
ccactgcgggatcgtcaccgtaatctgcttgcacgtagatcacataagcaccaagcgcgttggcctcatgcttg-
aggagattgat
gagcgcggtggcaatgccctgcctccggtgctcgccggagactgcgagatcatagatatagatctcactacgcg-
gctgctcaa
acctgggcagaacgtaagccgcgagagcgccaacaaccgcttcttggtcgaaggcagcaagcgcgatgaatgtc-
ttactacg
gagcaagttcccgaggtaatcggagtccggctgatgttgggagtaggtggctacgtctccgaactcacgaccga-
aaagatcaa
gagcagcccgcatggatttgacttggtcagggccgagcctacatgtgcgaatgatgcccatacttgagccacct-
aactttgtttta
gggcgactgccctgctgcgtaacatcgttgctgctgcgtaacatcgttgctgctccataacatcaaacatcgac-
ccacggcgtaa
cgcgcttgctgcttggatgcccgaggcatagactgtacaaaaaaacagtcataacaagccatgaaaaccgccac-
tgcgccgtta
ccaccgctgcgttcggtcaaggttctggaccagttgcgtgagcgcatacgctacttgcattacagtttacgaac-
cgaacaggctta
tgtcaactgggttcgtgccttcatccgtttccacggtgtgcgtccatgggcaaatattatacgcaaggcgacaa-
ggtgctgatgcc
gctggcgattcaggttcatcatgccgtttgtgatggcttccatgtcggcagaatgcttaatgaattacaacagt-
ttttatgcatgcgcc
caatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactgg-
aaagcgggc
agtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggc-
tcgtatgttgtgtg
gaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgccaagcgcgcaattaaccct-
cactaaaggg
aacaaaagctgggtaccgggccccccctcgagctgttgacaattaatcatccggctcgtataatgtgtggaatt-
gtgagcggata
acaatttcacacaggaaacagcgccgctgagaaaaagcgaagcggcactgctctttaacaatttatcagacaat-
ctgtgtgggca
ctcgaccggaattatcgattaactttattattaaaaattaaagaggtatatattaatgtatcgattaaataagg-
aggaataaaccatgg
atccgagctcaggaggtaaaaaaacatgaaaacagtagttattattgatgcattacgaacaccaattggaaaat-
ataaaggcagct
taagtcaagtaagtgccgtagacttaggaacacatgttacaacacaacttttaaaaagacattccactatttct-
gaagaaattgatca
agtaatctttggaaatgttttacaagctggaaatggccaaaatcccgcacgacaaatagcaataaacagcggtt-
tgtctcatgaaat
tcccgcaatgacggttaatgaggtctgcggatcaggaatgaaggccgttattttggcgaaacaattgattcaat-
taggagaagcg
gaagttttaattgctggcgggattgagaatatgtcccaagcacctaaattacaacgttttaattacgaaacaga-
aagctacgatgcg
cctttttctagtatgatgtatgatggattaacggatgcctttagtggtcaggcaatgggcttaactgctgaaaa-
tgtggccgaaaagt
atcatgtaactagagaagagcaagatcaattttctgtacattcacaattaaaagcagctcaagcacaagcagaa-
gggatattcgct
gacgaaatagccccattagaagtatcaggaacgcttgtggagaaagatgaagggattcgccctaattcgagcgt-
tgagaagcta
ggaacgcttaaaacagtttttaaagaagacggtactgtaacagcagggaatgcatcaaccattaatgatggggc-
ttctgctttgatt
attgcttcacaagaatatgccgaagcacacggtcttccttatttagctattattcgagacagtgtggaagtcgg-
tattgatccagcct
atatgggaatttcgccgattaaagccattcaaaaactgttagcgcgcaatcaacttactacggaagaaattgat-
ctgtatgaaatca
acgaagcatttgcagcaacttcaatcgtggtccaaagagaactggctttaccagaggaaaaggtcaacatttat-
ggtggcggtatt
tcattaggtcatgcgattggtgccacaggtgctcgtttattaacgagtttaagttatcaattaaatcaaaaaga-
aaagaaatatggag
tggcttctttatgtatcggcggtggcttaggactcgctatgctactagagagacctcagcaaaaaaaaaacagc-
cgattttatcaaa
tgagtcctgaggaacgcctggcttctcttcttaatgaaggccagatttctgctgatacaaaaaaagaatttgaa-
aatacggctttatc
ttcgcagattgccaatcatatgattgaaaatcaaatcagtgaaacagaagtgccgatgggcgttggcttacatt-
taacagtggacg
aaactgattatttggtaccaatggcgacagaagagccctcagttattgcggctttgagtaatggtgcaaaaata-
gcacaaggattta
aaacagtgaatcaacaacgcttaatgcgtggacaaatcgttttttacgatgttgcagatcccgagtcattgatt-
gataaactacaagt
aagagaagcggaagtttttcaacaagcagagttaagttatccatctatcgttaaacggggcggcggcttaagag-
atttgcaatatc
gtacttttgatgaatcatttgtatctgtcgactttttagtagatgttaaggatgcaatgggggcaaatatcgtt-
aacgctatgttggaag
gtgtggccgagttgttccgtgaatggtttgcggagcaaaagattttattcagtattttaagtaattatgccacg-
gagtcggttgttacg
atgaaaacggctattccagtttcacgtttaagtaaggggagcaatggccgggaaattgctgaaaaaattgtttt-
agcttcacgctat
gcttcattagatccttatcgggcagtcacgcataacaaaggaatcatgaatggcattgaagctgtagttttagc-
tacaggaaatgat
acacgcgctgttagcgcttcttgtcatgcttttgcggtgaaggaaggtcgctaccaaggcttgactagttggac-
gctggatggcga
acaactaattggtgaaatttcagttccgcttgctttagccacggttggcggtgccacaaaagtcttacctaaat-
ctcaagcagctgct
gatttgttagcagtgacggatgcaaaagaactaagtcgagtagtagcggctgttggtttggcacaaaatttagc-
ggcgttacggg
ccttagtctctgaaggaattcaaaaaggacacatggctctacaagcacgttctttagcgatgacggtcggagct-
actggtaaaga
agttgaggcagtcgctcaacaattaaaacgtcaaaaaacgatgaaccaagaccgagccatggctattttaaatg-
atttaagaaaa
caataaaggaggtaaaaaaacatgacaattgggattgataaaattagtttttttgtgcccccttattatattga-
tatgacggcactggc
tgaagccagaaatgtagaccctggaaaatttcatattggtattgggcaagaccaaatggcggtgaacccaatca-
gccaagatatt
gtgacatttgcagccaatgccgcagaagcgatcttgaccaaagaagataaagaggccattgatatggtgattgt-
cgggactgagt
ccagtatcgatgagtcaaaagcggccgcagttgtcttacatcgtttaatggggattcaacctttcgctcgctct-
ttcgaaatcaagg
aagcttgttacggagcaacagcaggcttacagttagctaagaatcacgtagccttacatccagataaaaaagtc-
ttggtcgtagcg
gcagatattgcaaaatatggcttaaattctggcggtgagcctacacaaggagctggggcggttgcaatgttagt-
tgctagtgaacc
gcgcattttggctttaaaagaggataatgtgatgctgacgcaagatatctatgacttttggcgtccaacaggcc-
acccgtatcctat
ggtcgatggtcctttgtcaaacgaaacctacatccaatcttttgcccaagtctgggatgaacataaaaaacgaa-
ccggtcttgatttt
gcagattatgatgctttagcgttccatattccttacacaaaaatgggcaaaaaagccttattagcaaaaatctc-
cgaccaaactgaa
gcagaacaggaacgaattttagcccgttatgaagaaagtatcgtctatagtcgtcgcgtaggaaacttgtatac-
gggttcactttat
ctgggactcatttcccttttagaaaatgcaacgactttaaccgcaggcaatcaaattggtttattcagttatgg-
ttctggtgctgtcgct
gaatttttcactggtgaattagtagctggttatcaaaatcatttacaaaaagaaactcatttagcactgctgga-
taatcggacagaact
ttctatcgctgaatatgaagccatgtttgcagaaactttagacacagacattgatcaaacgttagaagatgaat-
taaaatatagtattt
ctgctattaataataccgttcgttcttatcgaaactaaagatctgcagctggtaccatatgggaattcgaagct-
tgggcccgaacaa
aaactcatctcagaagaggatctgaatagcgccgtcgaccatcatcatcatcatcattgagtttaaacggtctc-
cagcttggctgttt
tggcggatgagagaagattttcagcctgatacagattaaatcagaacgcagaagcggtctgataaaacagaatt-
tgcctggcgg
cagtagcgcggtggtcccacctgaccccatgccgaactcagaagtgaaacgccgtagcgccgatggtagtgtgg-
ggtctcccc
atgcgagagtagggaactgccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttctagagcg-
gccgccac
cgcggtggagctccaattcgccctatagtgagtcgtattacgcgcgctcactggccgtcgttttacaacgtcgt-
gactgggaaaa
ccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggccc-
gcaccgatcg
cccttcccaacagttgcgcagcctgaatggcgaatggaaattgtaagcgttaatattttgttaaaattcgcgtt-
aaatttttgttaaatc
agctcattttttaaccaataggccgactgcgatgagtggcagggcggggcgtaatttttttaaggcagttattg-
gtgcccttaaacg
cctggtgctacgcctgaataagtgataataagcggatgaatggcagaaattcgaaagcaaattcgacccggtcg-
tcggttcagg
gcagggtcgttaaatagccgcttatgtctattgctggtttaccggtttattgactaccggaagcagtgtgaccg-
tgtgcttctcaaat
gcctgaggccagtttgctcaggctctccccgtggaggtaataattgacgatatgatcatttattctgcctccca-
gagcctgataaaa
acggtgaatccgttagcgaggtgccgccggcttccattcaggtcgaggtggcccggctccatgcaccgcgacgc-
aacgcggg
gaggcagacaaggtatagggcggcgaggcggctacagccgatagtctggaacagcgcacttacgggttgctgcg-
caaccca
agtgctaccggcgcggcagcgtgacccgtgtcggcggctccaacggctcgccatcgtccagaaaacacggctca-
tcgggcat
cggcaggcgctgctgcccgcgccgttcccattcctccgtttcggtcaaggctggcaggtctggttccatgcccg-
gaatgccggg
ctggctgggcggctcctcgccggggccggtcggtagttgctgctcgcccggatacagggtcgggatgcggcgca-
ggtcgcc
atgccccaacagcgattcgtcctggtcgtcgtgatcaaccaccacggcggcactgaacaccgacaggcgcaact-
ggtcgcgg
ggctggccccacgccacgcggtcattgaccacgtaggccgacacggtgccggggccgttgagcttcacgacgga-
gatccag
cgctcggccaccaagtccttgactgcgtattggaccgtccgcaaagaacgtccgatgagcttggaaagtgtctt-
ctggctgacca
ccacggcgttctggtggcccatctgcgccacgaggtgatgcagcagcattgccgccgtgggtttcctcgcaata-
agcccggcc
cacgcctcatgcgctttgcgttccgtttgcacccagtgaccgggcttgttcttggcttgaatgccgatttctct-
ggactgcgtggcca
tgcttatctccatgcggtagggtgccgcacggttgcggcaccatgcgcaatcagctgcaacttttcggcagcgc-
gacaacaatta
tgcgttgcgtaaaagtggcagtcaattacagattttctttaacctacgcaatgagctattgcggggggtgccgc-
aatgagctgttgc
gtaccccccttttttaagttgttgatttttaagtctttcgcatttcgccctatatctagttctttggtgcccaa-
agaagggcacccctgcg
gggttcccccacgccttcggcgcggctccccctccggcaaaaagtggcccctccggggcttgttgatcgactgc-
gcggccttc
ggccttgcccaaggtggcgctgcccccttggaacccccgcactcgccgccgtgaggctcggggggcaggcgggc-
gggcttc
gccttcgactgcccccactcgcataggcttgggtcgttccaggcgcgtcaaggccaagccgctgcgcggtcgct-
gcgcgagc
cttgacccgccttccacttggtgtccaaccggcaagcgaagcgcgcaggccgcaggccggaggcttttccccag-
agaaaatta
aaaaaattgatggggcaaggccgcaggccgcgcagttggagccggtgggtatgtggtcgaaggctgggtagccg-
gtgggca
atccctgtggtcaagctcgtgggcaggcgcagcctgtccatcagcttgtccagcagggttgtccacgggccgag-
cgaagcgag
ccagccggtggccgctcgcggccatcgtccacatatccacgggctggcaagggagcgcagcgaccgcgcagggc-
gaagcc
cggagagcaagcccgtagggctggcaaatattctgaaatgagctgttgacaattaatcatcgaactagttaact-
agtacgcaagtt
cacgtaaaaagggtatcgacatggtatcctgttctgcgccgggtaagatttacctgttcggtgaacacgccgta-
gtttatggcgaa
actgcaattgcgtgtgcggtggaactgcgtacccgtgttcgcgcggaactcaatgactctatcactattcagag-
ccagatcggcc
gcaccggtctggatttcgaaaagcacccttatgtgtctgcggtaattgagaaaatgcgcaaatctattcctatt-
aacggtgttttcttg
accgtcgattccgacatcccggtgggctccggtctgggtagcagcgcagccgttactatcgcgtctattggtgc-
gctgaacgag
ctgttcggctttggcctcagcctgcaagaaatcgctaaactgggccacgaaatcgaaattaaagtacagggtgc-
cgcgtcccca
accgatacgtatgtttctaccttcggcggcgtggttaccatcccggaacgtcgcaaactgaaaactccggactg-
cggcattgtgat
tggcgataccggcgttttctcctccaccaaagagttagtagctaacgtacgtcagctgcgcgaaagctacccgg-
atttgatcgaa
ccgctgatgacctctattggcaaaatctctcgtatcggcgaacaactggttctgtctggcgactacgcatccat-
cggccgcctgat
gaacgtcaaccagggtctcctggacgccctgggcgttaacatcttagaactgagccagctgatctattccgctc-
gtgcggcaggt
gcgtttggcgctaaaatcacgggcgctggcggcggtggctgtatggttgcgctgaccgctccggaaaaatgcaa-
ccaagtggc
agaagcggtagcaggcgctggcggtaaagtgactatcactaaaccgaccgagcaaggtctgaaagtagattaat-
acaaataaa
aaaggcacgtcagatgacgtgccttttttcttggccgcagccgccgtaggcggtcacgactttgcgaagcaaag-
tctagtgagta
tactcaagcattgagtggcccgccggaggcaccgccttgcgctgcccccgtcgagccggttggacaccaaaagg-
gaggggc
aggcatggcggcatacgcgatcatgcgatgcaagaagctggcgaaaatgggcaacgtggcggccagtctcaagc-
acgccta
ccgcgagcgcgagacgcccaacgctgacgccagcaggacgccagagaacgagcactgggcggccagcagcaccg-
atga
agcgatgggccgactgcgcgagttgctgccagagaagcggcgcaaggacgctgtgttggcggtcgagtacgtca-
tgacggc
cagcccggaatggtggaagtcggccagccaagaacagcaggcggcgttcttcgagaaggcgcacaagtggctgg-
cggaca
agtacggggcggatcgcatcgtgacggccagcatccaccgtgacgaaaccagcccgcacatgaccgcgttcgtg-
gtgccgct
gacgcaggacggcaggctgtcggccaaggagttcatcggcaacaaagcgcagatgacccgcgaccagaccacgt-
ttgcggc
cgctgtggccgatctagggctgcaacggggcatcgagggcagcaaggcacgtcacacgcgcattcaggcgttct-
acgaggc
cctggagcggccaccagtgggccacgtcaccatcagcccgcaagcggtcgagccacgcgcctatgcaccgcagg-
gattggc
cgaaaagctgggaatctcaaagcgcgttgagacgccggaagccgtggccgaccggctgacaaaagcggttcggc-
aggggta
tgagcctgccctacaggccgccgcaggagcgcgtgagatgcgcaagaaggccgatcaagcccaagagacggccc-
gag
Construction of Strain MCM928, BL21 t pgl
FRT-cmp-FRT-Ptrc-PMK-MVD-yIDI
[0898] Construction of Integration Construct pMCM900
[0899] The GI1.6 promoter and yeast MVK gene of pMCM296 were
replaced with a chloramphenicol resistance cassette and Trc
promoter. The cmR resistance cassette-Ptrc fragment was created by
amplification from pMCM883 (GeneBridges cmR cassette) using primers
MCM127 and MCM375. 2, 50 uL reactions were created according the
manufacturer's protocol for Herculase II Fusion (Agilent #600679)
containing 35 uL water, 10 uL buffer, 0.5 uL dNTPs, 1.25 uL each
primer at 10 uM, 1 uL plasmid template, 1 uL polymerase. Reactions
were cycled as follows: 95.degree. C., 2:00; 30.times. (95.degree.
C., 0:20; 55.degree. C., 0:20; 72.degree. C., 1:00); 72.degree. C.,
3:00; 4.degree. C. until cold.
[0900] The .about.1.6 kb amplicon and plasmid pMCM296 (described
infra) were digested at 37.degree. C. for 2 hour in 10 uL reactions
containing 5 uL DNA, 1 uL EcoRV, 1 uL NotI (amplicon) or 1 uL StuI
(pMCM296), 1 uL Roche Buffer H, and 2 uL ddH2O. Reactions were
heat-killed at 65.degree. C. for 2 hr then digested DNA was
purified on Qiagen PCR columns and eluted in 30 uL EB. The eluted
DNAs were ligated 1 hr at room temperature in a 10 uL Roche Rapid
Ligation kit reaction containing 1 uL pMCM296, 3 uL cut amplicon, 5
uL buffer 3, and 1 uL ligase. Ligated DNA was transformed into
Invitrogen Pir1 chemically competent cells, recovered for 1 hr at
37.degree. C., plated on LB/cmp 25 ug/mL, then grown overnight at
37.degree. C. The resulting plasmids were purified and sequenced
across the promoter region. Clone four was frozen as pMCM900; see
FIG. 95.
Integration of cmR-Ptrc-KDyI into Host BL21 t pgl to Create
MCM928
[0901] Strain MCM865 is an aliquot of strain MD253 (BL21 t pgl
pRedET-carb). MCM865 was grown in LB+carb50 at 30.degree. C.
overnight and then diluted 1:100 into fresh LB+carb50 and cultured
at 30.degree. C. for 2 hr. 130 uL 10% arabinose was added and cells
cultured at 37.degree. C. for approximately 2 hours. Cells were
prepared for electroporation by washing 3.times. in one half
culture volume iced ddH2O and resuspended in one tenth culture
volume of the same. 100 uL of cell suspension was combined with 1
uL pMCM900 DNA in a 2 mm electroporation cuvette, electroporated at
25 uFD, 200 ohms, 2.5 kV, and immediately quenched with 500 uL LB.
Cells were recovered shaking at 37.degree. C. for 1-3 hrs and then
transformants selected overnight on LB cmp5 plates at 37.degree.
C.
[0902] After restreaking on LB cmp5, transformants were tested for
growth on LB cmp5, LB kan10 and LB carb50. A cmpR/carbS/kanS clone
was frozen as MCM928.
TABLE-US-00046 Primers MCM139 (SEQ ID NO: 184)
ttttgcggccgcaattaaccctcactaaagggcgg MCM375 (SEQ ID NO: 185)
gatcgatatccctgcaggaaattgttatccgctcacaattccacacatta
tacgagccggatgattaattgtcaacagctaatacgactcactatagggc tcg
TABLE-US-00047 Sequence of pMCM900 (SEQ ID NO: 186)
caagaaaaatgccccgcttacgcagggcatccatttattactcaaccgtaaccgattttgccaggttacgcggc-
tggtcaacgtcggtgcctttgat
cagcgcgacatggtaagccagcagctgcagcggaacggtgtagaagatcggtgcaatcacctcttccacatgcg-
gcatctcgatgatgtgcat
gttatcgctacttacaaaacccgcatcctgatcggcgaagacatacaactgaccgccacgcgcgcgaacttctt-
caatgttggatttcagtttttcca
gcaattcgttgttcggtgcaacaacaataaccggcatatcggcatcaattagcgccagcggaccgtgtttcagt-
tcgccagcagcgtaggcttca
gcgtgaatgtaagagatctctttcaacttcaatgcgccttccagcgcgattgggtactgatcgccacggcccag-
gaacagcgcgtgatgtttgtca
gagaaatcttctgccagcgcttcaatgcgtttgtcctgagacagcatctgctcaatacggctcggcagcgcctg-
cagaccatgcacgatgtcatgt
tcaatggaggcatccagacctttcaggcgagacagcttcgccaccagcatcaacagcacagttaactgagtggt-
gaatgctttagtggatgccac
gccgatttctgtacccgcgttggtcattagcgccagatcggattcgcgcaccagagaagaacccggaacgttac-
agattgccagtgaaccaagg
taacccagctctttcgacagacgcaggccagccagggtatccgcggtttcgccagactgtgacacgatcgccct-
tcccaacagttgcgcagcct
atacgtacggcagtttaaggtttacacctataaaagagagagccgttatcgtctgtttgtggatgtacagagtg-
atattattgacacgccggggcga
cggatggtgatccccctggccagtgcacgtctgctgtcagataaagtctcccgtgaactttacccggtggtgca-
tatcggggatgaaagctggcg
catgatgaccaccgatatggccagtgtgccggtctccgttatcggggaagaagtggctgatctcagccaccgcg-
aaaatgacatcaaaaacgc
cattaacctgatgttctggggaatataaatgtcaggcatgagattatcaaaaaggatcttcacctagatccttt-
tcacgtagaaagccagtccgcag
aaacggtgctgaccccggatgaatgtcagctactgggctatctggacaagggaaaacgcaagcgcaaagagaaa-
gcaggtagcttgcagtgg
gcttacatggcgatagctagactgggcggttttatggacagcaagcgaaccggaattgccagctggggcgccct-
ctggtaaggttgggaagcc
ctgcaaagtaaactggatggctttctcgccgccaaggatctgatggcgcaggggatcaagctctgatcaagaga-
caggatgaggatcgtttcgc
atgattgaacaagatggattgcacgcaggttctccggccgcttgggtggagaggctattcggctatgactgggc-
acaacagacaatcggctgct
ctgatgccgccgtgttccggctgtcagcgcaggggcgcccggttctttttgtcaagaccgacctgtccggtgcc-
ctgaatgaactgcaagacga
ggcagcgcggctatcgtggctggccacgacgggcgttccttgcgcagctgtgctcgacgttgtcactgaagcgg-
gaagggactggctgctatt
gggcgaagtgccggggcaggatctcctgtcatctcaccttgctcctgccgagaaagtatccatcatggctgatg-
caatgcggcggctgcatacg
cttgatccggctacctgcccattcgaccaccaagcgaaacatcgcatcgagcgagcacgtactcggatggaagc-
cggtcttgtcgatcaggatg
atctggacgaagagcatcaggggctcgcgccagccgaactgttcgccaggctcaaggcgagcatgcccgacggc-
gaggatctcgtcgtgac
ccatggcgatgcctgcttgccgaatatcatggtggaaaatggccgcttttctggattcatcgactgtggccggc-
tgggtgtggcggaccgctatca
ggacatagcgttggctacccgtgatattgctgaagagcttggcggcgaatgggctgaccgcttcctcgtgcttt-
acggtatcgccgctcccgattc
gcagcgcatcgccttctatcgccttcttgacgagttcttctgaattattaacgcttacaatttcctgatgcggt-
attttctccttacgcatctgtgcggtat
ttcacaccgcatacaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaataca-
ttcaaatatgtatccgctcatgagac
aataaccctgataaatgcttcaataatagcacgtgaggagggccaccatggccaagttgaccagtgccgttccg-
gtgctcaccgcgcgcgacgt
cgccggagcggtcgagttctggaccgaccggctcgggttctcccctagtaacggccgccagtgtgctggaattc-
aggcagttcaacctgttgat
agtacgtactaagctctcatgtttcacgtactaagctctcatgtttaacgtactaagctctcatgtttaacgaa-
ctaaaccctcatggctaacgtactaa
gctctcatggctaacgtactaagctctcatgtttcacgtactaagctctcatgtttgaacaataaaattaatat-
aaatcagcaacttaaatagcctctaa
ggttttaagttttataagaaaaaaaagaatatataaggcttttaaagcttttaaggtttaacggttgtggacaa-
caagccagggatgtaacgcactga
gaagcccttagagcctctcaaagcaattttcagtgacacaggaacacttaacggctgacagcctgaattctgca-
gatatctgtttttccactcttcgtt
cactttcgccaggtagctggtgaagacgaaggaagtcccggagccatctgcgcggcgtactacagcaatgtttt-
gtgaaggcagtttcagaccc
ggattcagtttggcgatggcttcatcatcccacttcttgattttgcccaggtagatgtcgccgagggttttacc-
atccagcaccagttcgccagacttc
agccctggaatgttaaccgccagcaccacgccgccaatcacggtcgggaactggaacagaccttcctgagccag-
tttttcgtcagacagcggc
gcgtcagaggcaccaaaatcaacggtattagcgataatctgttttacgccaccggaagaaccgataccctggta-
gttaactttattaccggtttcttt
ctggtaagtgtcagcccatttggcatacaccggcgcagggaaggttgcacctgcacctgtcaggcttgcttctg-
caaacacagagaaagcactc
atcgataaggtcgcggcgacaacagttgcgacggtggtacgcataactttcataatgtctcctgggaggattca-
taaagcattgtttgttggctacg
agaagcaaaataggacaaacaggtgacagttatatgtaaggaatatgacagttttatgacagagagataaagtc-
ttcagtctgatttaaataagcgt
tgatattcagtcaattacaaacattaataacgaagagatgacagaaaaattttcattctgtgacagagaaaaag-
tagccgaagatgacggtttgtca
catggagttggcaggatgtttgattaaaagcggccgcgaagttcctattctctagaaagtataggaacttcatt-
ctaccgggtaggggaggcgcttt
tcccaaggcagtctggagcatgcgctttagcagccccgctgggcacttggcgctacacaagtggcctctggcct-
cgcacacattccacatccac
cggtaggcgccaaccggctccgttctttggtggccccttcgcgccaccttccactcctcccctagtcaggaagt-
tcccccccgccccgcagctcg
cgtcgtgcaggacgtgacaaatggaagtagcacgtctcactagtctcgtgcagatggacagcaccgctgagcaa-
tggaagcgggtaggccttt
ggggcagcggccaatagcagctttgctccttcgctttctgggctcagaggctgggaaggggtgggtccgggggc-
gggctcaggggcgggct
caggggcggggcgggcgcccgaaggtcctccggaggcccggcattctgcacgcttcaaaagcgcacgtctgccg-
cgctgttctcctcttcctc
atctccgggcctttcgacctgcagcagcacgtgttgacaattaatcatcggcatagtatatcggcatagtataa-
tacgacaaggtgaggaactaaa
ccatggagaaaaaaatcactggatataccaccgttgatatatcccaatggcatcgtaaagaacattttgaggca-
tttcagtcagttgctcaatgtacc
tataaccagaccgttcagctggatattacggcctttttaaagaccgtaaagaaaaataagcacaagttttatcc-
ggcctttattcacattcttgcccgc
ctgatgaatgctcatccggaattccgtatggcaatgaaagacggtgagctggtgatatgggatagtgttcaccc-
ttgttacaccgttttccatgagc
aaactgaaacgttttcatcgctctggagtgaataccacgacgatttccggcagtttctacacatatattcgcaa-
gatgtggcgtgttacggtgaaaac
ctggcctatttccctaaagggtttattgagaatatgtttttcgtctcagccaatccctgggtgagtttcaccag-
ttttgatttaaacgtggccaatatgga
caacttcttcgcccccgttttcaccatgggcaaatattatacgcaaggcgacaaggtgctgatgccgctggcga-
ttcaggttcatcatgccgtttgt
gatggcttccatgtcggcagaatgcttaatgaattacaacagtactgcgatgagtggcagggcggggcgtaagc-
gggactctggggttcgaata
aagaccgaccaagcgacgtctgagagctccctggcgaattcggtaccaataaaagagctttattttcatgatct-
gtgtgttggtttttgtgtgcggcg
cggaagttcctattctctagaaagtataggaacttcctcgagccctatagtgagtcgtattagctgttgacaat-
taatcatccggctcgtataatgtgt
ggaattgtgagcggataacaatttcctgcagggatcctgcacccttaaggaggaaaaaaacatgtcagagttga-
gagccttcagtgccccaggg
aaagcgttactagctggtggatatttagttttagatacaaaatatgaagcatttgtagtcggattatcggcaag-
aatgcatgctgtagcccatccttac
ggttcattgcaagggtctgataagtttgaagtgcgtgtgaaaagtaaacaatttaaagatggggagtggctgta-
ccatataagtcctaaaagtggct
tcattcctgtttcgataggcggatctaagaaccctttcattgaaaaagttatcgctaacgtatttagctacttt-
aaacctaacatggacgactactgcaa
tagaaacttgttcgttattgatattttctctgatgatgcctaccattctcaggaggatagcgttaccgaacatc-
gtggcaacagaagattgagttttcatt
cgcacagaattgaagaagttcccaaaacagggctgggctcctcggcaggtttagtcacagttttaactacagct-
ttggcctccttttttgtatcggac
ctggaaaataatgtagacaaatatagagaagttattcataatttagcacaagttgctcattgtcaagctcaggg-
taaaattggaagcgggtttgatgt
agcggcggcagcatatggatctatcagatatagaagattcccacccgcattaatctctaatttgccagatattg-
gaagtgctacttacggcagtaaa
ctggcgcatttggttgatgaagaagactggaatattacgattaaaagtaaccatttaccttcgggattaacttt-
atggatgggcgatattaagaatggt
tcagaaacagtaaaactggtccagaaggtaaaaaattggtatgattcgcatatgccagaaagcttgaaaatata-
tacagaactcgatcatgcaaatt
ctagatttatggatggactatctaaactagatcgcttacacgagactcatgacgattacagcgatcagatattt-
gagtctcttgagaggaatgactgt
acctgtcaaaagtatcctgaaatcacagaagttagagatgcagttgccacaattagacgttcctttagaaaaat-
aactaaagaatctggtgccgata
tcgaacctcccgtacaaactagcttattggatgattgccagaccttaaaaggagttcttacttgcttaatacct-
ggtgctggtggttatgacgccattg
cagtgattactaagcaagatgttgatcttagggctcaaaccgctaatgacaaaagattttctaaggttcaatgg-
ctggatgtaactcaggctgactg
gggtgttaggaaagaaaaagatccggaaacttatcttgataaataacttaaggtagctgcatgcagaattcgcc-
cttaaggaggaaaaaaaaatg
accgtttacacagcatccgttaccgcacccgtcaacatcgcaacccttaagtattgggggaaaagggacacgaa-
gttgaatctgcccaccaattc
gtccatatcagtgactttatcgcaagatgacctcagaacgttgacctctgcggctactgcacctgagtttgaac-
gcgacactttgtggttaaatgga
gaaccacacagcatcgacaatgaaagaactcaaaattgtctgcgcgacctacgccaattaagaaaggaaatgga-
atcgaaggacgcctcattg
cccacattatctcaatggaaactccacattgtctccgaaaataactttcctacagcagctggtttagcttcctc-
cgctgctggctttgctgcattggtct
ctgcaattgctaagttataccaattaccacagtcaacttcagaaatatctagaatagcaagaaaggggtctggt-
tcagcttgtagatcgttgtttggc
ggatacgtggcctgggaaatgggaaaagctgaagatggtcatgattccatggcagtacaaatcgcagacagctc-
tgactggcctcagatgaaa
gcttgtgtcctagttgtcagcgatattaaaaaggatgtgagttccactcagggtatgcaattgaccgtggcaac-
ctccgaactatttaaagaaagaa
ttgaacatgtcgtaccaaagagatttgaagtcatgcgtaaagccattgttgaaaaagatttcgccacctttgca-
aaggaaacaatgatggattccaa
ctctttccatgccacatgtttggactctttccctccaatattctacatgaatgacacttccaagcgtatcatca-
gttggtgccacaccattaatcagtttta
cggagaaacaatcgttgcatacacgtttgatgcaggtccaaatgctgtgttgtactacttagctgaaaatgagt-
cgaaactctttgcatttatctataa
attgtttggctctgttcctggatgggacaagaaatttactactgagcagcttgaggctttcaaccatcaatttg-
aatcatctaactttactgcacgtgaa
ttggatcttgagttgcaaaaggatgttgccagagtgattttaactcaagtcggttcaggcccacaagaaacaaa-
cgaatctttgattgacgcaaaga
ctggtctaccaaaggaataagatcaattcgctgcatcgcccttaggaggtaaaaaaaaatgactgccgacaaca-
atagtatgccccatggtgcag
tatctagttacgccaaattagtgcaaaaccaaacacctgaagacattttggaagagtttcctgaaattattcca-
ttacaacaaagacctaatacccga
tctagtgagacgtcaaatgacgaaagcggagaaacatgtttttctggtcatgatgaggagcaaattaagttaat-
gaatgaaaattgtattgttttggat
tgggacgataatgctattggtgccggtaccaagaaagtttgtcatttaatggaaaatattgaaaagggtttact-
acatcgtgcattctccgtctttatttt
caatgaacaaggtgaattacttttacaacaaagagccactgaaaaaataactttccctgatctttggactaaca-
catgctgctctcatccactatgtat
tgatgacgaattaggtttgaagggtaagctagacgataagattaagggcgctattactgcggcggtgagaaaac-
tagatcatgaattaggtattcc
agaagatgaaactaagacaaggggtaagtttcactttttaaacagaatccattacatggcaccaagcaatgaac-
catggggtgaacatgaaattg
attacatcctattttataagatcaacgctaaagaaaacttgactgtcaacccaaacgtcaatgaagttagagac-
ttcaaatgggtttcaccaaatgatt
tgaaaactatgtttgctgacccaagttacaagtttacgccttggtttaagattatttgcgagaattacttattc-
aactggtgggagcaattagatgacctt
tctgaagtggaaaatgacaggcaaattcatagaatgctataacaacgcgtctacaaataaaaaaggcacgtcag-
atgacgtgccttttttcttggg gcc
Construction of REM H4.sub.--15, the Parent Background of Strain
REM A2.sub.--17
[0903] The chloramphenicol marked PTrc PMK-MVD-yIDI locus of strain
MCM928, described above, was introduced into strain WW103 (see,
e.g., Examples 29 and 30) via P1-mediated transduction. The
resulting chloramphenicol resistant strain was named REM
H4.sub.--15 (BL21 pgl.sup.+ PL.6-dxs, GI1.6-dxr, GI1.6 yIDI,
CMP::PTrc PMK-MVD-yIDI).
Strategy for Creating REM A2.sub.--17
[0904] REM A2.sub.--17 was created by subsequent plasmid
transformations of pDW33, PTrp mMVK/pDW15, Ptac Anabaena ispH aspA
term/pEWL454, and lastly GI1.6 fldA-ispG/pCL initially into strain
REM H4.sub.--15.
[0905] Water-washed REM H4.sub.--15 cells were transformed with
pDW33 via electroporation using the BIO RAD Gene Pulser system (0.1
cm cuvette cat.#165-2089) and a transformation protocol suggested
by the manufacturer (BIO RAD). The cells were recovered in L broth
for 1 hour at 37.degree. C. and then plated on L agar containing
carbenicillin (50 ug/ml). One carbenicillin resistant colony was
chosen, named REM A4.sub.--16, and subsequently transformed with
PTrp mMVK/pDW15 via the method described; in this case L agar
containing carbenicillin (50 ug/ml) and gentamicin (10 ug/ml) was
used as a selection, resulting in the carbenicillin and gentamicin
resistant strain REM I4.sub.--16. Similarly, REM I4.sub.--16 was
transformed with Ptac Anabaena ispH aspA term/pEWL454 resulting in
the carbenicillin (50 ug/ml), gentamicin (10 ug/ml) and kanamycin
(50 ug/ml) resistant strain REM C5.sub.--16. Lastly, strain REM
C5.sub.--16 was transformed with GI1.6 fldA-ispG/pCL resulting in
the carbenicillin (50 ug/ml), gentamicin (10 ug/ml) kanamycin (50
ug/ml), and spectinomycin (50 ug/ml) resistant strain REM
A2.sub.--17.
Example 33
Analysis of Strain REM A2.sub.--17 in the Presence and Absence of
Fosmidomycin for Growth, Isoprene Production, and DXP and MVA
Metabolite Accumulation Using Unlabeled, 1-.sup.13C Labeled, or
3-.sup.13C Labeled Glucose as the Sole Carbon Source
[0906] It was previously determined that the addition of 1 mM
fosmidomycin to the growth media of an E. coli BL21 strain
harboring the GI1.6-dxr locus common to the REM A2.sub.--17 strain
could inhibit isoprene production to an undetectable level.
Fosmidomycin inhibits the activity of the DXR enzyme that performs
the committed step of the endogenous E. coli DXP pathway (Kuzuyama
et al., 1998). Furthermore, the addition of 1 mM fosmidomycin to
the growth media of a dxr null E. coli BL21 strain that harbors the
same heterologous MVA isoprenoid biosynthetic pathway enzymes
present in REM A2.sub.--17 was found to maintain the same level of
isoprene production as that grown in the absence of fosmidomycin.
This data indicates that the DXR inhibitor (fosmidomycin) does not
adversely affect in vivo flux through the MVA isoprenoid
biosynthetic pathway.
Specific Productivity of Isoprene Generated by REM A2.sub.--17
Strain.
[0907] 2 mM fosmidomycin in combination with 1-.sup.13C (Isotec) or
3-.sup.13C glucose (Omicron Biochemicals, Inc) was used in small
scale headspace assays and corresponding DXP and MVA metabolite
determination studies to demonstrate the simultaneous flux to
isoprene via the dual MVA and DXP isoprenoid biosynthetic pathways
expressed within REM A2.sub.--17. See below for the rationale of
using 1-.sup.13C glucose and 3-.sup.13C glucose to generate
uniquely labeled isoprene derived from the DXP pathway that can be
differentiated from the isoprene generated via the MVA pathway.
Shown in FIGS. 98 and 99 are the results of the headspace assays
utilizing the 1-.sup.13C and 3-.sup.13C labeled glucose which
indicate a 57-58% MVA-flux and 42-43% DXP-flux contribution to the
isoprene generated by strain REM A2.sub.--17, as determined by
isoprene specific productivity. These results are nearly identical
to that observed in the unlabeled glucose experiment shown in FIG.
96 (58% MVA and 41% DXP). Interestingly, the results depicted in
FIGS. 101 and 102 obtained from the GC/MS analysis on the various
.sup.12C and .sup.13C isotope ratios present in the isoprene
produced by REM A2.sub.--17 suggest a 58-62% MVA and 42-38%
DXP-flux contribution to the isoprene generated, respectively. This
data is in agreement with that determined by the isoprene specific
productivity determination.
[0908] The dual flux of carbon to isoprene down the MVA and DXP
isoprenoid biosynthetic pathways harbored by REM A2.sub.--17 was
further support by use of tryptophan to repress expression of the
MVK enzyme common to the MVA pathway (see FIG. 94 for an
illustration of the PTrp-MVK containing vector). [The tryptophan
promoter, PTrp, governs expression of MVK in REM A2.sub.--17; the
Trp repressor inhibits activity of the Trp promoter when bound to
tryptophan; please see information about the trp operon available
through EcoCyc www.ecocyc.org]. The data in FIG. 98 indicates that
the proportion of MVA-flux to isoprene is reduced by approximately
8% when REM A2.sub.--17 is grown in the presence of 50 uM
tryptophan, resulting in a strain with nearly 1:1 MVA-flux:DXP-flux
contribution to isoprene.
Accumulation of DXP and MVA Pathway Metabolites in the REM
8A2.sub.--17 Strain.
[0909] FIG. 97 compares accumulation of DXP and MVA pathway
metabolites in the REM A2.sub.--17 strain grown in the presence and
in the absence of fosmidomycin. Among the metabolites that were
detected and quantified by LC-MS/MS were mevalonic acid (the MVA
pathway intermediate), DXP, MEP, CDP-ME, cMEPP, HDMAPP (the DXP
pathway intermediates), and IPP and DMAPP (intermediates of both
DXP and MVA pathways). Growing cells in the presence of
fosmidomycin, which inhibits DXP to MEP conversion, caused a
significant increase in the DXP concentration and a drop in the
concentration of MEP, CDP-ME and cMEPP, but did not change the
concentration of MVA. The observed decrease in HDMAPP in
fosmidomycin-treated samples was noticeably smaller that the
decrease in other DXP pathway metabolites, such as MEP, CDP-ME and
cMEPP, presumably due to a poor sensitivity of the LC-MS/MS method
to HDMAPP and a large error associated with HDMAPP measurements.
The cumulative amount of IPP and DMAPP decreased in the presence of
fosmidomycin in average by 55% that correlates with a 41% decrease
in the isoprene production rate. Taken altogether these data
demonstrate that both DXP and MVA pathways are functional in the
REM A2.sub.--17 strain and are consistent with the idea that the
two pathways are contributing to the isoprene production in cells
grown without fosmidomycin.
Rationale for Use of Labeled Glucose to Measure Contribution of DXP
and MVA Pathways to Isoprene Production
[0910] To demonstrate that in the REM A2.sub.--17 strain isoprene
is produced by the DXP and MVA pathways operating simultaneously,
the above strain was grown on glucose containing .sup.13C isotope
at specific positions. As illustrated in FIG. 100, when cells are
grown on 1-.sup.13C glucose, it is expected that isoprene molecules
synthesized by the MVA route will be more enriched in .sup.13C than
the molecules synthesized by the DXP route, whereas when cells are
grown on 3-.sup.13C glucose, the isoprene molecules synthesized by
the MVA route should contain less .sup.13C than the isoprene
molecules made by the DXP route because .sup.13C-labeled carbon is
released as .sup.13CO.sub.2 when pyruvate is converted to
acetyl-CoA. When both pathways are operating simultaneously,
.sup.13C labeling pattern of isoprene emitted by the cells should
be represented by superposition of the labeling patterns of
isoprene molecules produced by each of the two routes.
Isoprene Labeling Experiments
[0911] FIG. 101 shows calculated relative abundances of cMEPP and
isoprene cumomers (cumulative isotopomers) produced by the REM
A2.sub.--17 strain grown on: A) 1-.sup.13C or B) 3-.sup.13C
glucose. The cumomer abundances of cMEPP and isoprene can be
directly compared to each other because both compounds contain five
carbon atoms in their molecules, whereas differences in the number
of 0, P, and H atoms can be neglected due to a very low natural
abundance of isotopes other than .sup.16O, .sup.31P, and .sup.1H.
The measured distributions of cMEPP cumomer abundances should be
equivalent to the cumomer distributions in isoprene made
exclusively by the DXP pathway and were clearly different from the
calculated distributions of isoprene cumomers for cells grown in
the absence of fosmidomycin (compare the amplitudes of "Isoprene
(-FM)" and cMEPP (-FM) bars in FIGS. 101A and 9B) indicating that
the DXP and MVA pathways together contribute to the isoprene
synthesized by the REM A2.sub.--17 strain.
[0912] The distribution of cumomers of isoprene produced
exclusively via the MVA pathway by REM A2.sub.--17 cells grown on
1-.sup.13C or 3-.sup.13C glucose was estimated by measuring GC
spectra of isoprene emitted in the presence of 2 mM fosmidomycin
(FIGS. 101 and 102 and relative contribution of the DXP and MVA
pathways to the total isoprene production was calculated by
superimposing the "Isoprene (+FM)" and cMEPP cumomer spectra with
the coefficients .phi..sup.MVA and .phi..sup.DXP to fit the
"Isoprene (-FM)" spectra, as described in "Methods" section. Based
on these calculations, the relative contribution of the DXP pathway
to the total isoprene production was estimated to be 42% and 38%
for the experiments with 1-.sup.13C and 3-.sup.13C glucose,
respectively. These numbers are close to the DXP pathway
contributions of 42% and 43%, respectively, estimated from the
inhibition of total isoprene production rate by fosmidomycin.
Methods
Growth
[0913] Strains REM A2.sub.--17 was grown at 34.degree. C. in TM3
liquid media (13.6 g K.sub.2PO.sub.4, 13.6 g KH.sub.2PO.sub.4, 2.0
g MgSO.sub.4*7H.sub.2O), 2.0 g citric acid monohydrate, 0.3 g
ferric ammonium citrate, 3.2 g (NH.sub.4).sub.2SO.sub.4, 1.0 ml
1000.times. Modified Trace Metal Solution, adjusted to pH 6.8 and
q.s. to H.sub.2O, and filter sterilized) supplemented to a final
concentration with either 1% unlabeled glucose and 0.1% yeast
extract (FIGS. 96 and 97 experiment), or with 1% unlabeled glucose,
no yeast extract, and no tryptophan; 1.0% 1-.sup.13C glucose
(Isotec), no yeast extract, and with or without 50 uM tryptophan
(FIGS. 98 and 101), or with 1.0% 3-.sup.13C glucose (Omicron
Biochemicals, Inc.) and no yeast extract (FIGS. 99 and 10). All
growth media also contained carbenicillin (50 ug/ml), gentamicin
(10 ug/ml) kanamycin (50 ug/ml), and spectinomycin (50 ug/ml). The
culture was induced with 400 uM IPTG and later DXP flux inhibited
for half of the culture by the addition of 2 mM fosmidomycin
(Invitrogen). Growth was monitored periodically by recording each
of the culture's optical density measured at 600 nm using an
Eppendorf Biophotometer spectrometer (Eppendorf).
GC Measurements of Isoprene
[0914] Isoprene production was analyzed using a headspace assay.
For the headspace cultures, 100 uL to 200 ul of the cultures was
transferred from the shake flasks to 2 ml CTC headspace vials
(SUN-SRI 2 mL HS vials, VWR#66020-950, and caps, VWR#66008-170).
The cap was screwed on tightly and the vials incubated at the
equivalent temperature with shaking at 250 rpm. After approx. 30
min. to 1 hour the vials were removed from the incubator, heat
killed at 70.degree. C. for 7 min., and analyzed. The analysis was
performed using an Agilent 6890 GC/MS system interfaced with a CTC
Analytics (Switzerland) CombiPAL autosampler operating in headspace
mode. An Agilent HP-5MS GC/MS column (15 m.times.0.25 mm; 0.25
.mu.m film thickness) was used for separation of analytes. The
sampler was set up to inject 100 .mu.L of headspace gas. The GC/MS
method utilized helium as the carrier gas at a flow of 1 ml/minute.
The injection port was held at 250.degree. C. with a split ratio of
50:1. The oven temperature was held at 37.degree. C. for 0.6
minute, the duration of the analysis. The Agilent 5793N mass
selective detector was run in single ion monitoring (SIM) mode on
m/z 67 or in a full scan mode covering m/z from 25 to 80. The
detector was switched off from 0 to 0.42 minutes to allow the
elution of permanent gases. Under these conditions isoprene
(2-methyl-1,3-butadiene) standard (SCOTTY.RTM. Analyzed Gases) was
observed to elute at approx. 0.49 minutes. A calibration table was
used to quantify the absolute amount of isoprene and was found to
be linear from 1 .mu.g/L to 5000 .mu.g/L. The limit of detection
was estimated to be 50 to 100 ng/L using this method. The specific
productivity of each strain is reported as ug/L OD Hr. Note, ratio
of 1800 ul headspace:200 ul broth in assay vials for 1 hour
incubation results in the following conversion of isoprene ug/L of
culture to specific productivity: (isoprene/L determined by
GC-MS).times.(9)/(OD 600 nm of the culture). To quantify the amount
of isoprene produced from .sup.13C-labeled glucose, the
concentration obtained based on the calibration curve with the
non-labeled standard was multiplied by the conversion factor K to
compensate for isotopic effects. The conversion factors were
calculated as
K=(.SIGMA.(A.sub.i)/A.sub.67)/(.SIGMA.(P.sub.i)/P.sub.67), (Eq.
1)
where A.sub.i are the measured intensities of GC peaks produced by
.sup.13C-enriched isoprene and P.sub.i are the measured intensities
of GC peaks produced by the isoprene standard (subscript indices
i=60 . . . 72 indicate m/z values of corresponding peaks, which
include peaks A.sub.67 and P.sub.67). For the experiments referred
to in this document the conversion factors of 2.901 and 3.369 were
applied to no fosmidomycin and to 2 mM fosmidomycin conditions,
respectively, for cells grown on 1-.sup.13C glucose and the factors
of 1.476 and 1.315 were applied to no fosmidomycin and to 2 mM
fosmidomycin conditions, respectively, for cells grown on
3-.sup.13C glucose.
LC-MS/MS Analysis of Cellular Metabolites
[0915] For metabolite analysis 1.5 to 5 mL of cell culture was spun
down by centrifugation and 100 or 150 uL of dry ice-cold methanol
was added to pelleted cells after the centrifugation. The resulting
samples were then stored at -80.degree. C. until further
processing. To extract cellular metabolites, 10 or 15 .mu.L of
water was added to methanol-containing samples, the pellet was
resuspended in the resulting methanol/water mix and then cell
debris were spun down for 4-min at 4500.times.g. The pellet was
re-extracted two more times, first with 90 .mu.L of 75% methanol
buffered with 1 mM ammonium acetate in water (pH=8.0), then with
100 .mu.L of 50% methanol in the ammonium acetate buffer. After
each extraction, cell debris were spun down by centrifugation and
the supernatants from all three extractions were combined. During
the extraction procedure, samples were kept on ice or in a
refrigerated centrifuge whenever possible to minimize metabolites
degradation.
[0916] The extract was analyzed by LC-MS/MS on a TSQ Quantum triple
quadrupole mass spectrometer (Thermo Electron Corporation, San
Jose, Calif.) using electrospray ionization in the negative mode.
The system control, data acquisition, and mass spectral data
evaluation were performed using XCalibur and LCQuan software
(Thermo Electron Corp). LC separation was done on a Synergi 45
.mu.M Hydro-RP HPLC column (150.times.2 mm, Phenomenex, USA) at a
flow rate of 0.4 mL/min and the column temperature of 40.degree. C.
The LC gradient was t=0 min, 12% B; t=5 min, 12% B; t=9 min, 23% B;
t=20 min, 99% B; t=23 min, 99% B; t=24 min, 12% B; t=29 min, 12% B,
where solvent A was 10 mM tributylamine/15 mM acetic acid in water
and solvent B was LCMS-grade methanol. The sample injection volume
was 10 to 25 .mu.L.
[0917] Mass detection was carried out using electrospray ionization
in the negative mode. The following MS/MS transitions were chosen
to detect the metabolites of interest: 213.fwdarw.79 for DXP,
215.fwdarw.79 for MEP, 245.fwdarw.79 for IPP and DMAPP,
261.fwdarw.79 for HDMAPP, 277.fwdarw.79 for cMEPP, 520.1.fwdarw.79
for CDP-ME, 227.fwdarw.79 for MVP, 307.fwdarw.79 for MVPP, and
147.fwdarw.59 for MVA. Other mass spec settings were optimized to
obtain the highest sensitivity using corresponding standards
purchased from Echelon Biosciences Inc. or synthesized in house. To
quantify the absolute concentrations of cellular metabolites a
calibration table was constructed by injecting the known amounts of
these standards. Note that the LC-MS/MS method that was used for
metabolite analysis does not discriminate between structurally
similar IPP and DMAPP, therefore their amount in samples was
determined as a sum of concentrations of the two compounds.
[0918] Cumomer distribution analysis for cMEPP was done by
calculating relative intensities of peaks arising from
277.fwdarw.79, 278.fwdarw.79, 279.fwdarw.79, 280.fwdarw.79,
281.fwdarw.79 and 282.fwdarw.79 MS/MS transitions corresponding to
M0, M+1, M+2, M+3, M+4, and M+5 cumomers of this metabolite. In a
separate experiment it has been verified that at t z 14.3 min (the
retention time of cMEPP) extracts from E. coli cells grown on a
regular glucose do not generate detectable peaks with MS/MS
transitions 272.fwdarw.79, 273.fwdarw.79, 274.fwdarw.79,
275.fwdarw.79, 276.fwdarw.79. These control measurements exclude
the possibility that compounds potentially co-eluting with cMEPP
but having slightly lower molecular weight can contribute to the
MS/MS peaks generated by cMEPP when cells are grown on
.sup.13C-enriched glucose.
Cumomer Analysis of .sup.13C Labeled Isoprene
[0919] To measure .sup.13C enrichment of isoprene emitted by cells
grown on .sup.13C-glucose, GC spectra were monitored from m/z 58 to
m/z 68, i.e. over the range of mass to charge ratios that can
originate from five-carbon isoprene derivatives. FIG. 102 shows
typical GC spectra of synthetic isoprene containing the natural
abundance of .sup.13C and of isoprene emitted by REM A2.sub.--17
strain grown on 3-.sup.13C glucose and therefore enriched in
.sup.13C.
[0920] The data shown in FIG. 102A were used to calculate the
theoretical GC spectrum of isoprene containing no .sup.13C isotopes
(all-.sup.12C.sub.5 isoprene) according to the following set of
linear equations:
P.sub.60=1.00000*k.sub.60
P.sub.61=0.05561*k.sub.60+1.00000*k.sub.61
P.sub.62=0.01238*k.sub.60+0.05561*k.sub.61+1.00000*k.sub.62
P.sub.63=0.01238*k.sub.61+0.05561*k.sub.62+1.00000*k.sub.63
P.sub.64=0.01238*k.sub.62+0.05561*k.sub.63+1.00000*k.sub.64
P.sub.65=0.01238*k.sub.63+0.05561*k.sub.64+1.00000*k.sub.65
P.sub.66=0.01238*k.sub.64+0.05561*k.sub.65+1.00000*k.sub.66
P.sub.67=0.01238*k.sub.65+0.05561*k.sub.66+1.00000*k.sub.67
P.sub.68=0.01238*k.sub.66+0.05561*k.sub.67+1.00000*k.sub.68
P.sub.69=0.01238*k.sub.67+0.05561*k.sub.68+1.00000*k.sub.69
P.sub.70=0.01238*k.sub.68+0.05561*k.sub.69+1.00000*k.sub.70 (Eqs.
2),
[0921] where P.sub.60 . . . P.sub.70 are the measured intensities
of GC peaks produced by the isoprene standard (subscript indices
indicate m/z values of corresponding peaks), k.sub.60 . . .
k.sub.70 are the calculated intensities of GC peaks that would be
generated by all-.sup.12C.sub.5 isoprene (subscript indices
indicate m/z values of corresponding peaks), and the coefficients
1.00000, 0.05561, and 0.05561 are the estimated relative abundances
of three C.sub.5 cumomers containing zero, one or two .sup.13C
isotopes per molecule assuming that this C.sub.5 compound has
natural abundance of .sup.13C isotope equal to 1.1%. (Note that in
our calculations of all-.sup.12C.sub.5 isoprene spectrum it was
assumed that the natural abundance of deuterium is too small to
affect the final results). The positive values of k.sub.60 . . .
k.sub.70 were obtained using lsqlin solver (MATLAB 7.0, MathWorks).
The calculated values of k.sub.69 and k.sub.70 were effectively
zero indicating that GC spectrum of all-.sup.12C.sub.5 isoprene
should not have any peaks with m/z=69 and higher.
[0922] Cumomer distribution analysis of labeled isoprene samples
was done according to the following set of linear equations based
on the values of k.sub.62 . . . k.sub.68 obtained as described
above:
A.sub.67=k.sub.67*.chi..sub.M0+k.sub.66*.chi..sub.M+1+k.sub.65*.chi..sub-
.M+2+k.sub.64*.chi..sub.M+3+k.sub.63*.chi..sub.M+4+k.sub.62*.chi..sub.M+5
A.sub.68=k.sub.68*.chi..sub.M0+k.sub.67*.chi..sub.M+1+k.sub.66*.chi..sub-
.M+2+k.sub.65*.chi..sub.M+3+k.sub.64*.chi..sub.M+4+k.sub.63*.chi..sub.M+5
A.sub.69=k.sub.68*.chi..sub.M+1+k.sub.67*.chi..sub.M+2+k.sub.66*.chi..su-
b.M+3+k.sub.65*.chi..sub.M+4+k.sub.64*.chi..sub.M+5
A.sub.70=k.sub.68*.chi..sub.M+2+k.sub.67*.chi..sub.M+3+k.sub.66*.chi..su-
b.M+4+k.sub.65*.chi..sub.M+5
A.sub.70=k.sub.68*.chi..sub.M+3+k.sub.67*.chi..sub.M+4+k.sub.66*.chi..su-
b.M+5
A.sub.71=k.sub.68*.chi..sub.M+4+k.sub.67*.chi..sub.M+5
A.sub.72=k.sub.68*.chi..sub.M+5 (Eqs. 3),
[0923] where A.sub.67-A.sub.72 are the measured intensities of GC
peaks produced by .sup.13C-enriched isoprene (subscript indices
indicate m/z values of corresponding peaks) and .chi..sub.M0 . . .
.chi..sub.M+5 are the relative abundances of isoprene cumomers
having from zero to five .sup.13C atoms (.chi..sub.M0 corresponds
to the isoprene molecules in which all carbons atoms are
represented by the isotope .sup.12C). The non-negative values of
.chi..sub.M0 . . . .chi..sub.M+5 were obtained using the isqlin
solver (MATLAB 7.0, MathWorks).
Determination of Relative Contribution of DXP and MVA Pathways to
the Isoprene Production
[0924] The relative contribution of DXP and MVA pathways to the
total isoprene production (.phi..sup.DXP and .phi..sup.MVA,
respectively) was estimated by solving in MATLAB (MathWorks) the
following overdetermined system of linear equations:
.chi..sub.MO,Isp-FM=.phi..sup.DXP*.chi..sub.MO,cMEPP+.phi..sup.MVA*.chi.-
.sub.MO,Isp+FM
.chi..sub.M+1,Isp-FM=.phi..sup.DXP*.chi..sub.M+1,cMEPP+.phi..sup.MVA*.ch-
i..sub.M+1,Isp+FM
.chi..sub.M+2,Isp-FM=.phi..sup.DXP*.chi..sub.M+2,cMEPP+.phi..sup.MVA*.ch-
i..sub.M+2,Isp+FM
.chi..sub.M+3,Isp-FM=.phi..sup.DXP*.chi..sub.M+3,cMEPP+.phi..sup.MVA*.ch-
i..sub.M+3,Isp+FM
.chi..sub.M+4,Isp-FM=.phi..sup.DXP*.chi..sub.M+4,cMEPP+.phi..sup.MVA*.ch-
i..sub.M+4,Isp+FM (Eqs. 4),
[0925] where .chi..sub.M0,Isp-FM . . . .chi..sub.M+4,Isp-FM and
.chi..sub.M0,Isp+FM . . . .chi..sub.M+4,Isp+FM are the relative
abundances of isoprene cumomers containing from zero to four
.sup.13C atoms calculated according to Eqs. 3 (subscript indices
"Isp+FM" and "Isp-FM" indicate that calculations were done for
cells incubated with and without fosmidomycin, respectively),
.chi..sub.M0,cMEPP . . . .chi..sub.M+4,cMEPP are the relative
abundances of corresponding cMEPP cumomers measured by LC-MS/MS as
described above.
Example 34
.sup.13C NMR Method for the Determination of Carbon Fluxes Through
the MVA and MEP Pathways Leading to BioIsoprene.TM. Product
[0926] In this Example, BioIsoprene.TM. refers to isoprene that is
biologically produced, e.g., using the methods described herein.
BioIsoprene.TM. was obtained from bioisoprene composition. The
relative contributions of the two isoprenoid precursor pathways,
the MVA and MEP (DXP) pathways, to isoprene production in a REM
A2.sub.--17 dual pathway strain were determined by .sup.13C NMR
spectroscopy and the resulting information used to calculate the
MVA/MEP carbon ratio. Similar techniques have been used to
determine the respective contributions of the MVA and MEP pathways
to the biosynthesis of polyisoprenoids (Skorupinska-Tudek, K. et
al. (2008) J. Biol. Chem., 283(30), pp. 21024-21035.) and isoprene
(Wagner, W. P., Helmig, D. and Fall, R. (2000) J. Nat. Prod., 63,
pp. 37-40). The labeling patterns of isoprene derived from .sup.13C
enriched glucose labels differ according to which pathway was
utilized to channel carbon from the substrate to product. These
patterns are shown in FIG. 100A for a [1-.sup.13C]-D-glucose
substrate and FIG. 100B for a [3-.sup.13C]-D-glucose substrate.
[0927] As can be seen from FIG. 100A, carbon #3 (C-3) of isoprene
is not enriched from either pathway from a [1-.sup.13C]-D-glucose
substrate, with the extent of .sup.13C-enrichment equal to the
natural abundance of 1.1% relative to .sup.12C. In contrast, C-5 is
labeled in both cases, thus the enrichment of C-5/C-3 allows the
determination of the total extent of .sup.13C-label incorporation.
The maximum possible .sup.13C enrichment at C-5 of Biosoprene.TM.
product derived from [1-.sup.13C]-D-glucose is 50%, with less if
oxidative pentose phosphate pathway is operating at a significant
flux relative to glycolysis. The ratio of MVA/MEP pathways is
determined by comparing the enrichment of C-1 relative to C-2 and
C-4. This is shown in FIG. 103.
[0928] In the case where carbon flux though the MVA and MEP
pathways is equal (1:1 MVA/MEP ratio), the extent of labeling at
C-1 relative to C-2 and C-4 is also equivalent in the
Bioisoprene.TM. product, with a maximum enrichment of 25%. At a
MVA/MEP ratio of 9:1, C-1 is only enriched to the extent of 5%,
whereas C-2 and C-4 are enriched to a level of 45%.
[0929] A method for the small-scale generation, collection and
analysis of .sup.13C-labeled BioIsoprene.TM. product was developed
in order to determine the relative contributions of the MVA and MEP
(DXP) pathways to isoprene production in strain REM A2.sub.--17.
The strain was grown in HM-1 media with [1-.sup.13C]-D-glucose (10
mg/mL) as the sole carbon source in a stirred bottle format and the
resulting BioIsoprene.TM. product was adsorbed to a small carbon
filter consisting of 200 mg activated carbon (Koby filters, MA)
packed into a glass Pasteur pipette with cotton wool (Scheme xx-1).
After overnight growth at 34.degree. C., the carbon filter was
removed and desorbed directly into a glass NMR tube with CDCl.sub.3
(1 mL). A reference spectrum of unlabeled isoprene was obtained by
diluting an isoprene standard (5 uL) (Sigma-Aldrich) into 0.75 mL
of deuterochloroform (CDCl3) and acquiring a .sup.13C NMR
spectrum.
[0930] Relative .sup.13C-enrichment of isoprene at each carbon atom
was determined by .sup.13C nuclear magnetic resonance spectroscopy
(.sup.13C-NMR) by determining the relative intensities of the
signals corresponding to each carbon atom of isoprene and comparing
these values to the relative intensities of the carbon signals from
unlabeled (natural .sup.13C abundance) isoprene. .sup.13C NMR
spectra were obtained on a Varian 500 MHz VNMRS system operating at
125.7 MHz. Acquisition parameters were sw=30487, at=1.3 sec, dl=1,
nt=10000, do=H1, dm=yyy, dmm=w, dpr=42, dmf=12600. .sup.13C signal
intensity was determined by peak height and integrated peak area.
The .sup.13C-NMR spectrum of unlabeled isoprene (% .sup.13C=1.1%)
is shown in FIG. 105. The peak heights of carbons 1-4 are similar,
with aliphatic C-5 showing a more intense signal.
[0931] The .sup.13C NMR spectrum of the BioIsoprene.TM. product
derived from dual pathway strain REM A2.sub.--17 is shown in FIG.
106. The signals for C-1, 3, 4 and 5 are clearly evident, whereas
the C-2 signal is equal or less than, or equal to the noise level.
The relative peak heights of C-1, C-3 and C-4 indicate that the
ratio of MVA/DXP pathway flux is more than 1:1 and less than 2:1.
The enrichment of C-1, 3 and 4 relative to C-2 and C-5 indicate
that both the MVA and DXP pathways are operating in strain REM
A2.sub.--17 and contribute to overall carbon flux to isoprene.
Example 35
fkpB-ispH iscR
[0932] In this example, we show that when the promoter PL.6
replaced the native promoter of the operon fkpB-ispH in strain
WW119 to create strain REM D8.sub.--15, isoprene production drops
from .apprxeq.500 to 600 ug/L/H/OD seen in strain WW119 (see FIG.
108) to .apprxeq.50 ug/L/H/OD in strain REM D8.sub.--15 (see FIG.
108). Addition of .DELTA.ispR to WW119 showed a small decrease in
isoprene specific productivity. The result observed for the
introduction of PL.6 fkpB-ispH into WW119 was unanticipated. Our
hypothesis was that more ispH would yield higher isoprene titer. We
further show that when the iron sulfur cluster regulatory gene,
iscR, is deleted from the latter strain, REM D8.sub.--15, to create
strain REM D6.sub.--15 the .DELTA.iscR mutation substantially
restores isoprene production to strain REM D6.sub.--15. These
observations suggest a beneficial interaction between .DELTA.iscR
and fkpB-ispH that can improve the process of isoprene production
via the DXP pathway.
Construction of Strains for this Example. Generation of the
PL.6-fkpB Locus
[0933] Within the E. coli BL21 genome the ispH gene is located
immediately downstream of the fkpB gene which encodes a FKBP-type
peptidyl-prolyl cis-trans isomerase. Interestingly, the structure
of the E. coli IspH enzyme shows that the protein has 2 proline
residues that are isomerized (Grawert, T. et al., 2009). The idea
that FkpB could be involved in IspH function may also be reflected
by the fact that the fkpB and ispH orfs are separated by just one
nucleotide and together have been shown to be transcribed as the
last 2 genes of the ribF-ileS-lspA-fkpB-ispH 5-gene operon (see
www.ecocyc.org).
[0934] Further more, BLAST analysis of the 125 bases separating the
stop codon of lspA and the start codon of fkpB revealed a highly
conserved sequence that occurs many times throughout the E. coli
genome. This commonly found sequence is:
TABLE-US-00048 (SEQ ID NO: 187)
AATCGTAGGCCGGATAAGGCGTTTACGCCGCATCCGGCAA
[0935] This sequence harbors characteristics of a transcriptional
terminator, which includes the likely formation of a stem loop. The
bases with potential of hybridizing together to form the stem loop
are highlighted above in bold and underlined text (bold anneals to
bold; underlined anneals to underlined). The location of this
repeated sequence, in each instance observed, was always found just
downstream of the 3' end of a single gene or downstream of the 3'
ends of 2 genes transcribed toward one another. The repeated
sequence was not found within the coding region of over 40 regions
analyzed. Together, this information suggests that the sequence
functions as a transcriptional terminator and hints at the
possibility of fkpB and ispH being transcribed as an independent
2-gene operon.
[0936] Our in-house transcriptional analyses of BL21 14-L
fermentations show the ispH transcript to be present at almost
undetectable levels; a result inline with that previously reported
in the field. Similarly, the level of IspH protein accumulates to
low levels within these and small scale grown cells (for small
scale result see FIG. 108). Increased expression of endogenous BL21
ispH and its effect on isoprene production was an aim of the work
described here. The previously described PL.6-promoter is a strong
constitutive promoter chosen to up-regulate the expression of ispH.
Based on the speculation that FkpB and IspH as well as fkpB and
ispH potentially share a functional and a transcriptional
relationship, respectively (described above), the PL.6-promoter was
inserted immediately upstream of the fkpB orf.
[0937] The PL.6-promoter insertion and subsequent loopout of the
chloramphenicol resistance marker described in this example was
carried out using the Red/ET system from Gene Bridges GmbH
according to the manufacturer's instructions. The strain BL21
(Novagen) was used. P1 lysate preparations and transductions were
performed as previously described (Thomason et al., 2007). The BIO
RAD Gene Pulser system (0.1 cm cuvette cat.#165-2089) and a
transformation protocol suggested by the manufacturer (BIO RAD) was
used for the electroporations described.
TABLE-US-00049 Primers 5' CMP::80bp up of fkpB (SEQ ID NO: 188)
5'-AGATTGCTGCGAAATCGTAGGCCGGATAAGGCGTTTACGC
CGCATCCGGCAAAAATCCTTAAATATAAGAGCAAACCTGCAA
TTAACCCTCACTAAAGGGCGGCCGC 3' CMP::PL.6-fkpB (SEQ ID NO: 189)
5'-AGCGTGAAGTGCACCAGGACGGCGCTATTGCTCTGTACAGATTCAGA
CATGTTTTTACCTCCTTTGCAGTGCGTCCTGCTGATGTGCTCAGTATCA
CCGCCAGTGGTATTTATGTCAACACCGCCAGAGATAATTTATCACCGCA
GATGGTTATCTTAATACGACTCACTATAGGGCTCGAG 5' confirm CMP::80bp up of
fkpB (SEQ ID NO: 190) 5'-ACGCATCTTA TCCGGCCTACA 3' confirm
CMP::PL.6-fkpB (SEQ ID NO: 191) 5'-ACCGTTGTTGCGGGTAGACTC 5' primer
to PL.6 (SEQ ID NO: 162) 5'-AGATAACCATCTGCGGTGATAAATTATCTCTGGCGGTG
top Gb's CMP (SEQ ID NO: 144) 5'-ACTGAAACGTTTTCATCGCTC bottom Pgb2
(SEQ ID NO: 163) 5'-GGTTTAGTTCCTCACCTTGTC
[0938] The PL.6-promoter introduced upstream of the endogenous fkpB
coding region using the Gene Bridges GmbH methods is illustrated in
FIG. 107. The antibiotic resistance cassette GB-CMP was amplified
by PCR using the primer set 5' CMP::80 bp up of fkpB and 3'
CMP::PL.6-fkpB. The 5' CMP::80 bp up of fkpB primer contains 80
bases of homology to the region immediately 5' to the fkpB coding
region and the 3' CMP::PL.6-fkpB primer contains 50 bases of
homology to the 5' region of the fkpB orf (open reading frame) to
allow recombination at the specific locus upon electroporation of
the PCR product in the presence of the pRed-ET plasmid. The FRT
(Flipase recognition target) "scar" sequence remaining after
Flipase-mediated excision of the antibiotic marker is also depicted
in the figure.
Amplification of the CMP:: PL.6 fkpB Fragment
[0939] To amplify the GB-CmpR cassette for inserting the
PL.6-promoter immediately upstream of the fkpB locus the following
PCR reaction was set up:
1 ul template (100 ng GB-CmpR)
10 ul HerculaseII Buffer
[0940] 0.5 ul dNTP's (100 mM) 1.25 ul primer (10 uM) 5' CMP::80 bp
up of fkpB 1.25 ul primer (10 uM) 3' CMP::PL.6-fkpB 35 ul diH2O +1
ul of HerculaseII fusion from Stratagene
Cycle Parameter:
[0941] 95.degree. C..times.2 min., [95.degree. C..times.30 sec.,
60.degree. C..times.30 sec., 72.degree. C..times.3 min.].times.29
cycles; 72.degree. C..times.5 min., 4.degree. C. until cool
(Biometra T3000 Combi Thermocycler)
[0942] The resulting PCR fragment was separated on a 0.8% E-gel
(Invitrogen) for verification of successful amplification, and
purified using the QIAquick PCR Purification kits (Qiagen)
according to manufacturer's instructions. The resulting stock was
CMP::PL.6 fkpB fragment.
Integration of CMP::PL.6 fkpB Fragment PCR Product into
BL21/pRed-ET Strain
[0943] The pRed-ET vector (Gene Bridges kit) was transformed into
BL21 (Novagen) by electroporation resulting in strain REM
F7.sub.--13 (BL21/pRed-ET). The purified CMP::PL.6 fkpB PCR
fragment was electroporated into REM F7.sub.--13. The transformants
were recovered in L Broth and then plated on L agar containing
chloramphenicol (10 ug/ml). Chloramphenicol resistant colonies were
analyzed by PCR for the presence of the GB-CmpR cassette and the
PL.6-promoter upstream of fkpB using primers 5' confirm CMP::80 bp
up of fkpB and bottom Pgb2 as well as 3' confirm CMP::PL.6-fkpB and
top Gb's CMP. The PCR fragments from a number of transformants were
sequenced using the 3' confirm CMP::PL.6-fkpB and top GB's CMP
primers (Sequetech; Mountain View, Calif.) and PL.6 fkpB strain of
interest identified. The chloramphenicol resistant strain, BL21
CMP::PL.6 fkpB, was designated REM A4.sub.--14.
Strategy for Creating REM D1.sub.--14
[0944] Verification of the Presence of PL.6 fkpB within REM
D1.sub.--14
[0945] To verify the REM D1.sub.--14 strain harbored the PL.6 fkp
locus the following PCR reaction was set up:
Approx. 0.5 ul cells from a colony
5 ul HerculaseII Buffer
[0946] 0.25 ul dNTP's (100 mM) 0.625 ul primer (10 uM) 5' primer to
PL.6 0.625 ul primer (10 uM) 3' confirm CMP::PL.6-fkpB 17.5 ul
diH2O +0.5 ul of HerculaseII fusion from Stratagene
Cycle Parameter:
[0947] 95.degree. C..times.2 min., [95.degree. C..times.30 sec.,
60.degree. C..times.30 sec., 72.degree. C..times.2 min.].times.29
cycles; 72.degree. C..times.5 min., 4.degree. C. until cool
(Biometra T3000 Combi Thermocycler)
[0948] The resulting PCR fragment was separated on a 2% E-gel
(Invitrogen) for verification of successful amplification.
[0949] The chloramphenicol marked PL.6 fkpB locus of strain REM
A4.sub.--14, described above, was introduced into strain WW103 via
P1-mediated transduction. The resulting chloramphenicol resistant
strain was named REM A9.sub.--14. After Flipase-mediated excision
of the antibiotic cassette the resulting chloramphenicol sensitive
strain was designated REM D1.sub.--14 (BL21 pgl.sup.+ PL.6-dxs,
GI1.6-dxr, GI1.6 yIDI, PL.2 lower MVA pathway, CMP::PL.6 fkpB). The
presence of the PL.6-promoter upstream of fkpB within REM
D1.sub.--14 was verified by PCR using primers 5' primer to PL.6 and
3' confirm CMP::PL.6-fkpB, which are described above.
Strategy for Creating REM A8.sub.--15
[0950] The chloramphenicol marked .DELTA.iscR locus of strain
REM14::CMP, described previously, was introduced into strain WW103
via P1-mediated transduction. The resulting chloramphenicol
resistant strain was named REM A5.sub.--15. After Flipase-mediated
excision of the antibiotic cassette the resulting chloramphenicol
sensitive strain was designated REM A8.sub.--15 (BL21 pgl.sup.+
PL.6-dxs, GI1.6-dxr, GI1.6 yIDI, PL.2 lower MVA pathway,
.DELTA.iscR).
Strategy for Creating REM A7.sub.--15
[0951] The chloramphenicol marked .DELTA.iscR locus of strain
REM14::CMP was introduced into strain REM D1.sub.--14 via
P1-mediated transduction. The resulting chloramphenicol resistant
strain was named REM A2.sub.--15. After Flipase-mediated excision
of the antibiotic cassette the resulting chloramphenicol sensitive
strain was designated REM A7.sub.--15 (BL21 pgl.sup.+ PL.6-dxs,
GI1.6-dxr, GI1.6 yIDI, PL.2 lower MVA pathway, CMP::PL.6 fkpB,
.DELTA.iscR).
Verification of Increased Accumulation of IspH within REM
D1.sub.--14 and REM A7.sub.--15
Western Blot Method
[0952] REM D1.sub.--14, REM A7.sub.--15, REM A8.sub.--15, and WW103
cells were grown in TM3 medium (1% glucose, 0.1% yeast extract) to
limiting OD and cells were harvested by centrifugation and pellets
stored at -80 deg until analyzed. For analysis culture pellets were
resuspended in 0.05 M sodium phosphate, 0.3 M sodium chloride, 0.02
M imidazole, pH 8 with 0.2 mg/ml DNaseI to 100 OD/ml. Cells were
broken by repeated pass through the French Press. 8 ml of each
lysate was then clarified by ultracentrifugation at 50,000 rpm for
30 minutes. Soluble material was removed and the insoluble pellet
was resuspended in 8 ml of 0.05 M sodium phosphate, 0.3 M sodium
chloride, 0.02 M imidazole, pH 8 buffer. Analysis for E. coli ispH
expression was performed using Nitrocellulose western blot,
following transfer and development techniques recommended by
Invitrogen as described in iBlot.RTM. and WesternBreeze.RTM. user
manuals. The western blot was probed using primary polyclonal
antibody produced against purified E. coli ispH in rabbit by ProSci
Inc. The detection used a fluorescent secondary antibody from
Invitrogen, Alexa Fluor.RTM. 488 goat anti-rabbit IgG (H+L). The
raw data is shown in FIG. 109. Sample quantitation was performed
using ImageQuant 5.2 software and the results are presented in FIG.
110.
[0953] The increased expression of ispH driven by the PL.6-promoter
located upstream of the fkpB-ispH 2 gene operon of strains REM
D1.sub.--14 and REM A7.sub.--15 relative to strain REM A8.sub.--15
and WW103 was indirectly assessed by measuring the level of IspH
accumulation via a Western blot method (see FIGS. 109 and 110). An
approximately 5-fold increase in soluble IspH levels was determined
for the PL.6 fkpB harboring strains REM D1.sub.--14 and REM
A7.sub.--15 relative to the REM A8.sub.--15 and WW103 strains which
harbor the endogenous wild type fkpB-ispH locus.
Strategy for Creating REM D8.sub.--15, REM D7.sub.--15, and REM
D6.sub.--15
[0954] Strains WW119, REM D8.sub.--15, REM D7.sub.--15, and REM
D6.sub.--15 were created by transforming pDW33 into WW103, REM
D1.sub.--14, REM A8.sub.--15, and REM A7.sub.--15, respectively
(strains described above).
[0955] Water-washed REM D1.sub.--14, REM A8.sub.--15, and REM
A7.sub.--15 cells were transformed with pDW33 via electroporation
using the BIO RAD Gene Pulser system (0.1 cm cuvette cat.#165-2089)
and a transformation protocol suggested by the manufacturer (BIO
RAD). The cells were recovered in L broth for 1 hour at 37.degree.
C. and then plated on L agar containing carbenicillin (50 ug/ml).
One carbenicillin resistant colony was chosen for each strain. The
resulting carbenicillin resistant strains were named as such:
REM D8.sub.--15 (BL21 pgl.sup.+ PL.6-dxs, GI1.6-dxr, GI1.6 yIDI,
PL.2 lower MVA pathway, PL.6 fkpB, and pDW33); REM D7.sub.--15
(BL21 pgl.sup.+ PL.6-dxs, GI1.6-dxr, GI1.6 yIDI, PL.2 lower MVA
pathway, .DELTA.iscR, and pDW33); REM D6.sub.--15 15 (BL21
pgl.sup.+ PL.6-dxs, GI1.6-dxr, GI1.6 yIDI, PL.2 lower MVA pathway,
PL.6 fkpB, .DELTA.iscR, and pDW33). Method Section for Isoprene
Production and Quantitation of ispH
[0956] Isoprene Measurements.
[0957] Cultures to measure isoprene production were set up in a
48-deep-well plate (cat# P-5ML-48-C-S Axygen Scientific,
California, USA) with each well providing a 2 mL culture. The
culture medium, named TM3, is described below. The strains to be
compared were grown o/n at 30 degrees at 250 rpm in TM3 medium
supplemented with 1% glucose and 0.1% yeast extract. In the morning
the strains were inoculated at 1:100 in quadruplicate sets of wells
in the 48-deep well block. The cultures were covered with a "Breath
Easier".TM. membrane (Electron Microscopy Sciences Cat#70536-10)
and were continuously shaken at at 600 rpm and 30 deg C (Shel-Lab
Inc. Model SI6R Refrigerated Shaking Incubator; Oregon, USA).
Culture OD was determined after two hours and then at timed
intervals out to 6 hours. Induction with IPTG was after two hours
of growth by the addition of 50, 100, 200, and 400 uM IPTG to the
quadruplicated sets of wells, one through four. At two hours
post-induction and hourly thereafter out to six hours these
cultures were samples for isoprene production assays as follow: A
100 uL aliquot of each culture was transferred to a 98-deep well
glass block (cat#3600600 Zinsser; North America) which was
immediately sealed with an impermeable adhesive aluminum film and
incubated for 30 minutes with shaking at 450 rmp on an Eppendorf
thermomixer (Eppendorf; North America.). The isoprene assay
cultures were killed by heating at 70 deg C for 7 min on a second
Eppendorf thermomixer. The glass block was transferred to an
Agilent 6890 GC attached to an Agilent 5973 MS and outfitted with a
LEAP CTC CombiPAL autosampler for head space analysis. The column
was an Agilent HP-5 (5% Phenyl Methyl Siloxane (15 m.times.0.25
mm.times.0.25 um)). A 100 uL gas volume was injected on the column.
Other conditions were as follows. Oven Temperature: 37 C (held
isothermal for 0.6 mins); Carrier Gas: Helium (flow -1 mL/min),
split ratio of 50:1 at 250.degree. C. on the injection port; Single
Ion Monitoring mode (SIM) on mass 67; Detector off: 0.00 min-0.42
mins; Dectector on: 0.42 mins-0.60 mins; elution time for Isoprene
(2-methyl-1,3 butadiene) was .about.0.49 min for a total analysis
time of 0.6 mins. Calibration of the instrument was performed by
methods well known to those trained in the art.
[0958] Isoprene head space measurements were normalized by culture
OD.sub.600 to yield a measure of specific isoprene production in
units of ug/L/H/OD. All reactions were followed for 4 to 8 hours.
The surprising results from this experiment is that when the
.DELTA.iscR mutation is combined with the chromosomal mutation of
PL.6 fkpB-ispH isoprene activity is restored. This result is
consistent with that iscR in a background of overexpressed ispH
takes on a regulatory role or at least interferes with flux through
the DXP pathway. For high flux ispH needs to be overexpressed and
under these condition .DELTA.iscR expected to be beneficial for the
process.
[0959] Verification of Increased ispH Expression Level by Western
Blot.
[0960] The substitution of the PL.6 promoter for the native
promoter of the fkpB-ispH operon was expected to raise the level of
ispH. This was confirmed in strain REM A7.sub.--15, REM D1.sub.--14
by comparison to control strains REM A8.sub.--15 and WW103 by
western Blot with polyclonal antibody prepared against this enzyme
as described; the promoter swap resulted in a 5-fold increase of
soluble ispH. Cells were grown in TM3 medium (1% glucose, 0.1%
yeast extract) to limiting OD and were harvested by centrifugation
and the pellets were stored at -80 deg until the next day. For
analysis pellets were resuspended in 0.05 M sodium phosphate, 0.3 M
sodium chloride, 0.02 M imidazole, pH 8 with 0.2 mg/ml DNaseI to
100 OD/ml. Cells were broken by repeated passage through the French
press. Eight ml of each lysate was clarified by ultracentrifugation
at 100,000.times.g for 30 minutes. Supernatant was removed and the
pellet was resuspended in 8 ml of buffer pH8, 0.05 M sodium
phosphate, 0.3 M sodium chloride, 0.02 M imidazole. Western blot
was performed as described in the users manuals iBlot.RTM. and
WesternBreeze.RTM. (in Vitrogen). The primary polyclonal antibody
was against purified E. coli IspH overexpressed in E. coli and
raised in rabbit by ProSci Inc (Poway, Calif.). For detection a
fluorescent secondary antibody from Invitrogen (Alexa Fluor.RTM.
488 goat anti-rabbit IgG H+L), was used. The raw data is shown in
FIG. 109. Sample quantitation was performed using ImageQuant 5.2
software and the results are presented in FIG. 110.
TM3 (Per Liter Fermentation Medium):
[0961] K.sub.2HPO.sub.4 13.6 g, KH.sub.2PO.sub.4 13.6 g,
MgSO.sub.4*7H.sub.2O 2 g, citric acid monohydrate 2 g, ferric
ammonium citrate 0.3 g, yeast extract 1.0 g, 1000.times. Modified
Trace Metal stock solution 1 ml. All of the components were added
together and dissolved in Di H2O. The pH is adjusted to 6.8 with
NH.sub.4OH and the solution is filter sterilized over a 0.22 micron
membrane. Glucose was typically added at 1% and yeast extract was
typically boosted to 0.1%. Antibiotics were added post-sterile as
needed (TM3 medium was sometimes prepared w/o any MgSO4 as this
Mg++ led to precipitation over time. In this case MgSO4 was added
from a sterile 1M solution just prior to use).
1000.times. Modified Trace Metal Stock Solution (Per Liter):
[0962] Citric Acids*H.sub.2O 40 g, MnSO.sub.4*H.sub.2O 30 g, NaCl
10 g, FeSO.sub.4*7H.sub.2O 1 g, CoCl.sub.2*6H.sub.2O 1 g,
ZnSO.sub.4*7H.sub.2O 1 g, CuSO.sub.4*5H.sub.2O 100 mg,
H.sub.3BO.sub.3 100 mg, NaMoO.sub.4*2H.sub.2O 100 mg. Each
component was dissolved one at a time in Di H.sub.2O, pH was
adjusted to 3.0 with HCl/NaOH, and then the solution was q.s. to
volume and filter sterilized with a 0.22 micron filter.
APPENDIX 1
Exemplary 1-deoxy-D-xylulose-5-phosphate synthase nucleic acids and
polypeptides
TABLE-US-00050 [0963] ATH: AT3G21500(DXPS1) SEW: SeSA_A0482(dxs)
AT4G15560(CLA1) AT5G11380(DXPS3) SES: SARI_02505 OSA:
4338768(Os05g0408900) STM: STM0422(dxs) 4340090(Os06g0142900) YPE:
YPO3177(dxs) 4342614(Os07g0190000) YPK: y1008(dxs) PPP:
PHYPADRAFT_105028(DXS1) YPM: YP_0754(dxs) PHYPADRAFT_137710 YPA:
YPA_2671 PHYPADRAFT_175220 YPN: YPN_0911 PHYPADRAFT_73475 YPP:
YPDSF_2812 OLU: OSTLU_48774(DXS) YPG: YpAngola_A3074(dxs) CRE:
CHLREDRAFT_196568(DXS1) YPS: YPTB0939(dxs) CME: CMF089C YPI:
YpsIP31758_3112(dxs) PFA: MAL13P1.186 YPY: YPK_3253 PFD: PFDG_00954
YPB: YPTS_0980 PFH: PFHG_02940 YEN: YE3155(b0420) PYO: PY04970 SFL:
SF0357(dxs) TAN: TA20470 SFX: S0365(dxs) TPV: TP01_0516 SFV:
SFV_0385(dxs) ECO: b0420(dxs) SSN: SSON_0397(dxs) ECJ: JW0410(dxs)
SBO: SBO_0314(dxs) ECD: ECDH10B_0376(dxs) SBC: SbBS512_E0341(dxs)
ECE: Z0523(dxs) SDY: SDY_0310(dxs) ECS: ECs0474 ECA: ECA1131(dxs)
ECC: c0531(dxs) ETA: ETA_25270(dxs) ECI: UTI89_C0443(dxs) PLU:
plu3887(dxs) ECP: ECP_0479 BUC: BU464(dxs) ECV: APECO1_1590(dxs)
BAS: BUsg448(dxs) ECW: EcE24377A_0451(dxs) WBR: WGLp144(dxs) ECX:
EcHS_A0491(dxs) SGL: SG0656 ECM: EcSMS35_0456(dxs) ENT: Ent638_0887
ECL: EcolC_3213 ESA: ESA_02882 STY: STY0461(dxs) KPN:
KPN_00372(dxs) STT: t2441(dxs) CKO: CKO_02741 SPT: SPA2301(dxs)
SPE: Spro_1078 SPQ: SPAB_03161 BFL: Bfl238(dxs) SEC: SC0463(dxs)
BPN: BPEN_244(dxs) SEH: SeHA_C0524(dxs) HIN: HI1439(dxs) SEE:
SNSL254_A0469(dxs) HIT: NTHI1691(dxs) HIP: CGSHiEE_04795 PEN:
PSEEN0600(dxs) HIQ: CGSHiGG_01080 PMY: Pmen_3844 HDU: HD0441(dxs)
PSA: PST_3706(dxs) HSO: HS_0905(dxs) CJA: CJA_3336(dxs) HSM:
HSM_1383 PAR: Psyc_0221(dxs) PMU: PM0532(dxs) PCR: Pcryo_0245 MSU:
MS1059(dxs) PRW: PsycPRwf_0411 APL: APL_0207(dxs) ACI:
ACIAD3247(dxs) APJ: APJL_0208(dxs) ACB: A1S_3106 APA: APP7_0210
ABM: ABSDF0389(dxs) ASU: Asuc_1372 ABY: ABAYE0381 XFA: XF2249 ABC:
ACICU_03307 XFT: PD1293(dxs) SON: SO_1525(dxs) XFM: Xfasm12_1447
SDN: Sden_2571 XFN: XfasM23_1378 SFR: Sfri_2790 XCC: XCC2434(dxs)
SAZ: Sama_2436 XCB: XC_1678 SBL: Sbal_1357 XCV: XCV2764(dxs) SBM:
Shew185_1343 XAC: XAC2565(dxs) SBN: Sbal195_1382 XOO: XOO2017(dxs)
SLO: Shew_2771 XOM: XOO_1900(XOO1900) SPC: Sputcn32_1275 SML:
Smlt3355(dxs) SSE: Ssed_3329 SMT: Smal_2779 SPL: Spea_2991 VCH:
VC0889 SHE: Shewmr4_2731 VCO: VC0395_A0412(dxs) SHM: Shewmr7_2804
VVU: VV1_0315 SHN: Shewana3_2901 VVY: VV0868 SHW: Sputw3181_2831
VPA: VP0686 SHL: Shal_3080 VFI: VF0711 SWD: Swoo_3478 VHA:
VIBHAR_01173 ILO: IL2138(dxs) PPR: PBPRA0805 CPS: CPS_1088(dxs)
PAE: PA4044(dxs) PHA: PSHAa2366(dxs) PAU: PA14_11550(dxs) PAT:
Patl_1319 PAP: PSPA7_1057(dxs) SDE: Sde_3381 PPU: PP_0527(dxs) MAQ:
Maqu_2438 PPF: Pput_0561 AMC: MADE_01425 PPG: PputGB1_0572 PIN:
Ping_2240 PPW: PputW619_0579 MCA: MCA0817(dxs) PST: PSPTO_0698(dxs)
FTU: FTT1018c(dxs) PSB: Psyr_0604 FTF: FTF1018c(dxs) PSP:
PSPPH_0599(dxs) FTW: FTW_0925(dxs) PFL: PFL_5510(dxs) FTL: FTL_1072
PFO: PflO1_5007 FTH: FTH_1047(dxs) FTA: FTA_1131(dxs) BPL:
BURPS1106A_A2392(dxs) FTN: FTN_0896(dxs) BPD: BURPS668_A2534(dxs)
FTM: FTM_0932(dxs) BTE: BTH_II0614(dxs) FPH: Fphi_1718 BPH:
Bphy_3948 NOC: Noc_1743 PNU: Pnuc_1704 AEH: Mlg_1381 PNE: Pnec_1422
HHA: Hhal_0983 BPE: BP2798(dxs) HCH: HCH_05866(dxs) BPA:
BPP2464(dxs) CSA: Csal_0099 BBR: BB1912(dxs) ABO: ABO_2166(dxs)
BPT: Bpet3060(dxs) MMW: Mmwyl1_1145 BAV: BAV2177(dxs) AHA:
AHA_3321(dxs) RFR: Rfer_2875 ASA: ASA_0990(dxs) POL: Bpro_1747 BCI:
BCI_0275(dxs) PNA: Pnap_1501 RMA: Rmag_0386 AAV: Aave_2015 VOK:
COSY_0360(dxs) AJS: Ajs_1038 NME: NMB1867(dxs) VEI: Veis_3283 NMA:
NMA0589(dxs) DAC: Daci_2242 NMC: NMC0352(dxs) MPT: Mpe_A2631 NMN:
NMCC_0354 HAR: HEAR0279(dxs) NGO: NGO0036 MMS: mma_0331 NGK:
NGK_0044 LCH: Lcho_3373 CVI: CV_2692(dxs) NEU: NE1161(dxs) RSO:
RSc2221(dxs) NET: Neut_1501 REU: Reut_A0882 NMU: Nmul_A0236 REH:
H16_A2732(dxs) EBA: ebA4439(dxs) RME: Rmet_2615 AZO: azo1198(dxs)
BMA: BMAA0330(dxs) DAR: Daro_3061 BMV: BMASAVP1_1512(dxs) TBD:
Tbd_0879 BML: BMA10229_1706(dxs) MFA: Mfla_2133 BMN:
BMA10247_A0364(dxs) HPY: HP0354 BXE: Bxe_B2827 HPJ: jhp0328(dxs)
BVI: Bcep1808_4257 HPA: HPAG1_0349 BUR: Bcep18194_B2211 HPS:
HPSH_01830 BCN: Bcen_4486 HHE: HH0608(dxs) BCH: Bcen2424_3879 HAC:
Hac_0968(dxs) BCM: Bcenmc03_3648 WSU: WS1996 BAM: Bamb_3250 TDN:
Suden_0475 BAC: BamMC406_3776 CJE: Cj0321(dxs) BMU: Bmul_4820 CJR:
CJE0366(dxs) BMJ: BMULJ_03696(dxs) CJJ: CJJ81176_0343(dxs) BPS:
BPSS1762(dxs) CJU: C8J_0298(dxs) BPM: BURPS1710b_A0842(dxs) CJD:
JJD26997_1642(dxs) CFF: CFF8240_0264(dxs) OAN: Oant_0547 CCV:
CCV52592_1671(dxs) BJA: bll2651(dxs) CHA: CHAB381_1297(dxs) BRA:
BRADO2161(dxs) CCO: CCC13826_1594(dxs) BBT: BBta_2479(dxs) ABU:
Abu_2139(dxs) RPA: RPA0952(dxs) NIS: NIS_0391(dxs) RPB: RPB_4460
SUN: SUN_2055(dxs) RPC: RPC_1149 GSU: GSU0686(dxs-1) GSU1764(dxs-2)
RPD: RPD_4305 GME: Gmet_1934 Gmet_2822 RPE: RPE_1067 GUR: Gura_1018
Gura_2175 RPT: Rpal_1022 GLO: Glov_2182 Glov_2235 NWI: Nwi_0633
PCA: Pcar_1667(dxs) NHA: Nham_0778 PPD: Ppro_1191 Ppro_2403 BHE:
BH04350(dxs) DVU: DVU1350(dxs) BQU: BQ03540(dxs) DVL: Dvul_1718
BBK: BARBAKC583_0400(dxs) DDE: Dde_2200 BTR: Btr_0649 LIP:
LI0408(dsx) XAU: Xaut_4733 DPS: DP2700 AZC: AZC_3111 DOL: Dole_1662
MEX: Mext_1939 Mext_4309 ADE: Adeh_1097 MRD: Mrad2831_3459
Mrad2831_3992 AFW: Anae109_1136 MET: M446_6352 M446_6391 MXA:
MXAN_4643(dxs) BID: Bind_1811 SAT: SYN_02456 CCR: CC_2068 SFU:
Sfum_1418 CAK: Caul_3314 PUB: SAR11_0611(dxs) SIL: SPO0247(dxs)
MLO: mlr7474 SIT: TM1040_2920 MES: Meso_0735 RSP: RSP_0254(dxsA)
RSP_1134(dxs) PLA: Plav_0781 RSH: Rsph17029_1897 Rsph17029_2795
SME: SMc00972(dxs) RSQ: Rsph17025_2027 Rsph17025_2792 SMD:
Smed_0492 JAN: Jann_0088 Jann_0170 ATU: Atu0745(dxs) RDE:
RD1_0101(dxs) RD1_0548(dxs) ATC: AGR_C_1351 PDE: Pden_0400 RET:
RHE_CH00913(dxs) DSH: Dshi_3294 Dshi_3526 REC:
RHECIAT_CH0001005(dxs) MMR: Mmar10_0849 RLE: RL0973(dxs) HNE:
HNE_1838(dxs) BME: BMEI1498 ZMO: ZMO1234(dxs) ZMO1598(dxs) BMF:
BAB1_0462(dxs) NAR: Saro_0161 BMB: BruAb1_0458(dxs) SAL: Sala_2354
BMC: BAbS19_I04270 SWI: Swit_1461 BMS: BR0436(dxs) ELI: ELI_12520
BMT: BSUIS_A0462(dxs) GOX: GOX0252 BOV: BOV_0443(dxs) GBE:
GbCGDNIH1_0221 BCS: BCAN_A0440(dxs) GbCGDNIH1_2404 ACR: Acry_1833
LRE: Lreu_0958 GDI: GDI1860(dxs) LRF: LAR_0902 RRU: Rru_A0054
Rru_A2619 LFE: LAF_1005 MAG: amb2904 STH: STH1842 MGM: Mmc1_1048
CAC: CAC2077 CA_P0106(dxs) SUS: Acid_1783 CPE: CPE1819 SWO:
Swol_0582 CPF: CPF_2073(dxs) CSC: Csac_1853 CPR: CPR_1787(dxs) BSU:
BSU24270(dxs) CTC: CTC01575 BHA: BH2779 CNO: NT01CX_1983 BAN:
BA4400(dxs) CTH: Cthe_0828 BAR: GBAA4400(dxs) CDF: CD1207(dxs) BAA:
BA_4853 CBO: CBO1881(dxs) BAT: BAS4081 CBA: CLB_1818(dxs) BCE:
BC4176 CBH: CLC_1825(dxs) BCA: BCE_4249(dxs) CBL: CLK_1271(dxs)
BCZ: BCZK3930(dxs) CBK: CLL_A1441 CLL_A2401(dxs) BCY: Bcer98_2870
CBB: CLD_2756(dxs) BTK: BT9727_3919(dxs) CBF: CLI_1945(dxs) BTL:
BALH_3785(dxs) CBE: Cbei_1706 BWE: BcerKBAB4_4029 CKL:
CKL_1231(dxs) BLI: BL01523(dxs) CPY: Cphy_2511 BLD: BLi02598(dxs)
AMT: Amet_2508 BCL: ABC2462(dxs) AOE: Clos_1607 BAY: RBAM_022600
CHY: CHY_1985(dxs) BPU: BPUM_2159 DSY: DSY2348 GKA: GK2392 DRM:
Dred_1078 GTN: GTNG_2322 PTH: PTH_1196(dxs) LSP: Bsph_3509 DAU:
Daud_1027 ESI: Exig_0908 HMO: HM1_0295(dxs) LMO: lmo1365(tktB) TTE:
TTE1298(dxs) LMF: LMOf2365_1382(dxs) TEX: Teth514_1540 LIN: lin1402
TPD: Teth39_1103 LWE: lwe1380(tktB) MTA: Moth_1511 LLA:
L108911(dxsA) L123365(dxsB) MPE: MYPE730 LLC: LACR_1572 LACR_1843
MGA: MGA_1268(dxs) LLM: llmg_0749(dxsB) MTU: Rv2682c(dxs1)
Rv3379c(dxs2) SAK: SAK_0263 MTC: MT2756(dxs) LPL: lp_2610(dxs) MRA:
MRA_2710(dxs1) MRA_3419(dxs2) LJO: LJ0406 MTF: TBFG_12697
TBFG_13415 LAC: LBA0356 MBO: Mb2701c(dxs1) Mb3413c(dxs2) LSL:
LSL_0209(dxs) MBB: BCG_2695c(dxs1) BCG_3450c(dxs2) LGA: LGAS_0350
MLE: ML1038(dxs) MPA: MAP2803c(dxs) BLO: BL1132(dxs) MAV:
MAV_3577(dxs) BLJ: BLD_0889(dxs) MSM: MSMEG_2776(dxs) BAD:
BAD_0513(dxs) MUL: MUL_3319(dxs1) FNU: FN1208 FN1464 MVA: Mvan_2477
RBA: RB2143(dxs) MGI: Mflv_3923 OTE: Oter_2780 MAB: MAB_2990c MIN:
Minf_1537(dxs) MMC: Mmcs_2208 AMU: Amuc_0315 MKM: Mkms_2254 CTR:
CT331(dxs) MJL: Mjls_2197 CTA: CTA_0359(dxs) MMI: MMAR_0276(dxs2)
CTB: CTL0585 MMAR_2032(dxs1) CTL: CTLon_0582(dxs) CGL:
NCgl1827(cgl1902) CMU: TC0608(dxs) CGB: cg2083(dxs) CPN:
CPn1060(tktB_2) CGT: cgR_1731 CPA: CP0790 CEF: CE1796 CPJ:
CPj1060(tktB_2) CDI: DIP1397(dxs) CPT: CpB1102 CJK: jk1078(dxs)
CCA: CCA00304(dxs) CUR: cu0909 CAB: CAB301(dxs) NFA: nfa37410(dxs)
CFE: CF0699(dxs) RHA: RHA1_ro06843 PCU: pc0619(dxs) SCO:
SCO6013(SC1C3.01) TPA: TP0824 SCO6768(SC6A5.17) TPP:
TPASS_0824(dxs) SMA: SAV1646(dxs1) SAV2244(dxs2) TDE: TDE1910(dxs)
SGR: SGR_1495(dxs) LIL: LA3285(dxs) TWH: TWT484 LIC: LIC10863(dxs)
TWS: TW280(Dxs) LBJ: LBJ_0917(dxs) LXX: Lxx10450(dxs) LBL:
LBL_0932(dxs) CMI: CMM_1660(dxsA) LBI: LEPBI_I2605(dxs) AAU:
AAur_1790(dxs) LBF: LBF_2525(dxs) RSA: RSal33209_2392 SYN:
sll1945(dxs) KRH: KRH_14140(dxs) SYW: SYNW1292(Dxs) PAC: PPA1062
SYC: syc1087_c(dxs) NCA: Noca_2859 SYF: Synpcc7942_0430 TFU:
Tfu_1917 SYD: Syncc9605_1430 FRA: Francci3_1326 SYE: Syncc9902_1069
FRE: Franean1_5184 SYG: sync_1410(dxs) FAL: FRAAL2088(dxs) SYR:
SynRCC307_1390(dxs) ACE: Acel_1393 SYX: SynWH7803_1223(dxs)
KRA: Krad_1452 Krad_1578 SYP: SYNPCC7002_A1172(dxs) SEN:
SACE_1815(dxs) CYA: CYA_1701(dxs) STP: Strop_1489 CYB:
CYB_1983(dxs) SAQ: Sare_1454 TEL: tll0623 MAR: MAE_62650 EMI:
Emin_0268 CYT: cce_1401(dxs) DRA: DR_1475 GVI: gll0194 DGE:
Dgeo_0994 ANA: alr0599 TTH: TTC1614 NPU: Npun_F5466 TTJ: TTHA0006
AVA: Ava_4532 AAE: aq_881 PMA: Pro0928(dxs) HYA: HY04AAS1_1061 PMM:
PMM0907(Dxs) SUL: SYO3AOP1_0652 PMT: PMT0685(dxs) TMA: TM1770 PMN:
PMN2A_0300 TPT: Tpet_1058 PMI: PMT9312_0893 TLE: Tlet_2013 PMB:
A9601_09541(dxs) TRQ: TRQ2_1054 PMC: P9515_09901(dxs) TME:
Tmel_0252 PMF: P9303_15371(dxs) FNO: Fnod_1517 PMG:
P9301_09521(dxs) PMO: Pmob_1001 PMH: P9215_09851 PMJ: P9211_08521
PME: NATL1_09721(dxs) TER: Tery_3042 AMR: AM1_5186(dxs) BTH:
BT_1403 BT_4099 BFR: BF0873 BF4306 BFS: BF0796(dxs) BF4114 BVU:
BVU_1763 BVU_3090 PGI: PG2217(dxs) PGN: PGN_2081 PDI: BDI_2664 CHU:
CHU_3643(dxs) GFO: GFO_3470(dxs) FJO: Fjoh_1523 FPS: FP0279(dxs)
CTE: CT0337(dxs) CPC: Cpar_1696 CPH: Cpha266_0671 CPB: Cphamn1_1826
PVI: Cvib_0498 PLT: Plut_0450 PPH: Ppha_2222 CTS: Ctha_0174 PAA:
Paes_1686 DET: DET0745(dxs) DEH: cbdb_A720(dxs) DEB:
DehaBAV1_0675
Exemplary 1-deoxy-D-xylulose-5-phosphate reductoisomerase nucleic
acids and polypeptides
TABLE-US-00051 [0964] ATH: AT5G62790(DXR) YPG: YpAngola_A3431(dxr)
OSA: 4326153(Os01g0106900) YPS: YPTB2999(dxr) PPP:
PHYPADRAFT_127023 YPI: YpsIP31758_1017(dxr) PHYPADRAFT_128953 YPY:
YPK_1070 OLU: OSTLU_31255(DXR) YPB: YPTS_3119 CRE:
CHLREDRAFT_196606(DXR1) YEN: YE3280(b0173) CME: CMG148C SFL:
SF0163(yaeM) PFA: PF14_0641 SFX: S0166(yaeM) PFD: PFDG_00980 SFV:
SFV_0156(yaeM) PYO: PY05578 SSN: SSON_0185(yaeM) TAN: TA14290 SBO:
SBO_0161(yaeM) TPV: TP02_0073 SBC: SbBS512_E0166(dxr) ECO:
b0173(dxr) SDY: SDY_0189(yaeM) ECJ: JW0168(dxr) ECA: ECA1035(dxr)
ECD: ECDH10B_0153(dxr) ETA: ETA_08940(dxr) ECE: Z0184(yaeM) PLU:
plu0676(dxr) ECS: ECs0175 BUC: BU235(dxr) ECI: UTI89_C0188(dxr)
BAS: BUsg229(dxr) ECP: ECP_0181 WBR: WGLp388(yaeM) ECV:
APECO1_1814(dxr) SGL: SG1939 ECW: EcE24377A_0177(dxr) ENT:
Ent638_0711 ECX: EcHS_A0175(dxr) ESA: ESA_03169 ECM:
EcSMS35_0184(dxr) KPN: KPN_00186(ispC) ECL: EcolC_3487 CKO:
CKO_03194 STY: STY0243(dxr) SPE: Spro_3786 STT: t0221(dxr) BFL:
Bfl275(dxr) SPT: SPA0227(dxr) BPN: BPEN_283(dxr) SPQ: SPAB_00282
HIN: HI0807 SEC: SC0220(dxr) HIT: NTHI0971(dxr) SEH:
SeHA_C0258(dxr) HIP: CGSHiEE_08025 SEE: SNSL254_A0242(dxr) HIQ:
CGSHiGG_07530 SEW: SeSA_A0245(dxr) HDU: HD1186(dxr) SES: SARI_02782
HSO: HS_0985(dxr) STM: STM0220(dxr) HSM: HSM_1463 YPE: YPO1048(dxr)
PMU: PM1988(dxr) YPK: y3131 MSU: MS1928(dxr) YPM: YP_2802(dxr) APL:
APL_0406(dxr) YPA: YPA_0524 APJ: APJL_0428(dxr) YPN: YPN_2952 APA:
APP7_0430 YPP: YPDSF_1664 ASU: Asuc_0657 XFA: XF1048 ABC:
ACICU_02094 XFT: PD0328(dxr) SON: SO_1635(dxr) XFM: Xfasm12_0359
SDN: Sden_1560 XFN: XfasM23_0324 SFR: Sfri_1276 XCC: XCC1367(dxr)
SAZ: Sama_1145 XCB: XC_2871 SBL: Sbal_1456 XCV: XCV1472(dxr) SBM:
Shew185_1451 XAC: XAC1415(dxr) SBN: Sbal195_1487 XOO: XOO1970(dxr)
SLO: Shew_2629 XOM: XOO_1860(XOO1860) SPC: Sputcn32_1354 SML:
Smlt1500(dxr) SSE: Ssed_3155 SMT: Smal_1259 SPL: Spea_2879 VCH:
VC2254 SHE: Shewmr4_2635 VCO: VC0395_A1845(dxr) SHM: Shewmr7_2702
VVU: VV1_1866 SHN: Shewana3_2809 VVY: VV2551 SHW: Sputw3181_2749
VPA: VP2312 SHL: Shal_2975 VFI: VF1956 SWD: Swoo_3275 VHA:
VIBHAR_03231 ILO: IL0839 PPR: PBPRA2962 CPS: CPS_1559(dxr) PAE:
PA3650(dxr) PHA: PSHAa2030(dxr) PAU: PA14_17130(dxr) PAT: Patl_1255
PAP: PSPA7_1489(dxr) SDE: Sde_2591 PPU: PP_1597(dxr) MAQ: Maqu_2542
PPF: Pput_4180 AMC: MADE_01379 PPG: PputGB1_1152 PIN: Ping_2970
PPW: PputW619_4076 MCA: MCA0573(dxr) PST: PSPTO_1540(dxr) FTU:
FTT1574c(dxr) PSB: Psyr_1349 FTF: FTF1574c(dxr) PSP:
PSPPH_3834(dxr) FTW: FTW_0352(dxr) PFL: PFL_1182(dxr) FTL: FTL_0534
PFO: PflO1_1107 FTH: FTH_0536(dxr) PEN: PSEEN4214(dxr) FTA:
FTA_0567(dxr) PMY: Pmen_3047 FTN: FTN_1483(dxr) PSA: PST_1543(dxr)
FTM: FTM_0324(dxr) CJA: CJA_1118(dxr) FPH: Fphi_1195 PAR:
Psyc_1531(dxr) NOC: Noc_0814 PCR: Pcryo_1710 AEH: Mlg_1857 PRW:
PsycPRwf_1798 HHA: Hhal_1460 ACI: ACIAD1376(dxr) HCH:
HCH_05246(dxr) ACB: A1S_1971 CSA: Csal_0569 ABM: ABSDF1684(dxr)
ABO: ABO_1149(dxr) ABY: ABAYE1581 MMW: Mmwyl1_1278 AHA:
AHA_1179(dxr) RFR: Rfer_1994 ASA: ASA_3154(dxr) POL: Bpro_2689 BCI:
BCI_0531(dxr) PNA: Pnap_1764 RMA: Rmag_0025 AAV: Aave_1829 VOK:
COSY_0025(dxr) AJS: Ajs_2579 NME: NMB0184(dxr) VEI: Veis_1444 NMA:
NMA0083(dxr) DAC: Daci_4942 NMC: NMC0175(dxr) MPT: Mpe_A1973 NMN:
NMCC_1968 HAR: HEAR1341(dxr) NGO: NGO1799 MMS: mma_2052 NGK:
NGK_2475 LCH: Lcho_2844 CVI: CV_2202(dxr) NEU: NE1712(dxr) RSO:
RSc1410(dxr) NET: Neut_2029 REU: Reut_A1875 NMU: Nmul_A0663 REH:
H16_A2049(dxp) EBA: ebA5994(dxr) RME: Rmet_1441 AZO: azo1903(dxr)
BMA: BMA1549(dxr) DAR: Daro_1748 BMV: BMASAVP1_A2050(dxr) TBD:
Tbd_0791 BML: BMA10229_A3261(dxr) MFA: Mfla_1524 BMN:
BMA10247_1322(dxr) HPY: HP0216 BXE: Bxe_A1688 HPJ: jhp0202 BVI:
Bcep1808_1919 HPA: HPAG1_0217 BUR: Bcep18194_A5323 HPS: HPSH_01115
BCN: Bcen_6064 HHE: HH0524(dxr) BCH: Bcen2424_2013 HAC:
Hac_1502(dxr_fragment_2) BCM: Bcenmc03_2033
Hac_1503(dxr_fragment_1) BAM: Bamb_2046 WSU: WS0812 BAC:
BamMC406_1915 TDN: Suden_0126 BMU: Bmul_1263 CJE: Cj1346c(dxr) BMJ:
BMULJ_01984(dxr) CJR: CJE1535(dxr) BPS: BPSL2153(dxr) CJJ:
CJJ81176_1345(dxr) BPM: BURPS1710b_2577(dxr) CJU: C8J_1262(dxr)
BPL: BURPS1106A_2487(dxr) CJD: JJD26997_0364(dxr) BPD:
BURPS668_2431(dxr) CFF: CFF8240_0210(dxr) BTE: BTH_I2033(dxr) CCV:
CCV52592_0594(dxr) BPH: Bphy_1332 CHA: CHAB381_0121(dxr) PNU:
Pnuc_1445 CCO: CCC13826_0420(dxr) PNE: Pnec_0513 ABU: Abu_0161(dxr)
BPE: BP1425(dxr) NIS: NIS_1666(ispC) BPA: BPP1533(dxr) SUN:
SUN_0144 BBR: BB2611(dxr) GSU: GSU1915(dxr) BPT: Bpet2529(dxr) GME:
Gmet_1256 BAV: BAV1740(dxr) GUR: Gura_3727 GLO: Glov_2714 NHA:
Nham_1700 PCA: Pcar_1915(dxr) XAU: Xaut_4433 PPD: Ppro_2050 AZC:
AZC_1699 DVU: DVU0866(dxr) MEX: Mext_2083 DVL: Dvul_2116 MRD:
Mrad2831_3444 DDE: Dde_1123 MET: M446_0636 LIP: LI0386(dxr) BID:
Bind_0297 DPS: DP1160 CCR: CC_1917 DOL: Dole_0480 CAK: Caul_2799
ADE: Adeh_3583 SIL: SPO1667(dxr) AFW: Anae109_3704 SIT: TM1040_1410
SAT: SYN_00916 RSP: RSP_2709(dxr) SFU: Sfum_1784 RSH:
Rsph17029_1366 WOL: WD0992(dxr) RSQ: Rsph17025_2149 WBM: Wbm0179
JAN: Jann_2455 WPI: WP0113(dxr) RDE: RD1_2590(dxr) AMA: AM743(dxr)
PDE: Pden_3997 APH: APH_0440(dxr) DSH: Dshi_1497 ERU: Erum4750(dxr)
MMR: Mmar10_1386 ERW: ERWE_CDS_04970(dxr) HNE: HNE_1774(dxr) ERG:
ERGA_CDS_04870(dxr) ZMO: ZMO1150(dxr) ECN: Ecaj_0473 NAR: Saro_1375
ECH: ECH_0557(dxr) SAL: Sala_1954 NSE: NSE_0443(dxr) SWI: Swit_0466
PUB: SAR11_0912(yaeM) ELI: ELI_03805 PLA: Plav_3190 GOX: GOX1816
SME: SMc03105(dxr) GBE: GbCGDNIH1_0938 SMD: Smed_2879 ACR:
Acry_2557 ATU: Atu2612(dxr) GDI: GDI2147(dxr) ATC: AGR_C_4736 RRU:
Rru_A1592 RET: RHE_CH03839(dxr) MAG: amb2492 REC:
RHECIAT_CH0004120(dxr) MGM: Mmcl_1846 RLE: RL4372(dxr) ABA:
Acid345_1419 BJA: bll4855(dxr) SUS: Acid_7136 BRA: BRADO4134(dxr)
SWO: Swol_0889 BBT: BBta_4511(dxr) CSC: Csac_2353 RPA: RPA2916(dxr)
BSU: BSU16550(dxr) RPB: RPB_2822 BHA: BH2421 RPC: RPC_2442 BAN:
BA3409(dxr-1) BA3959(dxr-2) RPD: RPD_2851 BAR: GBAA3409(dxr-1)
GBAA3959(dxr-2) RPE: RPE_2559 BAA: BA_4429 RPT: Rpal_3262 BAT:
BAS3160 BAS3672 NWI: Nwi_1853 BCE: BC3341 BC3819 BCA: BCE_3862(dxr)
PTH: PTH_1260(dxr) BCZ: BCZK3054(dxr) BCZK3580(dxr) DAU: Daud_0615
BCY: Bcer98_2128 Bcer98_2473 HMO: HM1_2264(dxr) BTK:
BT9727_3144(dxr) BT9727_3562(dxr) TTE: TTE1402(dxr) BTL: BALH_3451
TEX: Teth514_1654 BWE: BcerKBAB4_3082 BcerKBAB4_3644 TPD:
Teth39_1218 BLI: BL01237(dxr) MTA: Moth_1041 BLD: BLi01876(dxr)
MPE: MYPE1470 BCL: ABC2236(dxr) MGA: MGA_0787(dxr) BAY: RBAM_016390
MTU: Rv2870c(dxr) BPU: BPUM_1554 MTC: MT2938(dxr) GKA: GK1255 MRA:
MRA_2895(dxr) GTN: GTNG_1109 MTF: TBFG_12886 LSP: Bsph_1590 MBO:
Mb2895c(dxr) ESI: Exig_1845 MBB: BCG_2892c(dxr) LMO: lmo1317 MLE:
ML1583 LMF: LMOf2365_1334(dxr) MPA: MAP2940c LIN: lin1354 MAV:
MAV_3727(dxr) LWE: lwe1332(dxr) MSM: MSMEG_2578(dxr) STH:
STH1499(dxr) MUL: MUL_2085(dxr) CAC: CAC1795 MVA: Mvan_2260 CPE:
CPE1694 MGI: Mflv_4083 CPF: CPF_1948(dxr) MAB: MAB_3171c CPR:
CPR_1666(dxr) MMC: Mmcs_2042 CTC: CTC01268 MKM: Mkms_2088 CNO:
NT01CX_2143 MJL: Mjls_2025 CTH: Cthe_0999 MMI: MMAR_1836(dxr) CDF:
CD2130(dxr) CGL: NCgl1940(cgl2016) CBO: CBO2426 CGB: cg2208(dxr)
CBA: CLB_2290(dxr) CGT: cgR_1844 CBH: CLC_2273(dxr) CEF: CE1905
CBL: CLK_1802(dxr) CDI: DIP1500(dxr) CBK: CLL_A1265(dxr) CJK:
jk1167(ispC) CBB: CLD_2214(dxr) CUR: cu0831 CBF: CLI_2482(dxr) NFA:
nfa41200(dxr) CBE: Cbei_1195 RHA: RHA1_ro06588(dxr) CKL:
CKL_1423(dxr) SCO: SCO5694(dxr) CPY: Cphy_2622 SMA: SAV2563(dxr)
AMT: Amet_2682 SGR: SGR_1823 AOE: Clos_1519 TWH: TWT089(dxr) CHY:
CHY_1778(dxr) TWS: TW099(dxr) DSY: DSY2539 LXX: Lxx12180(dxr) DRM:
Dred_1970 CMI: CMM_2160(dxrA) ART: Arth_1399 LBL: LBL_0925(dxr)
AAU: AAur_1543(dxr) LBI: LEPBI_I2611(dxr) RSA: RSal33209_0635 LBF:
LBF_2531(dxr) KRH: KRH_16160(dxr) SYN: sll0019(dxr) PAC: PPA1510
SYW: SYNW0698(dxr) NCA: Noca_3204 SYC: syc2498_d(dxr) TFU: Tfu_0747
SYF: Synpcc7942_1513 FRA: Francci3_3575 SYD: Syncc9605_1970 FRE:
Franean1_1168 SYE: Syncc9902_0689 FAL: FRAAL5774(dxr) SYG:
sync_0920(dxr) ACE: Acel_1524 SYR: SynRCC307_1674(dxr) KRA:
Krad_1427 Krad_4655 SYX: SynWH7803_1622(dxr) SEN: SACE_5994(dxr)
SYP: SYNPCC7002_A0818(dxr) STP: Strop_1350 CYA: CYA_0193(dxr) SAQ:
Sare_1302 CYB: CYB_1233(dxr) BLO: BL0097(ispC) TEL: tlr1040 BLJ:
BLD_0115(dxr) MAR: MAE_50310 BAD: BAD_1158(ispC) CYT: cce_2124(dxr)
RXY: Rxyl_1404 GVI: gll2252 FNU: FN1324 ANA: alr4351 RBA:
RB5568(dxr) NPU: Npun_R5970 OTE: Oter_4632 AVA: Ava_1300 MIN:
Minf_1972(dxr) PMA: Pro1236(dxr) AMU: Amuc_1737 PMM: PMM1142(dxr)
CTR: CT071(yaeM) PMT: PMT1161(dxr) CTA: CTA_0076(dxr) PMN:
PMN2A_0751 CTB: CTL0327 PMI: PMT9312_1238 CTL: CTLon_0322(dxr) PMB:
A9601_13171(dxr) CMU: TC0343(dxr) PMC: P9515_13061(dxr) CPN:
CPn0345(yaeM) PMF: P9303_08651(dxr) CPA: CP0415 PMG:
P9301_13311(dxr) CPJ: CPj0344(yaeM) PMH: P9215_13461 CPT: CpB0352
PMJ: P9211_12161 CCA: CCA00441(dxr) PME: NATL1_15911(dxr) CAB:
CAB427(dxr) TER: Tery_0416 CFE: CF0566(yaeM) AMR: AM1_0563(dxr)
PCU: pc0260(dxr) BTH: BT_2002
TPA: TP0601 BFR: BF3699 TPP: TPASS_0601(dxr) BFS: BF3492 TDE:
TDE2342(dxr) BVU: BVU_1651 LIL: LA3292(dxr) PGI: PG1364(dxr) LIC:
LIC10856(dxr) PGN: PGN_1151 LBJ: LBJ_0910(dxr) PDI: BDI_0480 SRU:
SRU_1849(dxr) CHU: CHU_2996(dxr) CTE: CT0125(dxr) CPC: Cpar_0071
CCH: Cag_0008 CPH: Cpha266_2680 CPB: Cphamn1_0098 PVI: Cvib_0138
PLT: Plut_0077 PPH: Ppha_0080 CTS: Ctha_1044 PAA: Paes_0121 DET:
DET0371(dxr) DEH: cbdb_A314(dxr) DEB: DehaBAV1_0353 EMI: Emin_0690
DRA: DR_1508 DGE: Dgeo_1044 TTH: TTC0504 TTJ: TTHA0856 AAE: aq_404
HYA: HY04AAS1_0095 SUL: SYO3AOP1_0479 TMA: TM0889 TPT: Tpet_0038
TLE: Tlet_0658 TRQ: TRQ2_0038 TME: Tmel_0037 FNO: Fnod_0950 PMO:
Pmob_1939
Exemplary 4-diphosphocytidyl-2C-methyl-D-erythritol synthase
nucleic acids and polypeptides
TABLE-US-00052 [0965] ATH: AT2G02500(ISPD) YPY: YPK_3431 OSA:
4324893(Os01g0887100) YPB: YPTS_0804 OLU: OSTLU_24843(CMS) YEN:
YE0769(ispD) CRE: CHLREDRAFT_196604(CMS) SFL: SF2770(ispD) CME:
CMH115C SFX: S2963(ispD) TAN: TA02505 SFV: SFV_2751(ispD) TPV:
TP03_0057 SSN: SSON_2895(ispD) ECO: b2747(ispD) SBO: SBO_2773(ispD)
ECJ: JW2717(ispD) SBC: SbBS512_E3127(ispD) ECD: ECDH10B_2915(ispD)
SDY: SDY_2946(ispD) ECE: Z4055(ispD) ECA: ECA3535(ispD) ECS:
ECs3601(ispD) ETA: ETA_27010(ispD) ECC: c3314(ispD) PLU:
plu0713(ispD) ECI: UTI89_C3118(ispD) BUC: BU420(ygbP) ECP:
ECP_2729(ispD) BAS: BUsg405(ygbP) ECV: APECO1_3776(ispD) WBR:
WGLp532(ygbP) ECW: EcE24377A_3048(ispD) SGL: SG0526 ECX:
EcHS_A2885(ispD) ENT: Ent638_3218(ispD) ECM: EcSMS35_2872(ispD)
ESA: ESA_00544 ECL: EcolC_0965 KPN: KPN_03109(ispD) STY:
STY3055(ispD) CKO: CKO_04108 STT: t2831(ispD) SPE: Spro_0826 SPT:
SPA2786(ispD) BPN: BPEN_171(ispD) SPQ: SPAB_03644 HIN: HI0672(ispD)
SEC: SC2862(ispD) HIT: NTHI0794(ispD) SEH: SeHA_C3120(ispD) HIP:
CGSHiEE_08815(ispD) SEE: SNSL254_A3136(ispD) HIQ:
CGSHiGG_06635(ispD) SEW: SeSA_A3081(ispD) HDU: HD1329(ispD) SES:
SARI_00026 HSO: HS_1496(ispD) STM: STM2930(ispD) HSM: HSM_0505 YPE:
YPO3361(ispD) PMU: PM1608(ispD) YPK: y0828(ispD) MSU: MS2275(ispD)
YPM: YP_0326(ispD) APL: APL_0802(ispD) YPA: YPA_2782(ispD) APJ:
APJL_0807(ispD) YPN: YPN_0732(ispD) APA: APP7_0861 YPP:
YPDSF_2999(ispD) ASU: Asuc_2032 YPG: YpAngola_A0964(ispD) XFA:
XF1293(ispD) YPS: YPTB0770(ispD) XFT: PD0545(ispD) YPI:
YpsIP31758_3299(ispD) XFM: Xfasm12_0618 XFN: XfasM23_0570 SFR:
Sfri_1054 XCC: XCC1702(ispD) SAZ: Sama_1038 XCB: XC_2529(ispD) SBL:
Sbal_3125 XCV: XCV1754(ispD) SBM: Shew185_3134 XAC: XAC1721(ispD)
SBN: Sbal195_3277 XOO: XOO2961(ispD) SLO: Shew_1207 XOM:
XOO_2812(ispD) SPC: Sputcn32_2755 SML: Smlt1717(ispD) SSE:
Ssed_1292 SMT: Smal_1454 SPL: Spea_1187 VCH: VC0528(ispD) SHE:
Shewmr4_1117 VCO: VC0395_A0056(ispD) SHM: Shewmr7_1188 VVU:
VV1_1582(ispD) SHN: Shewana3_1118 VVY: VV2816(ispD) SHW:
Sputw3181_1257 VPA: VP1320 VP2559(ispD) SHL: Shal_1224 VFI:
VF2073(ispD) SWD: Swoo_3348 VHA: VIBHAR_03523 ILO: IL0752(ispD)
PPR: PBPRA3077 CPS: CPS_1072(ispD) PAE: PA3633(ispD) PHA:
PSHAa0684(ispD) PAU: PA14_17340(ispD) PAT: Patl_3857 PAP:
PSPA7_1506(ispD) SDE: Sde_1247 PPU: PP_1614(ispD) MAQ: Maqu_0923
PPF: Pput_4163(ispD) AMC: MADE_03721 PPG: PputGB1_1168 PIN:
Ping_0672 PPW: PputW619_4061 MCA: MCA2517(ispD) PST:
PSPTO_1556(ispD) FTU: FTT0711(ispD) PSB: Psyr_1365(ispD) FTF:
FTF0711(ispD) PSP: PSPPH_3818(ispD) FTW: FTW_1530(ispD) PFL:
PFL_1198(ispD) FTL: FTL_1525 PFO: PflO1_1123(ispD) FTH:
FTH_1475(ispD) PEN: PSEEN4198(ispD) FTA: FTA_1609(ispD) PMY:
Pmen_3031(ispD) FTN: FTN_0623(ispD) PSA: PST_1559(ispD) FTM:
FTM_1371(ispD) CJA: CJA_2223(ispD) FPH: Fphi_0219 PAR: Psyc_1634
NOC: Noc_0854 PCR: Pcryo_1868 AEH: Mlg_1837 PRW: PsycPRwf_1662 HHA:
Hhal_1435 ACI: ACIAD1999(ispD) HCH: HCH_01869(ispD) ACB: A1S_1895
CSA: Csal_2638 ABM: ABSDF2025(ispD) ABO: ABO_1166(ispD) ABY:
ABAYE1672 MMW: Mmwyl1_1301 ABC: ACICU_02004 AHA: AHA_0823(ispD)
SON: SO_3438(ispD) ASA: ASA_3473(ispD) SDN: Sden_1198 BCI:
BCI_0211(ispD) RMA: Rmag_0755 AAV: Aave_1581 VOK: COSY_0697(ispD)
AJS: Ajs_3156 NME: NMB1513 VEI: Veis_4360 NMA: NMA1713 DAC:
Daci_2849 NMC: NMC1442 MPT: Mpe_A1570 NMN: NMCC_1418 HAR:
HEAR1912(ispD) NGO: NGO0972 MMS: mma_1409 NGK: NGK_0824 LCH:
Lcho_2295 CVI: CV_1258(ispD) NEU: NE1412 RSO: RSc1643(ispD) NET:
Neut_1525 REU: Reut_A1361(ispD) NMU: Nmul_A2127 REH:
H16_A1456(ispD) EBA: ebA6543(ispD) RME: Rmet_1954(ispD) AZO:
azo1682 BMA: BMA1490(ispD) DAR: Daro_1973 BMV: BMASAVP1_A1987(ispD)
TBD: Tbd_1003 BML: BMA10229_A3319(ispD) MFA: Mfla_1116 BMN:
BMA10247_1259(ispD) HPY: HP1020(ispDF) BXE: Bxe_A2312(ispD) HPJ:
jhp0404(ispDF) BVI: Bcep1808_1870(ispD) HPA: HPAG1_0427(ispDF) BUR:
Bcep18194_A5254(ispD) HHE: HH1582(ispDF) BCN: Bcen_6136(ispD) HAC:
Hac_1124(ispDF) BCH: Bcen2424_1943(ispD) WSU: WS1940(ispDF) BCM:
Bcenmc03_1967 TDN: Suden_1487(ispDF) BAM: Bamb_1931(ispD) CJE:
Cj1607(ispDF) BAC: BamMC406_1858 CJR: CJE1779(ispDF) BMU: Bmul_1328
CJJ: CJJ81176_1594(ispDF) BMJ: BMULJ_01918(ispD) CFF:
CFF8240_0409(ispDF) BPS: BPSL2099(ispD) GSU: GSU3368(ispD) BPM:
BURPS1710b_2512(ispD) GME: Gmet_0060 BPL: BURPS1106A_2401(ispD)
GUR: Gura_4163 BPD: BURPS668_2358(ispD) GLO: Glov_0872 BTE:
BTH_I2089(ispD) PCA: Pcar_0103(ispD) BPH: Bphy_0998 PPD: Ppro_2969
PNU: Pnuc_0930 DVU: DVU1454(ispD) PNE: Pnec_0911 DVL: Dvul_1625
BPE: BP0865(ispD) DDE: Dde_1726 BPA: BPP3366(ispD) LIP: LI0446 BBR:
BB3817(ispD) DPS: DP0257 BPT: Bpet1695(ispD) DOL: Dole_2147 BAV:
BAV1060(ispD) ADE: Adeh_1272 RFR: Rfer_1332 SAT: SYN_01401 POL:
Bpro_2716 SFU: Sfum_1637 PNA: Pnap_2549 WOL: WD1143 WBM: Wbm0409
HNE: HNE_2014(ispDF) AMA: AM1357(ispD) ZMO: ZMO1128(ispDF) APH:
APH_1277(ispD) NAR: Saro_1925(ispDF) ERU: Erum1030(ispD) SAL:
Sala_1278 ERW: ERWE_CDS_01000(ispD) ELI: ELI_06290(ispDF) ERG:
ERGA_CDS_00960(ispD) GOX: GOX1669 ECN: Ecaj_0103 GBE:
GbCGDNIH1_1019 ECH: ECH_0157(ispD) ACR: Acry_0551 NSE: NSE_0178
RRU: Rru_A1674 PUB: SAR11_0945(ispD) MAG: amb2363 MLO:
mll0395(ispDF) MGM: Mmc1_2672 MES: Meso_1621(ispDF) ABA:
Acid345_0188 SME: SMc01040(ispDF) SWO: Swol_2361 ATU: Atu1443(ispF)
CSC: Csac_2198 ATC: AGR_C_2659 BSU: BSU00900(ispD) RET:
RHE_CH01945(ispDF) BHA: BH0107(ispD) RLE: RL2254(ispDF) BAN:
BA0084(ispD) BME: BMEI0863(ispDF) BAR: GBAA0084(ispD) BMF:
BAB1_1143(ispDF) BAA: BA_0674 BMB: BruAb1_1126(ispDF) BAT:
BAS0085(ispD) BMS: BR1120(ispDF) BCE: BC0106(ispD) BJA: bll4485
BCA: BCE_0085(ispD) BRA: BRADO3869(ispDF) BCZ: BCZK0081(ispD) BBT:
BBta_4067(ispDF) BCY: Bcer98_0080 RPA: RPA2590(ispD) BTK:
BT9727_0082(ispD) RPB: RPB_2885 BTL: BALH_0085(ispD) RPC: RPC_2575
BWE: BcerKBAB4_0080 RPD: RPD_2587 BLI: BL03265(ispD) RPE: RPE_2755
BLD: BLi00108(ispD) NWI: Nwi_1442 BCL: ABC0125(ispD) NHA: Nham_1834
BAY: RBAM_001150(yacM) BHE: BH05820 BPU: BPUM_0075 BQU:
BQ04980(ispDF) GKA: GK0081(ispD) BBK: BARBAKC583_0540(ispDF) GTN:
GTNG_0081(ispD) BTR: Btr_0870 LSP: Bsph_4646 CCR: CC_1738(ispDF)
ESI: Exig_0071 Exig_0189 SIL: SPO2090(ispDF) SAU: SA0241(ispD)
SA0245(ispD) SIT: TM1040_1364 SAV: SAV0251(ispD) SAV0255(ispD) RSP:
RSP_2835(ispD) SAW: SAHV_0250 SAHV_0254 RSQ: Rsph17025_1485 SAM:
MW0227(ispD) MW0231(ispD) RDE: RD1_2766(ispD) SAR: SAR0246(ispD)
SAR0252(ispD) PDE: Pden_3667 SAS: SAS0227(ispD) SAS0232(ispD) MMR:
Mmar10_1439 SAC: SACOL0236(ispD) SACOL0240(ispD) SAB: SAB0190
SAB0194(ispD) CBH: CLC_3453(ispD) SAA: SAUSA300_0245 CBL:
CLK_2951(ispD) SAUSA300_0249(ispD) CBK: CLL_A0216(ispD) SAX:
USA300HOU_0262(ispD2) CBB: CLD_0997(ispD) USA300HOU_0266 CBF:
CLI_3691(ispD) SAO: SAOUHSC_00220 CBE: Cbei_0129(ispD)
SAOUHSC_00225(ispD) CKL: CKL_0200(ispD) SAJ: SaurJH9_0236
SaurJH9_0240(ispD) CPY: Cphy_0353 SAH: SaurJH1_0242
SaurJH1_0246(ispD) AMT: Amet_4506 SAE: NWMN_0185 NWMN_0189(ispD)
AOE: Clos_0463 SEP: SE0319 CHY: CHY_2342(ispD) SER: SERP0196(ispD)
DSY: DSY0443 DSY3011 SSP: SSP0354(ispD) DRM: Dred_0187 LMO:
lmo0235(ispD) lmo1086(ispD) PTH: PTH_0289(ispD) LMF:
LMOf2365_0247(ispD) DAU: Daud_0186 LMOf2365_1100(ispD) FMA:
FMG_1230 LIN: lin0267(ispD) lin1071(ispD) TTE: TTE2322(ispD) LWE:
lwe0199(ispD) lwe1061(ispD) TEX: Teth514_0839 SPN: SP_1271(ispD)
TPD: Teth39_0346 SPR: spr1149(ispD) MTA: Moth_2487 SPD:
SPD_1127(ispD) MPE: MYPE2770 SPV: SPH_1387 MTU: Rv3582c(ispD) SPW:
SPCG_1235(ispD) MTC: MT3688(ispD) SPX: SPG_1165 MRA: MRA_3621(ispD)
SAG: SAG1417 MTF: TBFG_13615(ispD) SAN: gbs1487 MBO: Mb3613c(ispD)
SAK: SAK_1452(ispD) MBB: BCG_3647c(ispD) SSA: SSA_2214 MLE:
ML0321(ispD) SGO: SGO_2017 MPA: MAP0476(ispD) LPL: lp_1816 MAV:
MAV_0571(ispD) LCA: LSEI_1098 MSM: MSMEG_6076(ispD) EFA:
EF2172(ispD) MUL: MUL_4158(ispD) STH: STH3123 MVA: Mvan_4129
Mvan_4130 CAC: CAC3184 MGI: Mflv_2528 Mflv_2529 CPE: CPE2429(ispD)
MAB: MAB_0569 CPF: CPF_2739(ispD) MMC: Mmcs_4739(ispD) CPR:
CPR_2426(ispD) MKM: Mkms_4825(ispD) CTC: CTC02626 MJL:
Mjls_5125(ispD) CNO: NT01CX_1092(ispD) MMI: MMAR_5082(ispD) CTH:
Cthe_2941 CGL: NCgl2570(ispD) CDF: CD0047(ispD) CGB: cg2945(ispD)
CBO: CBO3504(ispD) CGT: cgR_2564(ispD) CBA: CLB_3564(ispD) CEF:
CE2521(ispD) CDI: DIP1973(ispD) CFE: CF0845(ispD) CJK: jk0308(ispD)
PCU: pc0327(ispD) CUR: cu1675 TPA: TP0512 NFA: nfa4360(ispD) TDE:
TDE2291(ispD) RHA: RHA1_ro04460(ispD) LIL: LA1048(ygbP) SCO:
SCO4233(ispD) LIC: LIC12617(ispD) SMA: SAV3969(mecT) LBJ:
LBJ_0280(ispD) SGR: SGR_4012 LBL: LBL_2796(ispD) TWH: TWT348(ispDF)
LBI: LEPBI_I1435(ispD) TWS: TW422 LBF: LBF_1381(ispD) LXX:
Lxx18250(ispF) SYN: slr0951 AAU: AAur_0898(ispD) SYW:
SYNW1849(ispD) RSA: RSal33209_0409 SYC: syc0848_d(ispD) KRH:
KRH_18710(ispD) SYF: Synpcc7942_0681(ispD) PAC: PPA0353 SYD:
Syncc9605_0620(ispD) NCA: Noca_4038 SYE: Syncc9902_1742(ispD) FRA:
Francci3_3932 Francci3_4254 SYG: sync_2140(ispD) FRE: Franean1_0363
Franean1_0798 SYR: SynRCC307_0684(ispD) FAL: FRAAL6243
FRAAL6524(ispD) SYX: SynWH7803_1858(ispD) ACE: Acel_0080 Acel_1533
SYP: SYNPCC7002_A1905(ispD) KRA: Krad_0899 CYA: CYA_1505(ispD) SEN:
SACE_0439(ispD) CYB: CYB_2706(ispD) STP: Strop_4261 TEL: tlr0605
SAQ: Sare_4691 MAR: MAE_45830 BLO: BL0324(ispD) CYT: cce_0963(ispD)
BLJ: BLD_1082(ispD) GVI: glr2791 RXY: Rxyl_2176 ANA: all5167 FNU:
FN1580 NPU: Npun_F5020 RBA: RB9133(ispD) AVA: Ava_2414(ispD) OTE:
Oter_0455 Oter_2440 PMA: Pro0453(ispD) MIN: Minf_0787(ispD) PMM:
PMM0454(ispD) AMU: Amuc_0068 PMT: PMT1330(ispD) CTR: CT462(ispD)
PMN: PMN2A_1786(ispD) CTA: CTA_0505(ispD) PMI: PMT9312_0454(ispD)
CTB: CTL0722 PMB: A9601_05101(ispD) CTL: CTLon_0718(ispD) PMC:
P9515_05171(ispD) CMU: TC0747(ispD) PMF: P9303_06551(ispD) CPN:
CPn0579(ispD) PMG: P9301_04791(ispD)
CPA: CP0169(ispD) PMH: P9215_05341 CPJ: CPj0579(ispD) PMJ:
P9211_04551 CPT: CpB0603(ispD) PME: NATL1_05091(ispD) CCA:
CCA00162(ispD) TER: Tery_0609(ispD) CAB: CAB160(ispD) AMR:
AM1_3984(ispD) BTH: BT_2881 BT_3923(ispD) BFR: BF3962(ispD) BFS:
BF3735(ispD) BVU: BVU_0472(ispD) BVU_2951 PGI: PG1434(ispD) PGN:
PGN_0841 PDI: BDI_1351 BDI_2700(ispD) BDI_3625 BDI_3828 SRU:
SRU_1652 CHU: CHU_3100(ispD) CTE: CT1317(ispD) CPC: Cpar_1335 CCH:
Cag_0929 CPH: Cpha266_1642 CPB: Cphamn1_1025 PVI: Cvib_1049 PPH:
Ppha_1615 CTS: Ctha_2474 PAA: Paes_1464 DET: DET0059(ispD) DEH:
cbdb_A74(ispD) DEB: DehaBAV1_0053 DRA: DR_2604 DGE: Dgeo_0181 TTH:
TTC1815 TTJ: TTHA0171 AAE: aq_1323 HYA: HY04AAS1_1287 SUL:
SYO3AOP1_0708 TMA: TM1393 TPT: Tpet_1390 TLE: Tlet_0798 TRQ:
TRQ2_1436 TME: Tmel_1925 FNO: Fnod_0183 PMO: Pmob_1218 HMA:
rrnAC1932(ispD) NMR: Nmar_1581
Exemplary 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase nucleic
acids and polypeptides
TABLE-US-00053 [0966] ATH: AT2G26930(ATCDPMEK) YPS: YPTB2002(ipk)
OSA: 4327968(Os01g0802100) YPI: YpsIP31758_2069(ispE) PPP:
PHYPADRAFT_190580 YPY: YPK_2182 OLU: OSTLU_4287(CMK) YPB: YPTS_2060
CRE: CHLREDRAFT_137673(CMK1) YEN: YE2434(ipk) CME: CMS444C SFL:
SF1211(ychB) PFA: PFE0150c SFX: S1295(ychB) PFD: PFDG_01632 SFV:
SFV_1222(ychB) PFH: PFHG_02738 SSN: SSON_1970(ychB) PYO: PY04665
SBO: SBO_1859(ychB) ECO: b1208(ispE) SBC: SbBS512_E1372(ispE) ECJ:
JW1199(ispE) SDY: SDY_1257(ychB) ECD: ECDH10B_1261(ispE) ECA:
ECA2187(ispE) ECE: Z1979(ychB) ETA: ETA_18820(ispE) ECS: ECs1713
PLU: plu2067(ispE) ECC: c1666(ispE) BUC: BU170(ychB) ECI:
UTI89_C1402(ychB) BAS: BUsg164(ipk) ECP: ECP_1256 WBR:
WGLp348(ychB) ECV: APECO1_324(ychB) SGL: SG1879 ECW:
EcE24377A_1356(ispE) ENT: Ent638_2340 ECX: EcHS_A1313(ispE) ESA:
ESA_01495 ECM: EcSMS35_1934(ispE) KPN: KPN_02237(ispE) ECL:
EcolC_2418 CKO: CKO_01272 STY: STY1905(ipk) SPE: Spro_1987 STT:
t1097(ipk) BFL: Bfl347(ipk) SPT: SPA1094(ipk) BPN: BPEN_357(ispE)
SPQ: SPAB_01449 HIN: HI1608 SEC: SC1773(ipk) HIT: NTHI1434(ispE)
SEH: SeHA_C1975(ispE) HIP: CGSHiEE_05690 SEE: SNSL254_A1911(ispE)
HIQ: CGSHiGG_10080 SEW: SeSA_A1917(ispE) HDU: HD1628(ispE) SES:
SARI_01174 HSO: HS_0997(ispE) STM: STM1779(ipk) HSM: HSM_1475 YPE:
YPO2014(ipk) PMU: PM0245 YPK: y2293 MSU: MS1535(ispE) YPM:
YP_1862(ipk) APL: APL_0776(ispE) YPA: YPA_1398 APJ: APJL_0779(ispE)
YPN: YPN_1496 APA: APP7_0837 YPP: YPDSF_1104 ASU: Asuc_1751 YPG:
YpAngola_A2463(ispE) XFA: XF2645 XFT: PD2018(ispE) SFR: Sfri_0720
XFM: Xfasm12_2208 SAZ: Sama_2569 XFN: XfasM23_2119 SBL: Sbal_0693
XCC: XCC0871(ipk) SBM: Shew185_3617 XCB: XC_3359 SBN: Sbal195_3740
XCV: XCV0979(ispE) SLO: Shew_2915 XAC: XAC0948(ipk) SPC:
Sputcn32_0798 XOO: XOO3604(ipk) SSE: Ssed_3462 XOM:
XOO_3406(XOO3406) SPL: Spea_3129 SML: Smlt0874(ipk) SHE:
Shewmr4_3172 SMT: Smal_0725 SHM: Shewmr7_0794 VCH: VC2182 SHN:
Shewana3_0766 VCO: VC0395_A1759 SHW: Sputw3181_3377 VVU: VV1_0256
SHL: Shal_3214 VVY: VV0928 SWD: Swoo_3688 VPA: VP0740 ILO:
IL0928(ispE) VFI: VF0765 CPS: CPS_3556(ispE) VHA: VIBHAR_01247 PHA:
PSHAa1055(ispE) PPR: PBPRA2848 PAT: Patl_2566 PAE: PA4669(ipk) SDE:
Sde_3255 PAU: PA14_61750(ipk) MAQ: Maqu_2364 PAP: PSPA7_5318(ispE)
AMC: MADE_02576 PPU: PP_0723(ipk) PIN: Ping_0912 PPF:
Pput_0757(ipk) MCA: MCA1055(ispE) PPG: PputGB1_0767 FTU:
FTT0271(ispE) PPW: PputW619_4460 FTF: FTF0271(ispE) PST:
PSPTO_1105(ispE) FTW: FTW_1830(ispE) PSB: Psyr_0945(ipk) FTL:
FTL_0151 PSP: PSPPH_0993(ipk) FTH: FTH_0144(ispE) PFL:
PFL_5163(ipk) FTA: FTA_0164(ispE) PFO: PflO1_4752(ipk) FTN:
FTN_0146(ispE) PEN: PSEEN0858(ipk) FTM: FTM_1592(ispE) PMY:
Pmen_1056(ipk) FPH: Fphi_0678 PSA: PST_3186(ipk) NOC: Noc_0513 CJA:
CJA_0646(ispE) AEH: Mlg_0282 PAR: Psyc_0173(ispE) HHA: Hhal_0990
PCR: Pcryo_0186 HCH: HCH_01727(ispE) PRW: PsycPRwf_2104 CSA:
Csal_1525 ACI: ACIAD2903(ispE) ABO: ABO_0519(ispE) ACB: A1S_0834
MMW: Mmwyl1_3603 ABC: ACICU_00788 AHA: AHA_3152(ispE) SON:
SO_3836(ispE) ASA: ASA_1172(ispE) SDN: Sden_0917 BCI:
BCI_0292(ispE) RMA: Rmag_0110 PNA: Pnap_0900 VOK: COSY_0115(ispE)
AAV: Aave_3609 NME: NMB0874 AJS: Ajs_0896 NMA: NMA1092 VEI:
Veis_0952 NMC: NMC0815 DAC: Daci_5432 NMN: NMCC_0833 MPT: Mpe_A3230
NGO: NGO0440 HAR: HEAR2892(ispE) NGK: NGK_0610 MMS: mma_3127 CVI:
CV_4059(ispE) LCH: Lcho_3497 RSO: RSc0396(ipk) NEU: NE1827(ipk)
REU: Reut_A0343 NET: Neut_1139 REH: H16_A0374 NMU: Nmul_A0588 RME:
Rmet_0290 EBA: ebA1405(ispE) CTI: RALTA_A0318(ispE) AZO:
azo0756(ispE) BMA: BMA3118(ispE) DAR: Daro_3729 BMV:
BMASAVP1_A0086(ispE) TBD: Tbd_0386 BML: BMA10229_A1504(ispE) MFA:
Mfla_0679 BMN: BMA10247_2932(ispE) HPY: HP1443 BXE: Bxe_A4132 HPJ:
jhp1336 BVI: Bcep1808_2906 HPA: HPAG1_1369 BUR: Bcep18194_A6131
HPS: HPSH_07385 BCN: Bcen_2187 HHE: HH0122 BCH: Bcen2424_2801 HAC:
Hac_0175(ipk) BCM: Bcenmc03_2812 WSU: WS0881 BAM: Bamb_2861 TDN:
Suden_0440 BAC: BamMC406_2719 CJE: Cj1104 BMU: Bmul_0514 CJR:
CJE1247(ispE) BMJ: BMULJ_02745(ispE) CJJ: CJJ81176_1122(ispE) BPS:
BPSL0523 CJU: C8J_1045 BPM: BURPS1710b_0755(ispE) CJD:
JJD26997_0618(ispE) BPL: BURPS1106A_0587(ispE) CFF: CFF8240_0713
BPD: BURPS668_0571(ispE) CCV: CCV52592_0696(ispE) BTE:
BTH_I0476(ispE) CHA: CHAB381_1110 BPH: Bphy_0316 CCO:
CCC13826_0061(ispE) PNU: Pnuc_1919 ABU: Abu_2083(ispE) PNE:
Pnec_1624 NIS: NIS_1475 BPE: BP3126(ispE) SUN: SUN_0381 BPA:
BPP0816(ispE) GSU: GSU0660(ispE) BBR: BB0900(ispE) GME: Gmet_2849
BPT: Bpet4003(ispE) GUR: Gura_3683 BAV: BAV0536(ispE) GLO:
Glov_2596 RFR: Rfer_1659 PCA: Pcar_2005(ispE) POL: Bpro_1294 PPD:
Ppro_0738 DVU: DVU1576(ispE) BBT: BBta_2348(ispE) DVL: Dvul_1557
RPA: RPA1039(ispE) DDE: Dde_2125 RPB: RPB_1086 LIP: LI0735(ychB)
RPC: RPC_4356 DPS: DP2735 RPD: RPD_1213 DOL: Dole_2816 RPE:
RPE_4419 ADE: Adeh_0123 RPT: Rpal_1231 AFW: Anae109_0127 NWI:
Nwi_2593 SAT: SYN_03046 NHA: Nham_3216 SFU: Sfum_3651 BHE:
BH04210(thrB1) WOL: WD0360(ispE) BQU: BQ03230(thrB) WBM: Wbm0173
BBK: BARBAKC583_0387(ispE) WPI: WP0174(ispE) BTR: Btr_0633 AMA:
AM493(ispE) XAU: Xaut_1381 APH: APH_0574(ispE) AZC: AZC_0910 ERU:
Erum3340(ispE) MEX: Mext_3109 ERW: ERWE_CDS_03410(ispE) MRD:
Mrad2831_5351 ERG: ERGA_CDS_03370(ispE) MET: M446_2748 ECN:
Ecaj_0317 BID: Bind_0858 ECH: ECH_0757(ispE) CCR: CC_1336 NSE:
NSE_0720 CAK: Caul_2169 PUB: SAR11_0105(ispE) SIL: SPO0318(ispE)
MLO: mll7422 SIT: TM1040_3743 MES: Meso_0706 RSP: RSP_1779(ispE)
PLA: Plav_0721 RSH: Rsph17029_0426 SME: SMc00862(ipk) RSQ:
Rsph17025_2471 SMD: Smed_0456 JAN: Jann_0486 ATU: Atu0632(ipk) RDE:
RD1_3402(ispE) ATC: AGR_C_1122 PDE: Pden_0423 RET:
RHE_CH00873(ispE) DSH: Dshi_3073 REC: RHECIAT_CH0000963(ispE) MMR:
Mmar10_2186 RLE: RL0935 HNE: HNE_0676(ispE) BME: BMEI1537 ZMO:
ZMO1182(ispE) BMF: BAB1_0423(ispE) NAR: Saro_1782 BMB:
BruAb1_0418(ispE) SAL: Sala_1187 BMC: BAbS19_I03890 SWI: Swit_4106
BMS: BR0394(ispE) ELI: ELI_06920 BMT: BSUIS_A0420(ispE) GOX:
GOX1559 BOV: BOV_0403(ispE) GBE: GbCGDNIH1_1848 BCS:
BCAN_A0398(ispE) ACR: Acry_2663 OAN: Oant_0512 GDI: GDI0728 BJA:
blr2526(ipk) RRU: Rru_A0263 BRA: BRADO2022(ispE) MAG: amb4435 MGM:
Mmc1_0819 SHA: SH2516 ABA: Acid345_4541 SSP: SSP2261 SUS: Acid_7097
LMO: lmo0190 SWO: Swol_0064 LMF: LMOf2365_0201(ispE) CSC: Csac_2225
LIN: lin0229 BSU: BSU00460(ispE) LWE: lwe0159(ispE) BHA: BH0061
SPZ: M5005_Spy_0074 M5005_Spy_0075 BAN: BA0043(ispE) M5005_Spy_0076
BAR: GBAA0043(ispE) SPH: MGAS10270_Spy0077 BAA: BA_0633
MGAS10270_Spy0078 BAT: BAS0044 SPI: MGAS10750_Spy0082 BCE: BC0050
MGAS10750_Spy0083 BCA: BCE_0043(ispE) SPJ: MGAS2096_Spy0077 BCZ:
BCZK0040(ispE) MGAS2096_Spy0078 MGAS2096_Spy0079 BCY: Bcer98_0040
SPK: MGAS9429_Spy0074 BTK: BT9727_0040(ispE) MGAS9429_Spy0075
MGAS9429_Spy0076 BTL: BALH_0040(ispE) SPA: M6_Spy0123 M6_Spy0124
BWE: BcerKBAB4_0040 SPB: M28_Spy0073 M28_Spy0074 BLI: BL00525(ispE)
SAG: SAG0153(ispE) BLD: BLi00059(ispE) SAN: gbs0149 BCL:
ABC0074(ispE) SAK: SAK_0216(ispE) BAY: RBAM_000550 SMU:
SMU.1996(ipk) BPU: BPUM_0030 SEZ: Sez_0102(ispE) OIH: OB0055 LPL:
lp_0460(ispE) GKA: GK0039 LSA: LSA1652(ispE) GTN: GTNG_0039 LSL:
LSL_0234(ispE) LSP: Bsph_0065 LBR: LVIS_0460 ESI: Exig_0038 LCA:
LSEI_2591 SAU: SA0453 LCB: LCABL_27570(ispE) SAV: SAV0495 LRE:
Lreu_0215 SAW: SAHV_0492 LRF: LAR_0206 SAM: MW0450 LFE: LAF_0190
SAR: SAR0496 EFA: EF0051(ispE) SAS: SAS0452 STH: STH3246 SAC:
SACOL0538(ispE) CAC: CAC2902 SAB: SAB0444 CPE: CPE2212(ipk) SAA:
SAUSA300_0472(ispE) CPF: CPF_2476(ipk) SAO: SAOUHSC_00466 CPR:
CPR_2186(ipk) SAJ: SaurJH9_0516 CTC: CTC00283 SAH: SaurJH1_0529
CNO: NT01CX_0566(ipk) SAE: NWMN_0458 CTH: Cthe_2403(ipk) SEP:
SE2288 CDF: CD3566(ipk) SER: SERP0133(ispE) CBO: CBO0121(ipk) CBA:
CLB_0157(ispE) CGL: NCgl0874(cg0911) CBH: CLC_0169(ispE) CGB:
cg1039 CBL: CLK_3296(ispE) CGT: cgR_1012 CBK: CLL_A0471(ispE) CEF:
CE0973 CBB: CLD_0665(ispE) CDI: DIP0876 CBF: CLI_0176(ispE) CJK:
jk1510(ispE) CBE: Cbei_0394(ipk) CUR: cu0564 CKL: CKL_3724(ispE)
NFA: nfa49010(cmeK) CPY: Cphy_3793 RHA: RHA1_ro05684 AMT: Amet_4604
SCO: SCO3148(SCE66.27c) AOE: Clos_0285 SMA: SAV3586(cmeK) CHY:
CHY_0188(ispE) SGR: SGR_4357 DSY: DSY0148 TWH: TWT605(ispE) DRM:
Dred_0094 TWS: TW159(ispE) PTH: PTH_0096(ispE) LXX: Lxx17480(ispE)
DAU: Daud_0058 CMI: CMM_2367(ispE) HMO: HM1_0738(ispE) AAU:
AAur_1338(ispE) FMA: FMG_0552 RSA: RSal33209_2993 TTE:
TTE2559(ispE) KRH: KRH_17370(ispE) TEX: Teth514_0599 PAC: PPA0527
TPD: Teth39_0176 NCA: Noca_3855 MTA: Moth_0072 TFU: Tfu_0407 MPE:
MYPE10380 FRA: Francci3_3958 MGA: MGA_0635 FRE: Franean1_0773 UUR:
UU600 FAL: FRAAL6276(ispE) MTU: Rv1011(ispE) ACE: Acel_0181 MTC:
MT1040 KRA: Krad_1046 MRA: MRA_1020(ispE) SEN: SACE_0807(ispE) MTF:
TBFG_11030 STP: Strop_0783 MBO: Mb1038(ispE) SAQ: Sare_0727 MBB:
BCG_1068(ispE) BLO: BL0656(ispE) MLE: ML0242 BAD: BAD_1616(ispE)
MPA: MAP0976 RXY: Rxyl_0893 MAV: MAV_1149(ispE) FNU: FN0021 MSM:
MSMEG_5436(ispE) RBA: RB10537(ispE) MUL: MUL_4649(ispE) OTE:
Oter_2442 MVA: Mvan_4799 MIN: Minf_1286(ispE)
MGI: Mflv_1934 AMU: Amuc_1195 MAB: MAB_1139 CTR: CT804(ychB) MMC:
Mmcs_4262 CTA: CTA_0876(ispE) MKM: Mkms_4348 CTB: CTL0173 MJL:
Mjls_4641 CTL: CTLon_0174(ispE) MMI: MMAR_4477(ispE) CMU: TC0187
CPJ: CPj0954 CPj0955 PMH: P9215_09581 CPT: CpB0991 CpB0992 PMJ:
P9211_07121 CCA: CCA00815(ispE) PME: NATL1_09481(ispE) CAB: CAB784
TER: Tery_4700 CFE: CF0199(ispE) AMR: AM1_1752(ispE) PCU: pc1589
BTH: BT_0624 TPA: TP0371 BFR: BF2589 TPP: TPASS_0371 BFS: BF2610
TDE: TDE1338(ispE) BVU: BVU_3466 LIL: LA3824(ychB) PGI:
PG0935(ispE) LIC: LIC10426(ispE) PGN: PGN_1012 LBJ: LBJ_2584(ispE)
PDI: BDI_0715 LBL: LBL_0528(ispE) SRU: SRU_0689(ispE) LBI:
LEPBI_I0238(ispE) CHU: CHU_1210(ispE) LBF: LBF_0232(ispE) CTE:
CT1495(ispE) SYN: sll0711(ipk) CPC: Cpar_1582 SYW: SYNVV1053(ispE)
CCH: Cag_1333 SYC: syc1203_d(ispE) CPH: Cpha266_1884 SYF:
Synpcc7942_0310 CPB: Cphamn1_0845 SYD: Syncc9605_1188 PVI:
Cvib_1321 SYE: Syncc9902_1282 PLT: Plut_1496 SYG: sync_1593(ispE)
PPH: Ppha_1063 SYR: SynRCC307_1314(ispE) CTS: Ctha_0721 SYX:
SynWH7803_1365(ispE) PAA: Paes_1591 SYP: SYNPCC7002_A2416(ispE)
DET: DET0405(ispE) CYA: CYA_0285(ispE) DEH: cbdb_A356(ispE) CYB:
CYB_1390(ispE) DEB: DehaBAV1_0384 TEL: tll0500 EMI: Emin_0501 MAR:
MAE_04520 DRA: DR_2605 CYT: cce_1317(ispE) DGE: Dgeo_0180 GVI:
gll0102 TTH: TTC1816 ANA: alr3230 TTJ: TTHA0170 NPU: Npun_R4911
AAE: aq_915 AVA: Ava_4887 HYA: HY04AAS1_1414 PMA: Pro0764(ispE)
SUL: SYO3AOP1_0238 PMM: PMM0932(ispE) TMA: TM1383 PMT:
PMT0620(ispE) TPT: Tpet_1400 PMN: PMN2A_0279 TLE: Tlet_1489 PMI:
PMT9312_0867 TRQ: TRQ2_1446 PMB: A9601_09281(ispE) TME: Tmel_0318
PMC: P9515_10151(ispE) FNO: Fnod_1663 PMF: P9303_16181(ispE) PMO:
Pmob_0160 PMG: P9301_09261(ispE)
Exemplary 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase
nucleic acids and polypeptides
TABLE-US-00054 [0967] ATH: AT1G63970(ISPF) YPN: YPN_0733(ispF) OSA:
4330320(Os02g0680600) YPP: YPDSF_3000(ispF) PPP: PHYPADRAFT_150209
YPG: YpAngola_A0963(ispF) OLU: OSTLU_44114(MCS) YPS: YPTB0771(ispF)
CRE: CHLREDRAFT_188593 YPI: YpsIP31758_3298(ispF) CME: CMT435C YPY:
YPK_3430 PFA: PFB0420w YPB: YPTS_0805 PFH: PFHG_00813 YEN:
YE0770(ispF) PYO: PY00321 SFL: SF2769(ispF) TAN: TA04155 SFX:
S2962(ispF) TPV: TP03_0365 SFV: SFV_2752(ispF) TET: TTHERM_01003920
SSN: SSON_2894(ispF) ECO: b2746(ispF) SBO: SBO_2774(ispF) ECJ:
JW2716(ispF) SBC: SbBS512_E3128(ispF) ECD: ECDH10B_2914(ispF) SDY:
SDY_2945(ispF) ECE: Z4054(ispF) ECA: ECA3534(ispF) ECS:
ECs3600(ispF) ETA: ETA_27000(ispF) ECC: c3313(ispF) PLU:
plu0714(ispF) ECI: UTI89_C3117(ispF) BUC: BU419(ygbB) ECP:
ECP_2728(ispF) BAS: BUsg404(ygbB) ECV: APECO1_3777(ispF) WBR:
WGLp531(ygbB) ECW: EcE24377A_3047(ispF) SGL: SG0527(ispF) ECX:
EcHS_A2884(ispF) ENT: Ent638_3217(ispF) ECM: EcSMS35_2871(ispF)
ESA: ESA_00545 ECL: EcolC_0966 KPN: KPN_03108(ispF) STY:
STY3054(ispF) CKO: CKO_04107 STT: t2830(ispF) SPE: Spro_0827 SPT:
SPA2785(ispF) BPN: BPEN_172(ispF) SPQ: SPAB_03643 HIN: HI0671(ispF)
SEC: SC2861(ispF) HIT: NTHI0793(ispF) SEH: SeHA_C3119(ispF) HIP:
CGSHiEE_08820(ispF) SEE: SNSL254_A3135(ispF) HIQ:
CGSHiGG_06630(ispF) SEW: SeSA_A3080(ispF) HDU: HD1328(ispF) SES:
SARI_00027 HSO: HS_1498(ispF) STM: STM2929(ispF) HSM: HSM_0503 YPE:
YPO3360(ispF) PMU: PM1609 YPK: y0829(ispF) MSU: MS2274(ispF) YPM:
YP_0327(ispF) APL: APL_0803(ispF) YPA: YPA_2783(ispF) APJ:
APJL_0808(ispF) APA: APP7_0862 ABM: ABSDF1672(ispF) ASU: Asuc_2031
ABY: ABAYE1569 XFA: XF1294(ispF) ABC: ACICU_02105 XFT: PD0546(ispF)
SON: SO_3437(ispF) XFM: Xfasm12_0619 SDN: Sden_1199 XFN:
XfasM23_0571 SFR: Sfri_1055 XCC: XCC1703(ispF) SAZ: Sama_1039 XCB:
XC_2528(ispF) SBL: Sbal_3124 XCV: XCV1755(ispF) SBM: Shew185_3133
XAC: XAC1722(ispF) SBN: Sbal195_3276 XOO: XOO2960(ispF) SLO:
Shew_1208 XOM: XOO_2811(ispF) SPC: Sputcn32_2754 SML:
Smlt1718(ispF) SSE: Ssed_1293 SMT: Smal_1455 SPL: Spea_1188 VCH:
VC0529(ispF) SHE: Shewmr4_1118 VCO: VC0395_A0057(ispF) SHM:
Shewmr7_1189 VVU: VV1_1583(ispF) SHN: Shewana3_1119 VVY:
VV2814(ispF) SHW: Sputw3181_1258 VPA: VP2558(ispF) SHL: Shal_1225
VFI: VF2072(ispF) SWD: Swoo_3347 VHA: VIBHAR_03522 ILO:
IL0751(ispF) PPR: PBPRA3076(ispF) CPS: CPS_1073(ispF) PAE:
PA3627(ispF) PHA: PSHAa0685(ispF) PAU: PA14_17420(ispF) PAT:
Patl_3858 PAP: PSPA7_1512(ispF) SDE: Sde_1248 PPU: PP_1618(ispF)
MAQ: Maqu_0924 PPF: Pput_4159(ispF) AMC: MADE_03722 PPG:
PputGB1_1172 PIN: Ping_0673 PPW: PputW619_4057 MCA: MCA2518(ispF)
PST: PSPTO_1560(ispF) FTU: FTT1128(ispF) PSB: Psyr_1369(ispF) FTF:
FTF1128(ispF) PSP: PSPPH_3814(ispF) FTW: FTW_1161(ispF) PFL:
PFL_1202(ispF) FTL: FTL_0833 PFO: PflO1_1127(ispF) FTH:
FTH_0823(ispF) PEN: PSEEN4194(ispF) FTA: FTA_0882(ispF) PMY:
Pmen_3026(ispF) FTN: FTN_1110(ispF) PSA: PST_1566(ispF) FTM:
FTM_1296(ispF) CJA: CJA_2222(ispF) FPH: Fphi_1496 PAR:
Psyc_1243(ispF) NOC: Noc_0855 PCR: Pcryo_1149 AEH: Mlg_1836 PRW:
PsycPRwf_0962 HHA: Hhal_1434 ACI: ACIAD1996(ispF) HCH:
HCH_01870(ispF) ACB: A1S_1982 CSA: Csal_2637 ABO: ABO_1167(ispF)
BPT: Bpet1696(ispF) MMW: Mmwyl1_1302 BAV: BAV1059(ispF) AHA:
AHA_0824(ispF) RFR: Rfer_1332 ASA: ASA_3472(ispF) POL: Bpro_2715
BCI: BCI_0210(ispF) PNA: Pnap_2548 RMA: Rmag_0756(ispF) AAV:
Aave_1582 VOK: COSY_0698(ispF) AJS: Ajs_3155 NME: NMB1512(ispF)
VEI: Veis_4361 NMA: NMA1712(ispF) DAC: Daci_2850 NMC: NMC1441(ispF)
MPT: Mpe_A1571 NMN: NMCC_1417 HAR: HEAR1911(ispF) NGO:
NGO0971(ispF) MMS: mma_1410 NGK: NGK_0825 LCH: Lcho_2293 CVI:
CV_1259(ispF) NEU: NE1402 RSO: RSc1644(RS04019) NET: Neut_1300 REU:
Reut_A1362 NMU: Nmul_A2126 REH: H16_A1457 EBA: ebA6542(ispF) RME:
Rmet_1953 AZO: azo1683(ispF) BMA: BMA1489(ispF) DAR:
Daro_1974(ispF) BMV: BMASAVP1_A1986(ispF) TBD: Tbd_1004 BML:
BMA10229_A3320(ispF) MFA: Mfla_1117 BMN: BMA10247_1258(ispF) HPY:
HP1020(ispDF) BXE: Bxe_A2311 HPJ: jhp0404(ispDF) BVI: Bcep1808_1869
HPA: HPAG1_0427(ispDF) BUR: Bcep18194_A5253 HPS: HPSH_02215(ispDF)
BCN: Bcen_6137 HHE: HH1582(ispDF) BCH: Bcen2424_1942 HAC:
Hac_1124(ispDF) BCM: Bcenmc03_1966 WSU: WS1940(ispDF) BAM:
Bamb_1930 TDN: Suden_1487(ispDF) BAC: BamMC406_1857 CJE:
Cj1607(ispDF) BMU: Bmul_1329 CJR: CJE1779(ispDF) BMJ:
BMULJ_01917(ispF) CJJ: CJJ81176_1594(ispDF) BPS: BPSL2098(ispF)
CJU: C8J_1508 BPM: BURPS1710b_2511(ispF) CJD: JJD26997_1961 BPL:
BURPS1106A_2400(ispF) CFF: CFF8240_0409(ispDF) BPD:
BURPS668_2357(ispF) CCV: CCV52592_0202 BTE: BTH_I2090(ispF) CHA:
CHAB381_0932 BPH: Bphy_0999 CCO: CCC13826_1467 PNU: Pnuc_0931 ABU:
Abu_0126(ispDF) PNE: Pnec_0910 NIS: NIS_0595 BPE: BP0866(ispF) SUN:
SUN_0522 BPA: BPP3365(ispF) GSU: GSU3367(ispF) BBR: BB3816(ispF)
GME: Gmet_0059 GUR: Gura_4164 OAN: Oant_2069 GLO: Glov_3480 BJA:
bll4485 PCA: Pcar_0102(ispF) BRA: BRADO3869(ispDF) PPD: Ppro_0012
BBT: BBta_4067(ispDF) DVU: DVU1454(ispD) RPA: RPA2590(ispD) DVL:
Dvul_1625 RPB: RPB_2885 DDE: Dde_1726 RPC: RPC_2575 LIP: LI0446
RPD: RPD_2587 DPS: DP0257 RPE: RPE_2755 DOL: Dole_1666 RPT:
Rpal_2860 ADE: Adeh_1272 NWI: Nwi_1442 AFW: Anae109_2497 NHA:
Nham_1834 SAT: SYN_01400 BHE: BH05820 SFU: Sfum_1636 BQU:
BQ04980(ispDF) WOL: WD1143 BBK: BARBAKC583_0540 WBM: Wbm0409
(ispDF) WPI: WP0969 BTR: Btr_0870 AMA: AM1356(ispF) XAU: Xaut_4402
APH: APH_1276(ispF) AZC: AZC_3089 ERU: Erum1020(ispF) MEX:
Mext_2817 ERW: ERWE_CDS_00990(ispF) MRD: Mrad2831_2171 ERG:
ERGA_CDS_00950(ispF) MET: M446_5927 ECN: Ecaj_0102 BID: Bind_1516
ECH: ECH_0156(ispF) CCR: CC_1738(ispDF) NSE: NSE_0134(ispF) CAK:
Caul_2603 MLO: mll0395(ispDF) SIL: SPO2090(ispDF) MES:
Meso_1621(ispDF) SIT: TM1040_1364 PLA: Plav_3132 RSP:
RSP_6071(ispF) SME: SMc01040(ispDF) RSH: Rsph17029_1460 SMD:
Smed_1087(ispDF) RSQ: Rsph17025_1484 ATU: Atu1443(ispF) RDE:
RD1_2767(ispF) ATC: AGR_C_2659 PDE: Pden_3667 RET:
RHE_CH01945(ispDF) DSH: Dshi_1577 REC: RHECIAT_CH0002043(ispDF)
MMR: Mmar10_1439 RLE: RL2254(ispDF) ZMO: ZMO1128(ispDF) BME:
BMEI0863(ispDF) NAR: Saro_1925(ispDF) BMF: BAB1_1143(ispDF) SAL:
Sala_1278 BMB: BruAb1_1126(ispDF) SWI: Swit_0244(ispDF) BMC:
BAbS19_I10610 ELI: ELI_06290(ispDF) BMS: BR1120(ispDF) GOX: GOX1669
BMT: BSUIS_A1169(ispF) GBE: GbCGDNIH1_1019 BOV: BOV_1078(ispDF)
ACR: Acry_2031 BCS: BCAN_A1139(ispF) GDI: GDI2269 MAG: amb2363 RRU:
Rru_A1674 MGM: Mmc1_2673 CBL: CLK_3243(ispF) ABA: Acid345_0187 CBK:
CLL_A0353(ispF) SUS: Acid_1861 CBB: CLD_0719(ispF) SWO: Swol_2360
CBF: CLI_0123(ispF) CSC: Csac_1587 CBE: Cbei_0297 BSU:
BSU00910(ispF) CKL: CKL_3774(ispF) BHA: BH0108(ispF) CPY: Cphy_3326
BAN: BA0085(ispF) AMT: Amet_4505 BAR: GBAA0085(ispF) AOE: Clos_0464
BAA: BA_0675(ygbB) CHY: CHY_2341(ispF) BAT: BAS0086(ispF) DSY:
DSY0444 BCE: BC0107(ispF) DRM: Dred_0188 BCA: BCE_0086(ispF) PTH:
PTH_0290(ispF) BCZ: BCZK0082(ispF) HMO: HM1_1354(ispD) BCY:
Bcer98_0081 FMA: FMG_1229 BTK: BT9727_0083(ispF) TTE: TTE2320(ispF)
BTL: BALH_0086(ispF) TEX: Teth514_0841 BWE: BcerKBAB4_0081 TPD:
Teth39_0348 BLI: BL03266(ispF) MTA: Moth_2486 BLD: BLi00109(ispF)
MPE: MYPE10270 BCL: ABC0126(ispF) MGA: MGA_0657 BAY:
RBAM_001160(yacN) MTU: Rv3581c(ispF) BPU: BPUM_0076 MTC:
MT3687(ispF) GKA: GK0082(ispF) MRA: MRA_3620(ispF) GTN:
GTNG_0082(ispF) MTF: TBFG_13614(ispF) LSP: Bsph_4645 MBO:
Mb3612c(ispF) ESI: Exig_0072 MBB: BCG_3646c(ispF) LMO:
lmo0236(ispF) MLE: ML0322(ispF) LMF: LMOf2365_0248(ispF) MPA:
MAP0477(ispF) LIN: lin0268(ispF) MAV: MAV_0572(ispF) EFA:
EF0042(ispF) MSM: MSMEG_6075(ispF) CAC: CAC0434 MUL: MUL_4157(ispF)
CPE: CPE2316(ispF) MVA: Mvan_5339(ispF) CPF: CPF_2616(ispF) MGI:
Mflv_1445(ispF) CPR: CPR_2302(ispF) MAB: MAB_0570 CTC: CTC00232
MMC: Mmcs_4738(ispF) CNO: NT01CX_0736(ispF) MKM: Mkms_4824(ispF)
CTH: Cthe_2946(ispF) MJL: Mjls_5124(ispF) CDF: CD0048(ispF) MMI:
MMAR_5081(ispF) CBO: CBO0066(ispF) CGL: NCgl2569(ispF) CBA:
CLB_0102(ispF) CGB: cg2944(ispF) CBH: CLC_0114(ispF) CGT:
cgR_2563(ispF) CDI: DIP1972(ispF) CEF: CE2520(ispF) CJK:
jk0309(ispF) CPT: CpB0568(ispF) CUR: cu1674 CCA: CCA00195(ispF)
NFA: nfa4370(ispF) CAB: CAB191(ispF) RHA: RHA1_ro04461(ispF) CFE:
CF0812(ispF) SCO: SCO4234(ispF) PCU: pc0227(ispF) SMA:
SAV3968(ispF) TPA: TP0512 SGR: SGR_4013 TPP: TPASS_0512 TWH:
TWT348(ispDF) TDE: TDE2292(ispF) TWS: TW422 LIL: LA3591(ygbB) LXX:
Lxx18250(ispF) LIC: LIC10610(ispF) CMI: CMM_2489(ispDF) LBJ:
LBJ_0323(ispF) ART: Arth_0728 LBL: LBL_2753(ispF) AAU:
AAur_0899(ispF) LBI: LEPBI_I0322(ispF) RSA: RSal33209_0410 LBF:
LBF_0313(ispF) KRH: KRH_18700(ispF) SYN: slr1542 PAC: PPA0354(ispF)
SYW: SYNW1610 NCA: Noca_4024(ispF) SYC: syc0380_d(ispF) TFU:
Tfu_2906(ispF) SYF: Synpcc7942_1170(ispF) FRA: Francci3_4253(ispF)
SYE: Syncc9902_1508 FRE: Franean1_0364 SYG: sync_0781(ispF) FAL:
FRAAL6523(ispF) SYR: SynRCC307_1730(ispF) ACE: Acel_0081 SYX:
SynWH7803_1723(ispF) KRA: Krad_0900 SYP: SYNPCC7002_A1166 SEN:
SACE_0440(ispF) (ispF) STP: Strop_4260 CYA: CYA_0267(ispF) SAQ:
Sare_4690 CYB: CYB_0783(ispF) BLO: BL0997(ispF) TEL: tlr2035 BAD:
BAD_0669(ispF) MAR: MAE_31930 RXY: Rxyl_2175 CYT: cce_0476(ispF)
FNU: FN1788 GVI: glr3547 RBA: RB3451(ispF) ANA: alr3883 OTE:
Oter_2439 NPU: Npun_F5826 MIN: Minf_0771(ispF) AVA: Ava_1811(ispF)
AMU: Amuc_1243 PMA: Pro1354(trmD) CTR: CT434(ispF) PMM: PMM1280
CTA: CTA_0474(ispF) PMT: PMT0356 CTB: CTL0693 PMN: PMN2A_0847
CTL: CTLon_0689(ispF) PMI: PMT9312_1374 CMU: TC0718(ispF) PMB:
A9601_14791(trmD) CPN: CPn0547(ispF) PMC: P9515_14411(trmD) CPA:
CP0205(ispF) PMF: P9303_19461(trmD) CPJ: CPj0547(ispF) PMG:
P9301_14651(trmD) PME: NATL1_17001(trmD) PMH: P9215_15051 TER:
Tery_4716(ispF) PMJ: P9211_13261 AMR: AM1_2915(ispF) BTH: BT_3884
BFR: BF4006 BFS: BF3780(ispF) BVU: BVU_1639 PGI: PG0028(ispF) PGN:
PGN_0024 PDI: BDI_2574 SRU: SRU_1651(ispF) CHU: CHU_3180(ispF) CTE:
CT1601(ispF) CPC: Cpar_1528 CCH: Cag_1782 CPH: Cpha266_0591 CPB:
Cphamn1_0610 PVI: Cvib_1388 PLT: Plut_1598 PPH: Ppha_0719 CTS:
Ctha_0565 PAA: Paes_0552 DET: DET0060(ispF) DEH: cbdb_A75(ispF)
DEB: DehaBAV1_0054 EMI: Emin_1165 DRA: DR_0230 DGE: Dgeo_0073 TTH:
TTC1438 TTJ: TTHA1790 AAE: aq_957 HYA: HY04AAS1_1161 SUL:
SYO3AOP1_1107 TMA: TM0647 TPT: Tpet_0283 TLE: Tlet_0532 TRQ:
TRQ2_0281 TME: Tmel_0239 FNO: Fnod_1503 PMO: Pmob_1172
Exemplary 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase
nucleic acids and polypeptides
TABLE-US-00055 [0968] ATH: AT5G60600(GcpE) YPN: YPN_1259 OSA:
4329911(Os02g0603800) YPP: YPDSF_2224(ispG) PPP:
PHYPADRAFT_130936(HDS3) YPG: YpAngola_A0418(ispG) PHYPADRAFT_55802
YPS: YPTB2841(ispG) OLU: OSTLU_12863(HDS) YPI:
YpsIP31758_1186(ispG) CRE: CHLREDRAFT_55268(HDS1) YPY: YPK_1293
CME: CML284C YPB: YPTS_2950 PFA: PF10_0221 YEN: YE1073(ispG) PFH:
PFHG_04116 SFL: SF2561(ispG) PYO: PY01664 SFX: S2733(ispG) TAN:
TA14455 SFV: SFV_2562(ispG) TPV: TP02_0667 SSN: SSON_2597(ispG)
ECO: b2515(ispG) SBO: SBO_2539(ispG) ECJ: JW2499(ispG) SBC:
SbBS512_E2890(ispG) ECD: ECDH10B_2681(ispG) SDY: SDY_2711(ispG)
ECE: Z3778(ispG) ECA: ECA3220(ispG) ECS: ECs3377(ispG) ETA:
ETA_10280(ispG) ECC: c3037(ispG) PLU: plu1376(ispG) ECI:
UTI89_C2836(ispG) BUC: BU287(gcpE) ECP: ECP_2520 BAS: BUsg276(gcpE)
ECV: APECO1_4009(ispG) WBR: WGLp573(gcpE) ECW: EcE24377A_2799(ispG)
SGL: SG1760(ispG) ECX: EcHS_A2666(ispG) ENT: Ent638_3009(ispG) ECM:
EcSMS35_2667(ispG) ESA: ESA_00745 ECL: EcolC_1162 KPN:
KPN_02845(ispG) STY: STY2768(ispG) CKO: CKO_00270 STT: t0333(ispG)
SPE: Spro_3609 SPT: SPA0344(ispG) BPN: BPEN_551(ispG) SPQ:
SPAB_00417 HIN: HI0368(ispG) SEC: SC2520(ispG) HIT: NTHI0488(ispG)
SEH: SeHA_C2781(ispG) HIP: CGSHiEE_01170 SEE: SNSL254_A2718(ispG)
HIQ: CGSHiGG_04650(ispG) SEW: SeSA_A2762(ispG) HDU: HD1037(ispG)
SES: SARI_00355 HSO: HS_0404(ispG) STM: STM2523(ispG) HSM: HSM_0729
YPE: YPO2879(ispG) PMU: PM2010(ispG) YPK: y1353(ispG) MSU:
MS1919(ispG) YPM: YP_2745(ispG) APL: APL_1176(ispG) YPA:
YPA_2319(ispG) APJ: APJL_1198(ispG) APA: APP7_1235 ABM:
ABSDF3001(ispG) ASU: Asuc_2027 ABY: ABAYE3263 XFA: XF2575(ispG)
ABC: ACICU_00511 XFT: PD1956(ispG) SON: SO_3312(ispG) XFM:
Xfasm12_2147 SDN: Sden_1256(ispG) XFN: XfasM23_2062 SFR:
Sfri_1116(ispG) XCC: XCC1781(ispG) SAZ: Sama_2365(ispG) XCB:
XC_2455(ispG) SBL: Sbal_2990 XCV: XCV1829(ispG) SBM: Shew185_3005
XAC: XAC1799(ispG) SBN: Sba1195_3148 XOO: XOO2229(ispG) SLO:
Shew_1290 XOM: XOO_2095(ispG) SPC: Sputcn32_2652(ispG) SML:
Smlt1786 SSE: Ssed_1432 SMT: Smal_1524 SPL: Spea_1305 VCH:
VC0759(ispG) SHE: Shewmr4_1228(ispG) VCO: VC0395_A0288(ispG) SHM:
Shewmr7_1299(ispG) VVU: VV1_0427(ispG) SHN: Shewana3_1229(ispG)
VVY: VV0766(ispG) SHW: Sputw3181_1355 VPA: VP0608(ispG) SHL:
Shal_1367 VFI: VF0629(ispG) SWD: Swoo_1544 VHA: VIBHAR_01067 ILO:
IL2034(ispG) PPR: PBPRA0763(ispG) CPS: CPS_4252(ispG) PAE:
PA3803(ispG) PHA: PSHAb0138(ispG) PAU: PA14_14880(ispG) PAT:
Patl_3126 PAP: PSPA7_1311(ispG) SDE: Sde_1434(ispG) PPU:
PP_0853(ispG) MAQ: Maqu_1127(ispG) PPF: Pput_0883(ispG) AMC:
MADE_02981 PPG: PputGB1_0896 PIN: Ping_1168 PPW: PputW619_4325 MCA:
MCA2483(ispG) PST: PSPTO_1434(ispG) FTU: FTT0607(ispG) PSB:
Psyr_1248(ispG) FTF: FTF0607(ispG) PSP: PSPPH_1320(ispG) FTW:
FTW_1121(ispG) PFL: PFL_4954(ispG) FTL: FTL_0875(ispG) PFO:
PflO1_4601(ispG) FTH: FTH_0861(ispG) PEN: PSEEN1021(ispG) FTA:
FTA_0926(ispG) PMY: Pmen_3500 FTN: FTN_1076(ispG) PSA:
PST_3031(ispG) FTM: FTM_0682(ispG) CJA: CJA_1481(ispG) FPH:
Fphi_0034 PAR: Psyc_0682(gcpE) NOC: Noc_1749 PCR: Pcryo_0652 AEH:
Mlg_1461(ispG) PRW: PsycPRwf_1902 HHA: Hhal_0132(ispG) ACI:
ACIAD0561(ispG) HCH: HCH_04456(ispG) ACB: A1S_0502 CSA:
Csal_2854(ispG) ABO: ABO_1860(ispG) BPT: Bpet2019(ispG) MMW:
Mmwyl1_1356 BAV: BAV2344(gcpE) AHA: AHA_1759(ispG) RFR: Rfer_2307
ASA: ASA_2599(ispG) POL: Bpro_2608(ispG) BCI: BCI_0008(ispG) PNA:
Pnap_1872(ispG) RMA: Rmag_0384 AAV: Aave_1424(ispG) VOK:
COSY_0358(ispG) AJS: Ajs_1170(ispG) NME: NMB1310(ispG) VEI:
Veis_0080(ispG) NMA: NMA1524(ispG) DAC: Daci_5019 NMC:
NMC1247(ispG) MPT: Mpe_A1996(ispG) NMN: NMCC_1223 HAR:
HEAR1264(ispG) NGO: NGO0594(ispG) MMS: mma_2127 NGK: NGK_1324 LCH:
Lcho_2868 CVI: CV_3538(ispG) NEU: NE0148 NE0149 RSO: RSc1215(ispG)
NET: Neut_2168(ispG) REU: Reut_A2086(ispG) NMU: Nmul_A2377 REH:
H16_A2364(ispG) EBA: ebA1261(ispG) RME: Rmet_2106(ispG) AZO:
azo0927(ispG) BMA: BMA1345(ispG) DAR: Daro_2985(ispG) BMV:
BMASAVP1_A1835(ispG) TBD: Tbd_0594(ispG) BML: BMA10229_A0062(ispG)
MFA: Mfla_1620(ispG) BMN: BMA10247_1107(ispG) HPY: HP0625(ispG)
BXE: Bxe_A1594(ispG) HPJ: jhp0569(ispG) BVI: Bcep1808_1739 HPA:
HPAG1_0608(ispG) BUR: Bcep18194_A5113(ispG) HPS: HPSH_03735(ispG)
BCN: Bcen_6267(ispG) HHE: HH0807(ispG) BCH: Bcen2424_1812 HAC:
Hac_0735(ispG) BCM: Bcenmc03_1836 WSU: WS1302(ispG) BAM:
Bamb_1750(ispG) TDN: Suden_0376(ispG) BAC: BamMC406_1723 CJE:
Cj0686(ispG) BMU: Bmul_1463 CJR: CJE0785(ispG) BMJ:
BMULJ_01780(gcpE) CJJ: CJJ81176_0709(ispG) BPS: BPSL1513(ispG) CJU:
C8J_0654(ispG) BPM: BURPS1710b_2355(ispG) CJD: JJD26997_1321(ispG)
BPL: BURPS1106A_2228(ispG) CFF: CFF8240_0983(ispG) BPD:
BURPS668_2190(ispG) CCV: CCV52592_0322(ispG) BTE: BTH_I2234(ispG)
CHA: CHAB381_0996(ispG) BPH: Bphy_1420 CCO: CCC13826_0680(ispG)
PNU: Pnuc_1291(ispG) ABU: Abu_0656(ispG) PNE: Pnec_0664 NIS:
NIS_0337(ispG) BPE: BP2199(ispG) SUN: SUN_2134(ispG) BPA:
BPP2855(ispG) GSU: GSU1459(ispG) BBR: BB3176(ispG) GME: Gmet_1353
GUR: Gura_2799 BCS: BCAN_A1816(ispG) GLO: Glov_1907 OAN: Oant_1123
PCA: Pcar_2368(ispG) BJA: blr0936(ispG) PPD: Ppro_1751 BRA:
BRADO0546(ispG) DVU: DVU1344(ispG) BBT: BBta_7633(ispG) DVL:
Dvul_1724 RPA: RPA0519(ispG) DDE: Dde_2207 RPB: RPB_0522(ispG) LIP:
LI0024(gcpE) RPC: RPC_0491(ispG) DPS: DP1163 RPD: RPD_0317(ispG)
DOL: Dole_2059 RPE: RPE_0183(ispG) ADE: Adeh_3949 RPT: Rpal_0520
AFW: Anae109_0476 NWI: Nwi_0494(ispG) SAT: SYN_00906 NHA:
Nham_0620(ispG) SFU: Sfum_2112 BHE: BH15270(ispG) WOL: WD0116(ispG)
BQU: BQ12180(ispG) WBM: Wbm0782(ispG) BBK: BARBAKC583_0119 WPI:
WP0196(ispG) (ispG) AMA: AM741(ispG) BTR: Btr_2457(gcpE) APH:
APH_0442(ispG) XAU: Xaut_1889 ERU: Erum4730(ispG) AZC: AZC_4581
ERW: ERWE_CDS_04950(ispG) MEX: Mext_1597 ERG: ERGA_CDS_04850(ispG)
MRD: Mrad2831_3931 ECN: Ecaj_0471(ispG) MET: M446_5049 ECH:
ECH_0559(ispG) BID: Bind_0434 NSE: NSE_0799(ispG) CCR: CC_0851 PUB:
SAR11_0517(ispG) CAK: Caul_0957 MLO: mll3792(ispG) SIL:
SPO2594(ispG) MES: Meso_3337(ispG) SIT: TM1040_0862(ispG) PLA:
Plav_1746 RSP: RSP_2982(ispG) SME: SMc03888(ispG) RSH:
Rsph17029_1628 SMD: Smed_3133(ispG) RSQ: Rsph17025_1861 ATU:
Atu2723(gcpE) JAN: Jann_1935(ispG) ATC: AGR_C_4936 RDE:
RD1_2825(ispG) RET: RHE_CH04009(ispG) PDE: Pden_1820(ispG) REC:
RHECIAT_CH0004297(gcpE) DSH: Dshi_1184 RLE: RL4630(ispG) MMR:
Mmar10_2256(ispG) BME: BMEI0269(ispG) HNE: HNE_0621(ispG) BMF:
BAB1_1788(ispG) ZMO: ZMO0180(ispG) BMB: BruAb1_1761(ispG) NAR:
Saro_0417 BMC: BAbS19_I16710 SAL: Sala_1848(ispG) BMS: BR1778(ispG)
SWI: Swit_2126 BMT: BSUIS_B1254(ispG) ELI: ELI_10365(ispG) BOV:
BOV_1713(ispG) GOX: GOX0034(ispG) ACR: Acry_1012(ispG) GBE:
GbCGDNIH1_0604 GDI: GDI1913(ispG) (ispG) RRU: Rru_A0747(ispG) CBA:
CLB_2288(gcpE) MAG: amb1616(ispG) CBH: CLC_2271(gcpE) MGM:
Mmc1_3591 CBL: CLK_1800(gcpE) ABA: Acid345_1423(ispG) CBK:
CLL_A1267(ispG) SUS: Acid_1193 CBB: CLD_2216(gcpE) SWO: Swol_0891
CBF: CLI_2480(gcpE) CSC: Csac_2351 CBE: Cbei_1197(ispG) BSU:
BSU25070(ispG) CKL: CKL_1425(ispG) BHA: BH1401(ispG) CPY: Cphy_2620
BAN: BA4502(ispG) AMT: Amet_2680 BAR: GBAA4502(ispG) AOE: Clos_1521
BAA: BA_4950 CHY: CHY_1776(ispG) BAT: BAS4180(ispG) DSY: DSY2537
BCE: BC4276(ispG) DRM: Dred_1968 BCA: BCE_4358(ispG) PTH:
PTH_1262(gcpE) BCZ: BCZK4028(ispG) DAU: Daud_0617 BCY: Bcer98_3006
HMO: HM1_2266(ispG) BTK: BT9727_4018(ispG) FMA: FMG_0730 BTL:
BALH_3871(ispG) TTE: TTE1400(ispG) BWE: BcerKBAB4_4131 TEX:
Teth514_1652 BLI: BL03725(ispG) TPD: Teth39_1216 BLD:
BLi02683(ispG) MTA: Moth_1043 BCL: ABC1708(ispG) MPE: MYPE9400 BAY:
RBAM_023380 MGA: MGA_1156 BPU: BPUM_2235 MTU: Rv2868c(ispG) GKA:
GK2466(ispG) MTC: MT2936(ispG) GTN: GTNG_2403(ispG) MRA:
MRA_2893(ispG) LSP: Bsph_3646 MTF: TBFG_12884(ispG) ESI: Exig_0854
MBO: Mb2893c(ispG) LMO: lmo1441(ispG) MBB: BCG_2890c(ispG) LMF:
LMOf2365_1460(ispG) MLE: ML1581(ispG) STH: STH1501 MPA:
MAP2938c(ispG) CAC: CAC1797(gcpE) MAV: MAV_3725(ispG) CPE:
CPE1692(ispG) MSM: MSMEG_2580(ispG) CPF: CPF_1946(ispG) MUL:
MUL_2087(ispG) CPR: CPR_1664(ispG) MVA: Mvan_2262 CTC:
CTC01270(gcpE) MGI: Mflv_4081(ispG) CNO: NT01CX_2141(ispG) MAB:
MAB_3169c CTH: Cthe_0997 MMC: Mmcs_2044(ispG) CDF: CD2128(ispG)
MKM: Mkms_2090(ispG) CBO: CBO2424(ispG) MJL: Mjls_2027(ispG) CGL:
NCgl1938(ispG) MMI: MMAR_0275(gcpE_2) CGB: cg2206(ispG)
MMAR_1838(ispG) CGT: cgR_1842(ispG) CTL: CTLon_0308(gcpE) CEF:
CE1903(ispG) CMU: TC0327(gcpE) CDI: DIP1498(ispG) CPN:
CPn0373(gcpE) CJK: jk1165(ispG) CPA: CP0383 CUR: cu0833 CPJ:
CPj0373(gcpE) NFA: nfa41180(ispG) CPT: CpB0385(aarC) RHA:
RHA1_ro06590(ispG) CCA: CCA00423(gcpE) SCO: SCO5696(ispG)
SCO6767(ispG) CAB: CAB409 SMA: SAV1647(ispG) SAV2561(ispG) CFE:
CF0584(gcpE) SGR: SGR_1821(gcpE) PCU: pc0740(gcpE) TWH:
TWT186(ispG) TPA: TP0446 TWS: TW586(ispG) TPP: TPASS_0446(gcpE)
LXX: Lxx12200(ispG) TDE: TDE1265(ispG) CMI: CMM_2156(ispG) LIL:
LA3160(gcpE) ART: Arth_1404(ispG) LIC: LIC10955(gcpE) AAU:
AAur_1546(ispG) LBJ: LBJ_0737(gcpE) RSA: RSal33209_0641 LBL:
LBL_2341(gcpE) KRH: KRH_16120(ispG) LBI: LEPBI_I1285(ispG) PAC:
PPA1506(ispG) LBF: LBF_1231(gcpE) NCA: Noca_3202 SYN: slr2136(gcpE)
TFU: Tfu_0749(ispG) SYW: SYNW1174(ispG) FRA: Francci3_3573(ispG)
SYC: syc0817_d(ispG) FRE: Franean1_1170 SYF: Synpcc7942_0713(ispG)
FAL: FRAAL5772(ispG) SYD: Syncc9605_1298(ispG) ACE: Acel_1522 SYE:
Syncc9902_1179(ispG) KRA: Krad_1429 SYG: sync_1674(ispG) SEN:
SACE_5992(ispG) SYR: SynRCC307_1462(ispG) STP: Strop_1352(ispG)
SYX: SynWH7803_1475(ispG) SAQ: Sare_1304 SYP: SYNPCC7002_A0743 BLO:
BL0098(ispG) (ispG) BLJ: BLD_0116(gcpE) CYA: CYA_2387(ispG) BAD:
BAD_1157(ispG) CYB: CYB_0121(ispG) RXY: Rxyl_1406 TEL: tlr0996 FNU:
FN0478 MAR: MAE_28180 RBA: RB2118(gcpE) CYT: cce_2312(ispG) OTE:
Oter_4634 GVI: gll3622
MIN: Minf_1968(gcpE) ANA: all2501 AMU: Amuc_1388 NPU: Npun_F5054
CTR: CT057(gcpE) AVA: Ava_0433(ispG) CTA: CTA_0061(gcpE) PMA:
Pro1015(ispG) CTB: CTL0313(gcpE) PMM: PMM0676(ispG) PMB:
A9601_07311(ispG) PMT: PMT0777(ispG) PMC: P9515_07491(ispG) PMN:
PMN2A_0109(ispG) PMF: P9303_14341(ispG) PMI: PMT9312_0676(ispG)
PMG: P9301_07291(ispG) TME: Tmel_0263 PMH: P9215_07611(gcpE) FNO:
Fnod_0952 PMJ: P9211_07901(gcpE) PMO: Pmob_1941 PME:
NATL1_07341(ispG) TER: Tery_4522(ispG) AMR: AM1_0149(ispG) BTH:
BT_2517 BFR: BF4365 BFS: BF4164 BVU: BVU_1415 PGI: PG0952(ispG)
PGN: PGN_0998 PDI: BDI_3173 SRU: SRU_0682(ispG) CHU: CHU_2192(ispG)
CTE: CT0147(gcpE) CPC: Cpar_1891 CCH: Cag_0349 CPH: Cpha266_0225
CPB: Cphamn1_0311 PVI: Cvib_1613 PLT: Plut_1970 PPH: Ppha_2687 CTS:
Ctha_1524 PAA: Paes_0280 DET: DET0369(ispG) DEH: cbdb_A311(ispG)
DEB: DehaBAV1_0351 EMI: Emin_0687 DRA: DR_0386(ispG) DGE:
Dgeo_0704(ispG) TTH: TTC1677(ispG) TTJ: TTHA0305(ispG) AAE:
aq_1540(gcpE) HYA: HY04AAS1_1229 SUL: SYO3AOP1_0412 TMA: TM0891
TPT: Tpet_0036 TLE: Tlet_0656 TRQ: TRQ2_0036
Exemplary 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase
nucleic acids and polypeptides
TABLE-US-00056 [0969] ATH: AT4G34350(CLB6) YPA: YPA_4071(ispH) OSA:
4334003(Os03g0731900) YPN: YPN_0350 PPP: PHYPADRAFT_194018 YPP:
YPDSF_3154(ispH) PHYPADRAFT_206243 YPG: YpAngola_A0787(ispH) OLU:
OSTLU_32979(IDS) YPS: YPTB0620(ispH) CRE: CHLREDRAFT_59822(IDS1)
YPI: YpsIP31758_3457(ispH) CME: CMJ152C YPY: YPK_3585 PFA:
MAL1P1.35 YPB: YPTS_0644 PFD: PFDG_01560 YEN: YE0619(ispH) PFH:
PFHG_00328 SFL: SF0026(ispH) PYO: PY01243 SFX: S0028(ispH) TAN:
TA17670 SFV: SFV_0023(ispH) TPV: TP03_0674 SSN: SSON_0034(ispH)
ECO: b0029(ispH) SBO: SBO_0028(ispH) ECJ: JW0027(ispH) SBC:
SbBS512_E0033(ispH) ECD: ECDH10B_0030(ispH) SDY: SDY_0051(ispH)
ECE: Z0034(ispH) ECA: ECA3873(ispH) ECS: ECs0032(ispH) ETA:
ETA_07150(ispH) ECC: c0033(ispH) PLU: plu0594(ispH) ECI:
UTI89_C0031(ispH) BUC: BU147(lytB) ECP: ECP_0027 BAS: BUsg140(lytB)
ECV: APECO1_1954(ispH) WBR: WGLp292(lytB) ECW: EcE24377A_0029(ispH)
SGL: SG0417(ispH) ECX: EcHS_A0031(ispH) ENT: Ent638_0587(ispH) ECM:
EcSMS35_0027(ispH) ESA: ESA_03309 ECL: EcolC_3626 KPN:
KPN_00024(ispH) STY: STY0058(ispH) CKO: CKO_03363 STT: t0051(ispH)
SPE: Spro_0701 SPT: SPA0050(ispH) BPN: BPEN_124(ispH) SPQ:
SPAB_00059 HIN: HI1007(ispH) SEC: SC0043(ispH) HIT: NTHI1182(ispH)
SEH: SeHA_C0053(ispH) HIP: CGSHiEE_06935(ispH) SEE:
SNSL254_A0053(ispH) HIQ: CGSHiGG_08635(ispH) SEW: SeSA_A0054(ispH)
HDU: HD0064(ispH) SES: SARI_02945 HSO: HS_0184(ispH) STM:
STM0049(ispH) HSM: HSM_0050 YPE: YPO0477(ispH) PMU: PM1664(ispH)
YPK: y3697(ispH) MSU: MS1749(ispH) YPM: YP_3702(ispH) APL:
APL_1520(ispH) APJ: APJL_1546(ispH) ACI: ACIAD3322(ispH) APA:
APP7_1580 ACB: A1S_3169 ASU: Asuc_1874 ABM: ABSDF0323(ispH) XFA:
XF2416(ispH) ABY: ABAYE0313 XFT: PD1435(ispH) ABC: ACICU_03371 XFM:
Xfasm12_1576 SON: SO_3529(lytB) XFN: XfasM23_1519 SDN: Sden_2720
XCC: XCC1157(ispH) SFR: Sfri_2887 XCB: XC_3085(ispH) SAZ: Sama_0927
XCV: XCV1292(ispH) SBL: Sbal_1057 XAC: XAC1256(ispH) SBM:
Shew185_1124 XOO: XOO1628(ispH) SBN: Sbal195_1159 XOM:
XOO_1514(ispH) SLO: Shew_1102 SML: Smlt1342(ispH) SPC:
Sputcn32_1062 SMT: Smal_1127 SSE: Ssed_1197 VCH: VC0685(ispH) SPL:
Spea_1086 VCO: VC0395_A0217(ispH) SHE: Shewmr4_2954 VVU:
VV1_0504(ispH) SHM: Shewmr7_3036 VVY: VV0690(ispH) SHN:
Shewana3_3133 VPA: VP0537(ispH) SHW: Sputw3181_3103 VFI:
VF0470(ispH) SHL: Shal_1134 VHA: VIBHAR_00983 SWD: Swoo_1294 PPR:
PBPRA0594(ispH) ILO: IL1125(lytB) PAE: PA4557(ispH) CPS:
CPS_1211(ispH) PAU: PA14_60330(ispH) PHA: PSHAa0921(ispH) PAP:
PSPA7_3192(ispH2) PAT: Patl_3175 PSPA7_5197(ispH1) SDE: Sde_2563
PPU: PP_0606(ispH) MAQ: Maqu_0865 PPF: Pput_0647(ispH) AMC:
MADE_03027 PPG: PputGB1_0652 PIN: Ping_3268 PPW: PputW619_4556 MCA:
MCA1815(ispH) PST: PSPTO_0809(ispH) FTU: FTT0833(ispH) PSB:
Psyr_0713(ispH) FTF: FTF0833(ispH) PSP: PSPPH_0724(ispH) FTW:
FTW_1353(ispH) PFL: PFL_5318(ispH) FTL: FTL_0327 PFO:
PflO1_4849(ispH) FTH: FTH_0325 PEN: PSEEN4689(ispH) FTA:
FTA_0348(ispH) PMY: Pmen_0956 FTM: FTM_0425(lytB) PSA:
PST_0967(ispH) FPH: Fphi_0475 CJA: CJA_3214(ispH) NOC: Noc_1744
PAR: Psyc_1722(lytB) AEH: Mlg_0854 PCR: Pcryo_2002 HHA: Hhal_1834
PRW: PsycPRwf_0578 HCH: HCH_05930(ispH) CSA: Csal_0484 BPD:
BURPS668_0981(ispH) ABO: ABO_0462(lytB) BURPS668_A3054(ispH) MMW:
Mmwyl1_4227 BTE: BTH_I0783(ispH-1) BTH_II2243(ispH- AHA:
AHA_0685(ispH) 2) ASA: ASA_0687(lytB) BPH: Bphy_0587 Bphy_4130 BCI:
BCI_0558(ispH) PNU: Pnuc_1731 RMA: Rmag_1023 PNE: Pnec_1449 VOK:
COSY_0924(lytB) BPE: BP1237(ispH) NME: NMB1831(ispH) BPA:
BPP1852(ispH) NMA: NMA0624(ispH) BBR: BB3256(ispH) NMC:
NMC0385(ispH) BPT: Bpet3147(lytB) NMN: NMCC_0391(lytB) BAV:
BAV2403(ispH) NGO: NGO0072(ispH) RFR: Rfer_3248 NGK: NGK_0106 POL:
Bpro_0951 CVI: CV_3567(ispH) PNA: Pnap_3337 RSO: RSc2442(ispH) AAV:
Aave_3771 REU: Reut_A2730(ispH) Reut_B4898(ispH) AJS: Ajs_3448 REH:
H16_A3031(ispH) H16_B2169(ispH) VEI: Veis_1652 RME: Rmet_2868(ispH)
Rmet_4169(ispH) DAC: Daci_1906 BMA: BMA2228(ispH) BMAA1962(ispH)
MPT: Mpe_A2693 BMV: BMASAVP1_0980(ispH) HAR: HEAR2466(ispH)
BMASAVP1_A2644(ispH) MMS: mma_2549 BML: BMA10229_1267(ispH) LCH:
Lcho_0693 BMA10229_A1018(ispH) NEU: NE0649(lytB) BMN:
BMA10247_2097(ispH) NET: Neut_1903 BMA10247_A2242(ispH) NMU:
Nmul_A0089 BXE: Bxe_A0820(ispH) Bxe_B0018(ispH) EBA: ebA4444(ispH)
BVI: Bcep1808_2577 Bcep1808_3716 AZO: azo1202(ispH) BUR:
Bcep18194_A5831(ispH) DAR: Daro_3043 Bcep18194_B0106(ispH) TBD:
Tbd_1860 BCN: Bcen_1888(ispH) Bcen_5308(ispH) MFA: Mfla_2431 BCH:
Bcen2424_2499 Bcen2424_5552 HPY: HP0400(ispH) BCM: Bcenmc03_2524
Bcenmc03_4720 HPJ: jhp0981(ispH) BAM: Bamb_2546(ispH)
Bamb_4876(ispH) HPA: HPAG1_0992(ispH) BAC: BamMC406_2417
BamMC406_5423 HPS: HPSH_05405(ispH) BMU: Bmul_0795 Bmul_3253 HHE:
HH0138(ispH) BMJ: BMULJ_02464(lytB) HAC: Hac_0458(ispH)
BMULJ_05272(lytB) WSU: WS1310(ispH) BPS: BPSL0919(ispH)
BPSS2168(ispH) TDN: Suden_0872(ispH) BPM: BURPS1710b_1141(ispH)
CJE: Cj0894c(ispH) BURPS1710b_A1285(ispH) CJR: CJE0973(ispH) BPL:
BURPS1106A_0986(ispH) CJJ: CJJ81176_0903(ispH)
BURPS1106A_A2929(ispH) CJU: C8J_0831(lytB) CJD: JJD26997_0919(ispH)
RET: RHE_CH00961(ispH) CFF: CFF8240_1251(ispH) REC:
RHECIAT_CH0001056(ispH) CCV: CCV52592_0515(ispH) RLE: RL1030(ispH)
CHA: CHAB381_0483(ispH) BME: BMEI1459(ispH) CCO:
CCC13826_1566(ispH) BMF: BAB1_0501(ispH) ABU: Abu_2050(ispH) BMB:
BruAb1_0497(ispH) NIS: NIS_0662(ispH) BMC: BAbS19_I04640 SUN:
SUN_0548(ispH) BMS: BR0475(ispH) GSU: GSU2604(lytB) BMT:
BSUIS_A0502(ispH) GME: Gmet_0866 BOV: BOV_0480(ispH) GUR: Gura_1466
BCS: BCAN_A0482(ispH) GLO: Glov_2146 OAN: Oant_0589 PCA:
Pcar_1883(lytB) BJA: bll3007(ispH) blr1314 PPD: Ppro_1349 BRA:
BRADO2632(ispH) DVU: DVU0055(ispH) BRADO6588(ispH1) DVL: Dvul_2906
BBT: BBta_0948(ispH1) BBta_2972(ispH) DDE: Dde_0390 RPA:
RPA3734(ispH) RPA4271(lytB2) LIP: LI0728(lytB) RPB: RPB_1340
RPB_1729(ispH) DPS: DP2166 RPC: RPC_1726(ispH) RPC_4078 DOL:
Dole_0383 RPD: RPD_3570(ispH) RPD_4030 ADE: Adeh_1519 RPE:
RPE_1816(ispH) RPE_4130 AFW: Anae109_2302 RPT: Rpal_4255 Rpal_4751
SAT: SYN_02454 NWI: Nwi_2266(ispH) Nwi_2689 SFU: Sfum_1812 NHA:
Nham_2679(ispH) Nham_3745 WOL: WD1274(ispH) BHE: BH04410(ispH) WBM:
Wbm0046(ispH) BQU: BQ03600(ispH) WPI: WP0811(lytB) BBK:
BARBAKC583_0406(ispH) AMA: AM804(ispH) BTR: Btr_0655(lytB) APH:
APH_0380(ispH) XAU: Xaut_2355 ERU: Erum5180(ispH) AZC: AZC_1468
ERW: ERWE_CDS_05430(ispH) MEX: Mext_2593 ERG: ERGA_CDS_05330(ispH)
MRD: Mrad2831_4312 ECN: Ecaj_0526(ispH) MET: M446_6025 ECH:
ECH_0502(ispH) BID: Bind_1904 NSE: NSE_0438(ispH) CCR: CC_3361 PUB:
SAR11_0124(lytB) CAK: Caul_4391 MLO: mlr7502(ispH) SIL:
SPO3207(ispH) MES: Meso_0748(ispH) SIT: TM1040_2569 PLA: Plav_0686
RSP: RSP_1666(lytB) SME: SMc00016(ispH) RSH: Rsph17029_0299 SMD:
Smed_0527(ispH) RSQ: Rsph17025_2580 ATU: Atu0774(lytB) JAN:
Jann_0507 ATC: AGR_C_1414(lytB) RDE: RD1_1355(ispH) PDE: Pden_3619
CPF: CPF_1341(ispH) DSH: Dshi_0188 CPR: CPR_1152(ispH) MMR:
Mmar10_2215 CTC: CTC01314 HNE: HNE_2713(ispH) CNO: NT01CX_2096 ZMO:
ZMO0875(ispH) CTH: Cthe_0714 NAR: Saro_1087 CDF: CD1818(ispH) SAL:
Sala_1136 AMT: Amet_2625 SWI: Swit_2692 AOE: Clos_1562 ELI:
ELI_01560 DRM: Dred_1154 GOX: GOX0179 TTE: TTE1352(lytB) GBE:
GbCGDNIH1_1875 TEX: Teth514_1606 ACR: Acry_1832 TPD: Teth39_1169
GDI: GDI3102(ispH) MPE: MYPE1330 RRU: Rru_A0059 MTU: Rv1110(ispH)
Rv3382c(lytB1) MAG: amb0764 MTC: MT1141(ispH) MT3490(lytB-2) MGM:
Mmc1_3428 MRA: MRA_1121(ispH) MRA_3422(lytB1) ABA: Acid345_1739
MTF: TBFG_11132(ispH) TBFG_13416 SUS: Acid_1259 MBO: Mb1140(ispH)
Mb3414c(lytB1) BSU: BSU25160(ispH) MBB: BCG_1170(ispH)
BCG_3451c(lytB1) BHA: BH1382(ispH) MLE: ML1938(ispH) BAN:
BA4511(ispH) MPA: MAP2684c(ispH) BAR: GBAA4511(ispH) MAV:
MAV_1230(ispH) BAA: BA_4959 MSM: MSMEG_5224(ispH) BAT:
BAS4190(ispH) MUL: MUL_0168(ispH) BCA: BCE_4368(ispH) MVA:
Mvan_4631 BCZ: BCZK4038(ispH) MGI: Mflv_2079(ispH) BCY: Bcer98_3015
MAB: MAB_1257 BTK: BT9727_4028(ispH) MMC: Mmcs_4105(ispH) BTL:
BALH_3881(ispH) MKM: Mkms_4181(ispH) BWE: BcerKBAB4_4140 MJL:
Mjls_4336(ispH) BLI: BL03721(ispH) MMI: MMAR_0277(lytB2) BLD:
BLi02695(ispH) MMAR_4355(ispH) BCL: ABC1694(ispH) CGL:
NCgl0982(ispH) BAY: RBAM_023470(yqfP) CGB: cg1164(ispH) BPU:
BPUM_2249(yqfP) CGT: cgR_1109(ispH) GKA: GK2477(ispH) CEF:
CE1079(ispH) GTN: GTNG_2414(ispH) CDI: DIP0943(ispH) LSP: Bsph_3685
CJK: jk1449(ispH) ESI: Exig_0836 CUR: cu0618 LMO: lmo1451(ispH)
NFA: nfa47950(ispH) LMF: LMOf2365_1470(ispH) RHA:
RHA1_ro05870(ispH) STH: STH910(ispH) SCO: SCO5058(ispH) CPE:
CPE1085(lytB) SMA: SAV3210(ispH) SGR: SGR_2472 LIL: LA2420(lytB)
TWH: TWT642(ispH) LIC: LIC11529(lytB) TWS: TW664(ispH) LBJ:
LBJ_1807(lytB) LXX: Lxx16760(ispH) LBL: LBL_1476(lytB) CMI:
CMM_2228(ispH) LBI: LEPBI_I1588(ispH) ART: Arth_2833(ispH) LBF:
LBF_1537(lytB) RSA: RSal33209_1156 SYN: slr0348 KRH:
KRH_07120(ispH) SYW: SYNW0252(lytB) PAC: PPA0572(ispH) SYC:
syc1431_d(lytB) NCA: Noca_1075 SYF: Synpcc7942_0073 TFU:
Tfu_0471(ispH) SYD: Syncc9605_0246 FRA: Francci3_0824
Francci3_3881(ispH) SYE: Syncc9902_0275 FRE: Franean1_0845
Franean1_5712 SYG: sync_0292(ispH) FAL: FRAAL1433(ispH)
FRAAL6150(ispH) SYR: SynRCC307_2319(lytB) ACE: Acel_1858 SYX:
SynWH7803_0296(lytB) KRA: Krad_1123 SYP: SYNPCC7002_A0517(ispH)
SEN: SACE_0939(ispH) SACE_4326(ispH) CYA: CYA_1148(ispH) STP:
Strop_0879(ispH) CYB: CYB_2643(ispH) SAQ: Sare_0824 TEL: tlr1041
BLO: BL1361(ispH) MAR: MAE_16190 BLJ: BLD_0227(lytB) CYT: cce_1108
BAD: BAD_1081(ispH) GVI: glr3299 RXY: Rxyl_2212 ANA: all0985 RBA:
RB9288(lytB) NPU: Npun_R3286 OTE: Oter_3652 AVA: Ava_2949 MIN:
Minf_2119(lytB) PMA: Pro0296(lytB) AMU: Amuc_1646 PMM:
PMM0264(lytB) CTR: CT859(ispH) PMT: PMT1854(lytB) CTA:
CTA_0937(ispH) PMN: PMN2A_1630 CTB: CTL0234 PMI: PMT9312_0266 CTL:
CTLon_0234(ispH) PMB: A9601_02861(lytB) CMU: TC0249(ispH) PMC:
P9515_02971(lytB) CPN: CPn1017(ispH) PMF: P9303_24821(lytB) CPA:
CP0836(ispH) PMG: P9301_02871(lytB) CPJ: CPj1017(ispH) PMH:
P9215_02881(lytB) CPT: CpB1055(ispH) PMJ: P9211_02911(lytB) CCA:
CCA00744(ispH) PME: NATL1_03421(lytB) CAB: CAB711(ispH) TER:
Tery_4479
CFE: CF0272(ispH) AMR: AM1_4950(ispH) PCU: pc1078(ispH) BTH:
BT_2061(ispH) TPA: TP0547 BFR: BF3748(ispH) TPP: TPASS_0547(lytB)
BFS: BF3536(ispH) TDE: TDE1096(ispH) BVU: BVU_1936 PGI:
PG0604(ispH) PGN: PGN_0647 PDI: BDI_3740(ispH) SRU: SRU_1880(ispH)
CHU: CHU_0087(ispH) CTE: CT0283(ispH) CPC: Cpar_1751 CCH:
Cag_0579(ispH) CPH: Cpha266_0414(ispH) CPB: Cphamn1_0456 PVI:
Cvib_1518(ispH) PLT: Plut_1736(ispH) PPH: Ppha_0448 CTS: Ctha_0114
PAA: Paes_0419 DET: DET1344(ispH) DEH: cbdb_A1294(ispH) DEB:
DehaBAV1_1155 EMI: Emin_0409 DRA: DR_2164 DGE: Dgeo_1010 TTH:
TTC1983(lytB) TTJ: TTHA0015 AAE: aq_1739(lytB) HYA: HY04AAS1_1048
SUL: SYO3AOP1_1148 TMA: TM1444 TPT: Tpet_1350 TLE: Tlet_1650 TRQ:
TRQ2_1336 PMO: Pmob_1619
Exemplary Isopentenyl-Diphosphate Delta-Isomerase Nucleic Acids and
Polypeptides
TABLE-US-00057 [0970] HSA: 3422(IDI1) 91734(IDI2) CGR: CAGL0J06952g
PTR: 450262(IDI2) 450263(IDI1) YLI: YALI0F04015g MCC:
710052(LOC710052) SPO: SPBC106.15(idi1) 721730(LOC721730) NCR:
NCU07719 MMU: 319554(Idi1) PAN: PODANSg7228 RNO: 89784(Idi1) MGR:
MGG_07125 BTA: 514293(IDI1) FGR: FG09722.1 MDO:
100021550(LOC100021550) ANI: AN0579.2 100021613(LOC100021613) AFM:
AFUA_6G11160 100021638(LOC100021638) AOR: AO090023000500 OAA:
100080658(LOC100080658) ANG: An08g07570 GGA: 420459(IDI1) CNE:
CNA02550 XLA: 494671(LOC494671) CNB: CNBA2380 XTR: 496783(idi2)
LBC: LACBIDRAFT_291469 SPU: 586184(LOC586184) UMA: UM04838.1 NVE:
MGL: MGL_1929 NEMVE_v1g121175(NEMVEDRAFT_v1g1 ECU: ECU02_0230
21175) MBR: MONBRDRAFT_34433 DME: Dmel_CG5919(CG5919) GLA:
GL50803_6335 DPO: Dpse_GA19228 DDI: DDB_0191342(ipi) AGA:
AgaP_AGAP001704 TET: TTHERM_00237280 AAG: AaeL_AAEL006144
TTHERM_00438860 TCA: 660176(LOC660176) PTM: GSPATT00007643001 CEL:
K06H7.9(idi-1) GSPATT00011951001 CBR: CBG22969 TBR: Tb09.211.0700
BMY: Bm1_16940 TCR: 408799.19 510431.10 ATH:
AT3G02780(IDI2/IPIAT1/IPP2) LMA: LmjF35.5330 AT5G16440(IPP1) EHI:
EHI_194410 OSA: 4338791(Os05g0413400) TVA: TVAG_116230 TVAG_495540
4343523(Os07g0546000) ECO: b2889(idi) PPP: PHYPADRAFT_56143 ECJ:
JW2857(idi) OLU: OSTLU_13493 ECD: ECDH10B_3063(idi) CRE:
CHLREDRAFT_24471(IDI1) ECE: Z4227 CME: CMB062C ECS: ECs3761 SCE:
YPL117C(IDI1) ECC: c3467 AGO: AGOS_ADL268C ECI: UTI89_C3274 KLA:
KLLA0F00924g ECP: ECP_2882 DHA: DEHA0G20009g ECV: APECO1_3638 PIC:
PICST_68990(IDI1) ECW: EcE24377A_3215(idi) VPO: Kpol_479p9 ECX:
EcHS_A3048(idi) ECM: EcSMS35_3022(idi) EBA: ebA5678 p2A143 ECL:
EcolC_0820 DVU: DVU1679(idi) STY: STY3195 DDE: Dde_1991 STT: t2957
LIP: LI1134 SPT: SPA2907(idi) BBA: Bd1626 SPQ: SPAB_03786 AFW:
Anae109_4082 SEC: SC2979(idi) MXA: MXAN_5021(fni) SEH:
SeHA_C3270(idi) SCL: sce1761(idi) SEE: SNSL254_A3273(idi) RPR:
RP452 SEW: SeSA_A3207(idi) RTY: RT0439(idi) SES: SARI_04611 RCO:
RC0744 STM: STM3039(idi) RFE: RF_0785(fni) SFL: SF2875 RBE:
RBE_0731(fni) SFX: S3074 RBO: A1I_04760 SFV: SFV_2937 RAK:
A1C_04195 SSN: SSON_3042 SSON_3489(yhfK) RCM: A1E_02555 SBO:
SBO_3103 RRI: A1G_04195 SBC: SbBS512_E3308(idi) RRJ: RrIowa_0882
SDY: SDY_3193 RMS: RMA_0766(fni) ECA: ECA2789 MLO: mlr6371 ETA:
ETA_22390(idi) MES: Meso_4299 PLU: plu3987 RET:
RHE_PD00245(ypd00046) ENT: Ent638_3307 REC: RHECIAT_PB0000285 ESA:
ESA_00346 XAU: Xaut_4134 KPN: KPN_03317(idi) SIL: SPO0131 CKO:
CKO_04250 SIT: TM1040_3442 SPE: Spro_2201 RSP: RSP_0276 VPA:
VPA0278 RSH: Rsph17029_1919 VFI: VF0403 RSQ: Rsph17025_1019 VHA:
VIBHAR_04924 JAN: Jann_0168 PPR: PBPRA0469 RDE: RD1_0147(idi) PEN:
PSEEN4850 DSH: Dshi_3527 PSA: PST_3876 SWO: Swol_1341 CBU:
CBU_0607(mvaD) BSU: BSU22870(ypgA) CBS: COXBURSA331_A0720(mvaD)
BAN: BA1520 CBD: CBUD_0619(mvaD) BAR: GBAA1520 LPN: lpg2051 BAA:
BA_2041 LPF: lp12029 BAT: BAS1409 LPP: lpp2034 BCE: BC1499 LPC:
LPC_1537(fni) BCA: BCE_1626 TCX: Tcr_1718 BCZ: BCZK1380(fni) HHA:
Hhal_1623 BCY: Bcer98_1222 DNO: DNO_0798 BTK: BT9727_1381(fni) BTL:
BALH_1354 SPB: M28_Spy0665 BWE: BcerKBAB4_1422 SPN: SP_0384 BLI:
BL02217(fni) SPR: spr0341(fni) BLD: BLi02426 SPD: SPD_0349(fni)
BAY: RBAM_021020(fni) SPV: SPH_0491(fni) BPU: BPUM_2020(fni) SPW:
SPCG_0379(fni) OIH: OB0537 SPX: SPG_0349 SAU: SA2136(fni) SAG:
SAG1323 SAV: SAV2346(fni) SAN: gbs1393 SAW: SAHV_2330(fni) SAK:
SAK_1354(fni) SAM: MW2267(fni) SMU: SMU.939 SAR: SAR2431(fni) STC:
str0562(idi) SAS: SAS2237 STL: stu0562(idi) SAC: SACOL2341(fni)
STE: STER_0601 SAB: SAB2225c(fni) SSA: SSA_0336 SAA:
SAUSA300_2292(fni) SSU: SSU05_0292 SAX: USA300HOU_2327 SSV:
SSU98_0288 SAO: SAOUHSC_02623 SGO: SGO_0242 SAJ: SaurJH9_2370 SEZ:
Sez_1081 SAH: SaurJH1_2416 LPL: lp_1732(idi1) SAE: NWMN_2247(idi)
LJO: LJ1208 SEP: SE1925 LAC: LBA1171 SER: SERP1937(fni-2) LSA:
LSA0905(idi) SHA: SH0712(fni) LSL: LSL_0682 SSP: SSP0556 LDB:
Ldb0996(fni) LMO: lmo1383 LBU: LBUL_0903 LMF: LMOf2365_1402(fni)
LBR: LVIS_0861 LIN: lin1420 LCA: LSEI_1493 LWE: lwe1399(fni) LCB:
LCABL_17150(fni) LLA: L11083(yebB) LGA: LGAS_1036 LLC: LACR_0457
LRE: Lreu_0912 LLM: llmg_0428(fni) LRF: LAR_0859 SPY: SPy_0879 LHE:
lhv_1278 SPZ: M5005_Spy_0685 LFE: LAF_1195 SPM: spyM18_0940 EFA:
EF0901 SPG: SpyM3_0598 OOE: OEOE_1103 SPS: SPs1255 LME: LEUM_1388
SPH: MGAS10270_Spy0743 LCI: LCK_00620 SPI: MGAS10750_Spy0777 STH:
STH1674 SPJ: MGAS2096_Spy0756 DRM: Dred_0474 SPK: MGAS9429_Spy0740
HMO: HM1_1981(fni) SPF: SpyM51123(fni) FMA: FMG_1144 SPA:
M6_Spy0702 MTA: Moth_1328 ACL: ACL_0797(idi) BGA: BG0707 MTU:
Rv1745c(idi) BTU: BT0684 MTC: MT1787(idi) SYN: sll1556 MRA:
MRA_1756(idi) SYC: syc2161_c MTF: TBFG_11763 SYF: Synpcc7942_1933
MBO: Mb1774c(idi) SYP: SYNPCC7002_A1132(fni) MBB: BCG_1784c(idi)
CYA: CYA_2395(fni) MPA: MAP3079c CYB: CYB_2691(fni) MAV:
MAV_3894(fni) TEL: tll1403 MSM: MSMEG_1057(fni) MAR: MAE_56570
MSMEG_2337(fni) CYT: cce_1202 MUL: MUL_0380(idi2) ANA: all4591 MVA:
Mvan_1582 Mvan_2176 NPU: Npun_R1243 MGI: Mflv_1842 Mflv_4187 AVA:
Ava_2461 Ava_B0346 MAB: MAB_3242 TER: Tery_1589 MMC: Mmcs_1954 AMR:
AM1_4374(fni) MKM: Mkms_2000 SRU: SRU_1900(idi) MJL: Mjls_1934 CHU:
CHU_0674(idi) MMI: MMAR_3218(idi) MMAR_4812(idi2) GFO:
GFO_2363(idi) CGL: NCgl2223(cgl2305) FJO: Fjoh_0269 CGB:
cg2531(idi) FPS: FP1792(idi) CGT: cgR_2177 CTE: CT0257 CEF: CE2207
CPC: Cpar_1777 CDI: DIP1730(idi) CCH: Cag_1445 NFA: nfa19790
nfa22100 CPH: Cpha266_0385 RHA: RHA1_ro00239 CPB: Cphamn1_0417 SCO:
SCO6750(SC5F2A.33c) PVI: Cvib_1545 SMA: SAV1663(idi) PLT: Plut_1764
SGR: SGR_977 PPH: Ppha_0414 LXX: Lxx23810(idi) CTS: Ctha_0403 CMI:
CMM_2889(idiA) PAA: Paes_0384 AAU: AAur_0321(idi) RRS: RoseRS_2437
KRH: KRH_03040(idi) RCA: Rcas_2215 PAC: PPA2115 CAU: Caur_3877 FRA:
Francci3_4188 HAU: Haur_4687 FRE: Franean1_5570 DRA: DR_1087 FAL:
FRAAL6504(idi) DGE: Dgeo_1381 KRA: Krad_3991 TTH: TT_P0067 SEN:
SACE_2627(idiB_2) SACE_5210(idi) TTJ: TTHB110 STP: Strop_4438 MJA:
MJ0862 SAQ: Sare_4564 Sare_4928 MMP: MMP0043 RXY: Rxyl_0400 MMQ:
MmarC5_1637 BBU: BB0684 MMX: MmarC6_0906 MMZ: MmarC7_1040 PCL:
Pcal_0017 MAE: Maeo_1184 PAS: Pars_0051 MVN: Mevan_1058 CMA:
Cmaq_0231 Cmaq_1145 MAC: MA0604(idi) TNE: Tneu_0048 MBA: Mbar_A1419
TPE: Tpen_0272 MMA: MM_1764 NMR: Nmar_0313 MBU: Mbur_2397 KCR:
Kcr_1016 MTP: Mthe_0474 MHU: Mhun_2888 MLA: Mlab_0683 Mlab_1665
MEM: Memar_1814 MBN: Mboo_2211 MTH: MTH48 MST: Msp_0856(fni) MSI:
Msm_1441 MKA: MK0776(lldD) AFU: AF2287 HAL: VNG1818G(idi)
VNG6081G(crt_1) VNG6445G(crt_2) VNG7060 VNG7149 HSL: OE3560F(idiA)
OE6213R(idiB2) OE7093R(idiB1) HMA: rrnAC3484(idi) HWA:
HQ2772A(idiA) HQ2847A(idiB) NPH: NP0360A(idiB_1) NP4826A(idiA)
NP5124A(idiB_2) TAC: Ta0102 TVO: TVN0179 PTO: PTO0496 PHO: PH1202
PAB: PAB1662 PFU: PF0856 TKO: TK1470 RCI: LRC397(fni) APE:
APE_1765.1(fni) SMR: Smar_0822 IHO: Igni_0804 HBU: Hbut_0539 SSO:
SSO0063 STO: ST2059 SAI: Saci_0091 MSE: Msed_2136 PAI: PAE0801 PIS:
Pisl_1093
Exemplary Isoprene Synthase Nucleic Acids and Polypeptides
Genbank Accession Nos.
AY341431
AY316691
AY279379
AJ457070
[0971] AY182241
Sequence CWU 1
1
20511692DNAArtificial SequenceSynthetic Construct 1atgtgtgcga
cctcttctca atttactcag attaccgagc ataattcccg tcgttccgca 60aactatcagc
caaacctgtg gaatttcgaa ttcctgcaat ccctggagaa cgacctgaaa
120gtggaaaagc tggaggagaa agcgaccaaa ctggaggaag aagttcgctg
catgatcaac 180cgtgtagaca cccagccgct gtccctgctg gagctgatcg
acgatgtgca gcgcctgggt 240ctgacctaca aatttgaaaa agacatcatt
aaagccctgg aaaacatcgt actgctggac 300gaaaacaaaa agaacaaatc
tgacctgcac gcaaccgctc tgtctttccg tctgctgcgt 360cagcacggtt
tcgaggtttc tcaggatgtt tttgagcgtt tcaaggataa agaaggtggt
420ttcagcggtg aactgaaagg tgacgtccaa ggcctgctga gcctgtatga
agcgtcttac 480ctgggtttcg agggtgagaa cctgctggag gaggcgcgta
ccttttccat cacccacctg 540aagaacaacc tgaaagaagg cattaatacc
aaggttgcag aacaagtgag ccacgccctg 600gaactgccat atcaccagcg
tctgcaccgt ctggaggcac gttggttcct ggataaatac 660gaaccgaaag
aaccgcatca ccagctgctg ctggagctgg cgaagctgga ttttaacatg
720gtacagaccc tgcaccagaa agagctgcaa gatctgtccc gctggtggac
cgagatgggc 780ctggctagca aactggattt tgtacgcgac cgcctgatgg
aagtttattt ctgggcactg 840ggtatggcgc cagacccgca gtttggtgaa
tgtcgcaaag ctgttactaa aatgtttggt 900ctggtgacga tcatcgatga
cgtgtatgac gtttatggca ctctggacga actgcaactg 960ttcaccgatg
ctgtagagcg ctgggacgtt aacgctatta acaccctgcc ggactatatg
1020aaactgtgtt tcctggcact gtacaacacc gttaacgaca cgtcctattc
tattctgaaa 1080gagaaaggtc ataacaacct gtcctatctg acgaaaagct
ggcgtgaact gtgcaaagcc 1140tttctgcaag aggcgaaatg gtccaacaac
aaaattatcc cggctttctc caagtacctg 1200gaaaacgcca gcgtttcctc
ctccggtgta gcgctgctgg cgccgtctta cttttccgta 1260tgccagcagc
aggaagacat ctccgaccac gcgctgcgtt ccctgaccga cttccatggt
1320ctggtgcgtt ctagctgcgt tatcttccgc ctgtgcaacg atctggccac
ctctgcggcg 1380gagctggaac gtggcgagac taccaattct atcattagct
acatgcacga aaacgatggt 1440accagcgagg aacaggcccg cgaagaactg
cgtaaactga tcgacgccga atggaaaaag 1500atgaatcgtg aacgcgttag
cgactccacc ctgctgccta aagcgttcat ggaaatcgca 1560gttaacatgg
cacgtgtttc ccactgcacc taccagtatg gcgatggtct gggtcgccca
1620gactacgcga ctgaaaaccg catcaaactg ctgctgattg accctttccc
gattaaccag 1680ctgatgtatg tc 169226080DNAArtificial
SequenceSynthetic Construct 2gtttgacagc ttatcatcga ctgcacggtg
caccaatgct tctggcgtca ggcagccatc 60ggaagctgtg gtatggctgt gcaggtcgta
aatcactgca taattcgtgt cgctcaaggc 120gcactcccgt tctggataat
gttttttgcg ccgacatcat aacggttctg gcaaatattc 180tgaaatgagc
tgttgacaat taatcatccg gctcgtataa tgtgtggaat tgtgagcgga
240taacaatttc acacaggaaa cagcgccgct gagaaaaagc gaagcggcac
tgctctttaa 300caatttatca gacaatctgt gtgggcactc gaccggaatt
atcgattaac tttattatta 360aaaattaaag aggtatatat taatgtatcg
attaaataag gaggaataaa ccatgtgtgc 420gacctcttct caatttactc
agattaccga gcataattcc cgtcgttccg caaactatca 480gccaaacctg
tggaatttcg aattcctgca atccctggag aacgacctga aagtggaaaa
540gctggaggag aaagcgacca aactggagga agaagttcgc tgcatgatca
accgtgtaga 600cacccagccg ctgtccctgc tggagctgat cgacgatgtg
cagcgcctgg gtctgaccta 660caaatttgaa aaagacatca ttaaagccct
ggaaaacatc gtactgctgg acgaaaacaa 720aaagaacaaa tctgacctgc
acgcaaccgc tctgtctttc cgtctgctgc gtcagcacgg 780tttcgaggtt
tctcaggatg tttttgagcg tttcaaggat aaagaaggtg gtttcagcgg
840tgaactgaaa ggtgacgtcc aaggcctgct gagcctgtat gaagcgtctt
acctgggttt 900cgagggtgag aacctgctgg aggaggcgcg taccttttcc
atcacccacc tgaagaacaa 960cctgaaagaa ggcattaata ccaaggttgc
agaacaagtg agccacgccc tggaactgcc 1020atatcaccag cgtctgcacc
gtctggaggc acgttggttc ctggataaat acgaaccgaa 1080agaaccgcat
caccagctgc tgctggagct ggcgaagctg gattttaaca tggtacagac
1140cctgcaccag aaagagctgc aagatctgtc ccgctggtgg accgagatgg
gcctggctag 1200caaactggat tttgtacgcg accgcctgat ggaagtttat
ttctgggcac tgggtatggc 1260gccagacccg cagtttggtg aatgtcgcaa
agctgttact aaaatgtttg gtctggtgac 1320gatcatcgat gacgtgtatg
acgtttatgg cactctggac gaactgcaac tgttcaccga 1380tgctgtagag
cgctgggacg ttaacgctat taacaccctg ccggactata tgaaactgtg
1440tttcctggca ctgtacaaca ccgttaacga cacgtcctat tctattctga
aagagaaagg 1500tcataacaac ctgtcctatc tgacgaaaag ctggcgtgaa
ctgtgcaaag cctttctgca 1560agaggcgaaa tggtccaaca acaaaattat
cccggctttc tccaagtacc tggaaaacgc 1620cagcgtttcc tcctccggtg
tagcgctgct ggcgccgtct tacttttccg tatgccagca 1680gcaggaagac
atctccgacc acgcgctgcg ttccctgacc gacttccatg gtctggtgcg
1740ttctagctgc gttatcttcc gcctgtgcaa cgatctggcc acctctgcgg
cggagctgga 1800acgtggcgag actaccaatt ctatcattag ctacatgcac
gaaaacgatg gtaccagcga 1860ggaacaggcc cgcgaagaac tgcgtaaact
gatcgacgcc gaatggaaaa agatgaatcg 1920tgaacgcgtt agcgactcca
ccctgctgcc taaagcgttc atggaaatcg cagttaacat 1980ggcacgtgtt
tcccactgca cctaccagta tggcgatggt ctgggtcgcc cagactacgc
2040gactgaaaac cgcatcaaac tgctgctgat tgaccctttc ccgattaacc
agctgatgta 2100tgtctaactg cagctggtac catatgggaa ttcgaagctt
tctagaacaa aaactcatct 2160cagaagagga tctgaatagc gccgtcgacc
atcatcatca tcatcattga gtttaaacgg 2220tctccagctt ggctgttttg
gcggatgaga gaagattttc agcctgatac agattaaatc 2280agaacgcaga
agcggtctga taaaacagaa tttgcctggc ggcagtagcg cggtggtccc
2340acctgacccc atgccgaact cagaagtgaa acgccgtagc gccgatggta
gtgtggggtc 2400tccccatgcg agagtaggga actgccaggc atcaaataaa
acgaaaggct cagtcgaaag 2460actgggcctt tcgttttatc tgttgtttgt
cggtgaacgc tctcctgagt aggacaaatc 2520cgccgggagc ggatttgaac
gttgcgaagc aacggcccgg agggtggcgg gcaggacgcc 2580cgccataaac
tgccaggcat caaattaagc agaaggccat cctgacggat ggcctttttg
2640cgtttctaca aactcttttt gtttattttt ctaaatacat tcaaatatgt
atccgctcat 2700gagacaataa ccctgataaa tgcttcaata atattgaaaa
aggaagagta tgagtattca 2760acatttccgt gtcgccctta ttcccttttt
tgcggcattt tgccttcctg tttttgctca 2820cccagaaacg ctggtgaaag
taaaagatgc tgaagatcag ttgggtgcac gagtgggtta 2880catcgaactg
gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt
2940tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
gtgttgacgc 3000cgggcaagag caactcggtc gccgcataca ctattctcag
aatgacttgg ttgagtactc 3060accagtcaca gaaaagcatc ttacggatgg
catgacagta agagaattat gcagtgctgc 3120cataaccatg agtgataaca
ctgcggccaa cttacttctg acaacgatcg gaggaccgaa 3180ggagctaacc
gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga
3240accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
ctgtagcaat 3300ggcaacaacg ttgcgcaaac tattaactgg cgaactactt
actctagctt cccggcaaca 3360attaatagac tggatggagg cggataaagt
tgcaggacca cttctgcgct cggcccttcc 3420ggctggctgg tttattgctg
ataaatctgg agccggtgag cgtgggtctc gcggtatcat 3480tgcagcactg
gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag
3540tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
cactgattaa 3600gcattggtaa ctgtcagacc aagtttactc atatatactt
tagattgatt taaaacttca 3660tttttaattt aaaaggatct aggtgaagat
cctttttgat aatctcatga ccaaaatccc 3720ttaacgtgag ttttcgttcc
actgagcgtc agaccccgta gaaaagatca aaggatcttc 3780ttgagatcct
ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
3840agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
taactggctt 3900cagcagagcg cagataccaa atactgtcct tctagtgtag
ccgtagttag gccaccactt 3960caagaactct gtagcaccgc ctacatacct
cgctctgcta atcctgttac cagtggctgc 4020tgccagtggc gataagtcgt
gtcttaccgg gttggactca agacgatagt taccggataa 4080ggcgcagcgg
tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
4140ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
ttcccgaagg 4200gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
acaggagagc gcacgaggga 4260gcttccaggg ggaaacgcct ggtatcttta
tagtcctgtc gggtttcgcc acctctgact 4320tgagcgtcga tttttgtgat
gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa 4380cgcggccttt
ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
4440gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg
ataccgctcg 4500ccgcagccga acgaccgagc gcagcgagtc agtgagcgag
gaagcggaag agcgcctgat 4560gcggtatttt ctccttacgc atctgtgcgg
tatttcacac cgcatatggt gcactctcag 4620tacaatctgc tctgatgccg
catagttaag ccagtataca ctccgctatc gctacgtgac 4680tgggtcatgg
ctgcgccccg acacccgcca acacccgctg acgcgccctg acgggcttgt
4740ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg
catgtgtcag 4800aggttttcac cgtcatcacc gaaacgcgcg aggcagcaga
tcaattcgcg cgcgaaggcg 4860aagcggcatg catttacgtt gacaccatcg
aatggtgcaa aacctttcgc ggtatggcat 4920gatagcgccc ggaagagagt
caattcaggg tggtgaatgt gaaaccagta acgttatacg 4980atgtcgcaga
gtatgccggt gtctcttatc agaccgtttc ccgcgtggtg aaccaggcca
5040gccacgtttc tgcgaaaacg cgggaaaaag tggaagcggc gatggcggag
ctgaattaca 5100ttcccaaccg cgtggcacaa caactggcgg gcaaacagtc
gttgctgatt ggcgttgcca 5160cctccagtct ggccctgcac gcgccgtcgc
aaattgtcgc ggcgattaaa tctcgcgccg 5220atcaactggg tgccagcgtg
gtggtgtcga tggtagaacg aagcggcgtc gaagcctgta 5280aagcggcggt
gcacaatctt ctcgcgcaac gcgtcagtgg gctgatcatt aactatccgc
5340tggatgacca ggatgccatt gctgtggaag ctgcctgcac taatgttccg
gcgttatttc 5400ttgatgtctc tgaccagaca cccatcaaca gtattatttt
ctcccatgaa gacggtacgc 5460gactgggcgt ggagcatctg gtcgcattgg
gtcaccagca aatcgcgctg ttagcgggcc 5520cattaagttc tgtctcggcg
cgtctgcgtc tggctggctg gcataaatat ctcactcgca 5580atcaaattca
gccgatagcg gaacgggaag gcgactggag tgccatgtcc ggttttcaac
5640aaaccatgca aatgctgaat gagggcatcg ttcccactgc gatgctggtt
gccaacgatc 5700agatggcgct gggcgcaatg cgcgccatta ccgagtccgg
gctgcgcgtt ggtgcggata 5760tctcggtagt gggatacgac gataccgaag
acagctcatg ttatatcccg ccgtcaacca 5820ccatcaaaca ggattttcgc
ctgctggggc aaaccagcgt ggaccgcttg ctgcaactct 5880ctcagggcca
ggcggtgaag ggcaatcagc tgttgcccgt ctcactggtg aaaagaaaaa
5940ccaccctggc gcccaatacg caaaccgcct ctccccgcgc gttggccgat
tcattaatgc 6000agctggcacg acaggtttcc cgactggaaa gcgggcagtg
agcgcaacgc aattaatgtg 6060agttagcgcg aattgatctg
6080337DNAArtificial SequenceSynthetic Construct 3cgtgagatca
tatgtgtgcg acctcttctc aatttac 37438DNAArtificial SequenceSynthetic
Construct 4cggtcgacgg atccctgcag ttagacatac atcagctg
3857404DNAArtificial SequenceSynthetic Construct 5ttctcatgtt
tgacagctta tcatcgataa gctttaatgc ggtagtttat cacagttaaa 60ttgctaacgc
agtcaggcac cgtgtatgaa atctaacaat gcgctcatcg tcatcctcgg
120caccgtcacc ctggatgctg taggcatagg cttggttatg ccggtactgc
cgggcctctt 180gcgggatatc cggatatagt tcctcctttc agcaaaaaac
ccctcaagac ccgtttagag 240gccccaaggg gttatgctag ttattgctca
gcggtggcag cagccaactc agcttccttt 300cgggctttgt tagcagccgg
atccctgcag ttagacatac atcagctggt taatcgggaa 360agggtcaatc
agcagcagtt tgatgcggtt ttcagtcgcg tagtctgggc gacccagacc
420atcgccatac tggtaggtgc agtgggaaac acgtgccatg ttaactgcga
tttccatgaa 480cgctttaggc agcagggtgg agtcgctaac gcgttcacga
ttcatctttt tccattcggc 540gtcgatcagt ttacgcagtt cttcgcgggc
ctgttcctcg ctggtaccat cgttttcgtg 600catgtagcta atgatagaat
tggtagtctc gccacgttcc agctccgccg cagaggtggc 660cagatcgttg
cacaggcgga agataacgca gctagaacgc accagaccat ggaagtcggt
720cagggaacgc agcgcgtggt cggagatgtc ttcctgctgc tggcatacgg
aaaagtaaga 780cggcgccagc agcgctacac cggaggagga aacgctggcg
ttttccaggt acttggagaa 840agccgggata attttgttgt tggaccattt
cgcctcttgc agaaaggctt tgcacagttc 900acgccagctt ttcgtcagat
aggacaggtt gttatgacct ttctctttca gaatagaata 960ggacgtgtcg
ttaacggtgt tgtacagtgc caggaaacac agtttcatat agtccggcag
1020ggtgttaata gcgttaacgt cccagcgctc tacagcatcg gtgaacagtt
gcagttcgtc 1080cagagtgcca taaacgtcat acacgtcatc gatgatcgtc
accagaccaa acattttagt 1140aacagctttg cgacattcac caaactgcgg
gtctggcgcc atacccagtg cccagaaata 1200aacttccatc aggcggtcgc
gtacaaaatc cagtttgcta gccaggccca tctcggtcca 1260ccagcgggac
agatcttgca gctctttctg gtgcagggtc tgtaccatgt taaaatccag
1320cttcgccagc tccagcagca gctggtgatg cggttctttc ggttcgtatt
tatccaggaa 1380ccaacgtgcc tccagacggt gcagacgctg gtgatatggc
agttccaggg cgtggctcac 1440ttgttctgca accttggtat taatgccttc
tttcaggttg ttcttcaggt gggtgatgga 1500aaaggtacgc gcctcctcca
gcaggttctc accctcgaaa cccaggtaag acgcttcata 1560caggctcagc
aggccttgga cgtcaccttt cagttcaccg ctgaaaccac cttctttatc
1620cttgaaacgc tcaaaaacat cctgagaaac ctcgaaaccg tgctgacgca
gcagacggaa 1680agacagagcg gttgcgtgca ggtcagattt gttctttttg
ttttcgtcca gcagtacgat 1740gttttccagg gctttaatga tgtctttttc
aaatttgtag gtcagaccca ggcgctgcac 1800atcgtcgatc agctccagca
gggacagcgg ctgggtgtct acacggttga tcatgcagcg 1860aacttcttcc
tccagtttgg tcgctttctc ctccagcttt tccactttca ggtcgttctc
1920cagggattgc aggaattcga aattccacag gtttggctga tagtttgcgg
aacgacggga 1980attatgctcg gtaatctgag taaattgaga agaggtcgca
cacatatgac gaccttcgat 2040atggccgctg ctgtgatgat gatgatgatg
atgatgatga tggcccatgg tatatctcct 2100tcttaaagtt aaacaaaatt
atttctagag gggaattgtt atccgctcac aattccccta 2160tagtgagtcg
tattaatttc gcgggatcga gatctcgatc ctctacgccg gacgcatcgt
2220ggccggcatc accggcgcca caggtgcggt tgctggcgcc tatatcgccg
acatcaccga 2280tggggaagat cgggctcgcc acttcgggct catgagcgct
tgtttcggcg tgggtatggt 2340ggcaggcccc gtggccgggg gactgttggg
cgccatctcc ttgcatgcac cattccttgc 2400ggcggcggtg ctcaacggcc
tcaacctact actgggctgc ttcctaatgc aggagtcgca 2460taagggagag
cgtcgagatc ccggacacca tcgaatggcg caaaaccttt cgcggtatgg
2520catgatagcg cccggaagag agtcaattca gggtggtgaa tgtgaaacca
gtaacgttat 2580acgatgtcgc agagtatgcc ggtgtctctt atcagaccgt
ttcccgcgtg gtgaaccagg 2640ccagccacgt ttctgcgaaa acgcgggaaa
aagtggaagc ggcgatggcg gagctgaatt 2700acattcccaa ccgcgtggca
caacaactgg cgggcaaaca gtcgttgctg attggcgttg 2760ccacctccag
tctggccctg cacgcgccgt cgcaaattgt cgcggcgatt aaatctcgcg
2820ccgatcaact gggtgccagc gtggtggtgt cgatggtaga acgaagcggc
gtcgaagcct 2880gtaaagcggc ggtgcacaat cttctcgcgc aacgcgtcag
tgggctgatc attaactatc 2940cgctggatga ccaggatgcc attgctgtgg
aagctgcctg cactaatgtt ccggcgttat 3000ttcttgatgt ctctgaccag
acacccatca acagtattat tttctcccat gaagacggta 3060cgcgactggg
cgtggagcat ctggtcgcat tgggtcacca gcaaatcgcg ctgttagcgg
3120gcccattaag ttctgtctcg gcgcgtctgc gtctggctgg ctggcataaa
tatctcactc 3180gcaatcaaat tcagccgata gcggaacggg aaggcgactg
gagtgccatg tccggttttc 3240aacaaaccat gcaaatgctg aatgagggca
tcgttcccac tgcgatgctg gttgccaacg 3300atcagatggc gctgggcgca
atgcgcgcca ttaccgagtc cgggctgcgc gttggtgcgg 3360atatctcggt
agtgggatac gacgataccg aagacagctc atgttatatc ccgccgttaa
3420ccaccatcaa acaggatttt cgcctgctgg ggcaaaccag cgtggaccgc
ttgctgcaac 3480tctctcaggg ccaggcggtg aagggcaatc agctgttgcc
cgtctcactg gtgaaaagaa 3540aaaccaccct ggcgcccaat acgcaaaccg
cctctccccg cgcgttggcc gattcattaa 3600tgcagctggc acgacaggtt
tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat 3660gtaagttagc
tcactcatta ggcaccggga tctcgaccga tgcccttgag agccttcaac
3720ccagtcagct ccttccggtg ggcgcggggc atgactatcg tcgccgcact
tatgactgtc 3780ttctttatca tgcaactcgt aggacaggtg ccggcagcgc
tctgggtcat tttcggcgag 3840gaccgctttc gctggagcgc gacgatgatc
ggcctgtcgc ttgcggtatt cggaatcttg 3900cacgccctcg ctcaagcctt
cgtcactggt cccgccacca aacgtttcgg cgagaagcag 3960gccattatcg
ccggcatggc ggccgacgcg ctgggctacg tcttgctggc gttcgcgacg
4020cgaggctgga tggccttccc cattatgatt cttctcgctt ccggcggcat
cgggatgccc 4080gcgttgcagg ccatgctgtc caggcaggta gatgacgacc
atcagggaca gcttcaagga 4140tcgctcgcgg ctcttaccag cctaacttcg
atcactggac cgctgatcgt cacggcgatt 4200tatgccgcct cggcgagcac
atggaacggg ttggcatgga ttgtaggcgc cgccctatac 4260cttgtctgcc
tccccgcgtt gcgtcgcggt gcatggagcc gggccacctc gacctgaatg
4320gaagccggcg gcacctcgct aacggattca ccactccaag aattggagcc
aatcaattct 4380tgcggagaac tgtgaatgcg caaaccaacc cttggcagaa
catatccatc gcgtccgcca 4440tctccagcag ccgcacgcgg cgcatctcgg
gcagcgttgg gtcctggcca cgggtgcgca 4500tgatcgtgct cctgtcgttg
aggacccggc taggctggcg gggttgcctt actggttagc 4560agaatgaatc
accgatacgc gagcgaacgt gaagcgactg ctgctgcaaa acgtctgcga
4620cctgagcaac aacatgaatg gtcttcggtt tccgtgtttc gtaaagtctg
gaaacgcgga 4680agtcagcgcc ctgcaccatt atgttccgga tctgcatcgc
aggatgctgc tggctaccct 4740gtggaacacc tacatctgta ttaacgaagc
gctggcattg accctgagtg atttttctct 4800ggtcccgccg catccatacc
gccagttgtt taccctcaca acgttccagt aaccgggcat 4860gttcatcatc
agtaacccgt atcgtgagca tcctctctcg tttcatcggt atcattaccc
4920ccatgaacag aaatccccct tacacggagg catcagtgac caaacaggaa
aaaaccgccc 4980ttaacatggc ccgctttatc agaagccaga cattaacgct
tctggagaaa ctcaacgagc 5040tggacgcgga tgaacaggca gacatctgtg
aatcgcttca cgaccacgct gatgagcttt 5100accgcagctg cctcgcgcgt
ttcggtgatg acggtgaaaa cctctgacac atgcagctcc 5160cggagacggt
cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg
5220cgtcagcggg tgttggcggg tgtcggggcg cagccatgac ccagtcacgt
agcgatagcg 5280gagtgtatac tggcttaact atgcggcatc agagcagatt
gtactgagag tgcaccatat 5340atgcggtgtg aaataccgca cagatgcgta
aggagaaaat accgcatcag gcgctcttcc 5400gcttcctcgc tcactgactc
gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct 5460cactcaaagg
cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
5520tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
ggcgtttttc 5580cataggctcc gcccccctga cgagcatcac aaaaatcgac
gctcaagtca gaggtggcga 5640aacccgacag gactataaag ataccaggcg
tttccccctg gaagctccct cgtgcgctct 5700cctgttccga ccctgccgct
taccggatac ctgtccgcct ttctcccttc gggaagcgtg 5760gcgctttctc
atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
5820ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
cggtaactat 5880cgtcttgagt ccaacccggt aagacacgac ttatcgccac
tggcagcagc cactggtaac 5940aggattagca gagcgaggta tgtaggcggt
gctacagagt tcttgaagtg gtggcctaac 6000tacggctaca ctagaaggac
agtatttggt atctgcgctc tgctgaagcc agttaccttc 6060ggaaaaagag
ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
6120tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
tcctttgatc 6180ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
gttaagggat tttggtcatg 6240agattatcaa aaaggatctt cacctagatc
cttttaaatt aaaaatgaag ttttaaatca 6300atctaaagta tatatgagta
aacttggtct gacagttacc aatgcttaat cagtgaggca 6360cctatctcag
cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
6420ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
accgcgagac 6480ccacgctcac cggctccaga tttatcagca ataaaccagc
cagccggaag ggccgagcgc 6540agaagtggtc ctgcaacttt atccgcctcc
atccagtcta ttaattgttg ccgggaagct 6600agagtaagta gttcgccagt
taatagtttg cgcaacgttg ttgccattgc tgcaggcatc 6660gtggtgtcac
gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
6720cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
tcctccgatc 6780gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
ttatggcagc actgcataat 6840tctcttactg tcatgccatc cgtaagatgc
ttttctgtga
ctggtgagta ctcaaccaag 6900tcattctgag aatagtgtat gcggcgaccg
agttgctctt gcccggcgtc aacacgggat 6960aataccgcgc cacatagcag
aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg 7020cgaaaactct
caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
7080cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
aaaaacagga 7140aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
aatgttgaat actcatactc 7200ttcctttttc aatattattg aagcatttat
cagggttatt gtctcatgag cggatacata 7260tttgaatgta tttagaaaaa
taaacaaata ggggttccgc gcacatttcc ccgaaaagtg 7320ccacctgacg
tctaagaaac cattattatc atgacattaa cctataaaaa taggcgtatc
7380acgaggccct ttcgtcttca agaa 7404641DNAArtificial
SequenceSynthetic Construct 6catatgaaag cttgtatcga ttaaataagg
aggaataaac c 4176266DNAArtificial SequenceSynthetic Construct
7cccgtcttac tgtcgggaat tcgcgttggc cgattcatta atgcagctgg cacgacaggt
60ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtgagttag ctcactcatt
120aggcacccca ggctttacac tttatgcttc cggctcgtat gttgtgtgga
attgtgagcg 180gataacaatt tcacacagga aacagctatg accatgatta
cgccaagctt gtatcgatta 240aataaggagg aataaaccat gtgtgcgacc
tcttctcaat ttactcagat taccgagcat 300aattcccgtc gttccgcaaa
ctatcagcca aacctgtgga atttcgaatt cctgcaatcc 360ctggagaacg
acctgaaagt ggaaaagctg gaggagaaag cgaccaaact ggaggaagaa
420gttcgctgca tgatcaaccg tgtagacacc cagccgctgt ccctgctgga
gctgatcgac 480gatgtgcagc gcctgggtct gacctacaaa tttgaaaaag
acatcattaa agccctggaa 540aacatcgtac tgctggacga aaacaaaaag
aacaaatctg acctgcacgc aaccgctctg 600tctttccgtc tgctgcgtca
gcacggtttc gaggtttctc aggatgtttt tgagcgtttc 660aaggataaag
aaggtggttt cagcggtgaa ctgaaaggtg acgtccaagg cctgctgagc
720ctgtatgaag cgtcttacct gggtttcgag ggtgagaacc tgctggagga
ggcgcgtacc 780ttttccatca cccacctgaa gaacaacctg aaagaaggca
ttaataccaa ggttgcagaa 840caagtgagcc acgccctgga actgccatat
caccagcgtc tgcaccgtct ggaggcacgt 900tggttcctgg ataaatacga
accgaaagaa ccgcatcacc agctgctgct ggagctggcg 960aagctggatt
ttaacatggt acagaccctg caccagaaag agctgcaaga tctgtcccgc
1020tggtggaccg agatgggcct ggctagcaaa ctggattttg tacgcgaccg
cctgatggaa 1080gtttatttct gggcactggg tatggcgcca gacccgcagt
ttggtgaatg tcgcaaagct 1140gttactaaaa tgtttggtct ggtgacgatc
atcgatgacg tgtatgacgt ttatggcact 1200ctggacgaac tgcaactgtt
caccgatgct gtagagcgct gggacgttaa cgctattaac 1260accctgccgg
actatatgaa actgtgtttc ctggcactgt acaacaccgt taacgacacg
1320tcctattcta ttctgaaaga gaaaggtcat aacaacctgt cctatctgac
gaaaagctgg 1380cgtgaactgt gcaaagcctt tctgcaagag gcgaaatggt
ccaacaacaa aattatcccg 1440gctttctcca agtacctgga aaacgccagc
gtttcctcct ccggtgtagc gctgctggcg 1500ccgtcttact tttccgtatg
ccagcagcag gaagacatct ccgaccacgc gctgcgttcc 1560ctgaccgact
tccatggtct ggtgcgttct agctgcgtta tcttccgcct gtgcaacgat
1620ctggccacct ctgcggcgga gctggaacgt ggcgagacta ccaattctat
cattagctac 1680atgcacgaaa acgatggtac cagcgaggaa caggcccgcg
aagaactgcg taaactgatc 1740gacgccgaat ggaaaaagat gaatcgtgaa
cgcgttagcg actccaccct gctgcctaaa 1800gcgttcatgg aaatcgcagt
taacatggca cgtgtttccc actgcaccta ccagtatggc 1860gatggtctgg
gtcgcccaga ctacgcgact gaaaaccgca tcaaactgct gctgattgac
1920cctttcccga ttaaccagct gatgtatgtc taactgcagg tcgactctag
aggatccccg 1980ggtaccgagc tcgaattcac tggccgtcgt tttacaacgt
cgtgactggg aaaaccctgg 2040cgttacccaa cttaatcgcc ttgcagcaca
tccccctttc gccagctggc gtaatagcga 2100agaggcccgc accgatcgcc
cttcccaaca gttgcgcagc ctgaatggcg aatggcgcct 2160gatgcggtat
tttctcctta cgcatctgtg cggtatttca caccgcatat ggtgcactct
2220cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc
caacacccgc 2280tgacgagctt agtaaagccc tcgctagatt ttaatgcgga
tgttgcgatt acttcgccaa 2340ctattgcgat aacaagaaaa agccagcctt
tcatgatata tctcccaatt tgtgtagggc 2400ttattatgca cgcttaaaaa
taataaaagc agacttgacc tgatagtttg gctgtgagca 2460attatgtgct
tagtgcatct aacgcttgag ttaagccgcg ccgcgaagcg gcgtcggctt
2520gaacgaattg ttagacatta tttgccgact accttggtga tctcgccttt
cacgtagtgg 2580acaaattctt ccaactgatc tgcgcgcgag gccaagcgat
cttcttcttg tccaagataa 2640gcctgtctag cttcaagtat gacgggctga
tactgggccg gcaggcgctc cattgcccag 2700tcggcagcga catccttcgg
cgcgattttg ccggttactg cgctgtacca aatgcgggac 2760aacgtaagca
ctacatttcg ctcatcgcca gcccagtcgg gcggcgagtt ccatagcgtt
2820aaggtttcat ttagcgcctc aaatagatcc tgttcaggaa ccggatcaaa
gagttcctcc 2880gccgctggac ctaccaaggc aacgctatgt tctcttgctt
ttgtcagcaa gatagccaga 2940tcaatgtcga tcgtggctgg ctcgaagata
cctgcaagaa tgtcattgcg ctgccattct 3000ccaaattgca gttcgcgctt
agctggataa cgccacggaa tgatgtcgtc gtgcacaaca 3060atggtgactt
ctacagcgcg gagaatctcg ctctctccag gggaagccga agtttccaaa
3120aggtcgttga tcaaagctcg ccgcgttgtt tcatcaagcc ttacggtcac
cgtaaccagc 3180aaatcaatat cactgtgtgg cttcaggccg ccatccactg
cggagccgta caaatgtacg 3240gccagcaacg tcggttcgag atggcgctcg
atgacgccaa ctacctctga tagttgagtc 3300gatacttcgg cgatcaccgc
ttccctcatg atgtttaact ttgttttagg gcgactgccc 3360tgctgcgtaa
catcgttgct gctccataac atcaaacatc gacccacggc gtaacgcgct
3420tgctgcttgg atgcccgagg catagactgt accccaaaaa aacagtcata
acaagccatg 3480aaaaccgcca ctgcgccgtt accaccgctg cgttcggtca
aggttctgga ccagttgcgt 3540gagcgcatac gctacttgca ttacagctta
cgaaccgaac aggcttatgt ccactgggtt 3600cgtgccttca tccgtttcca
cggtgtgcgt cacccggcaa ccttgggcag cagcgaagtc 3660gaggcatttc
tgtcctggct ggcgaacgag cgcaaggttt cggtctccac gcatcgtcag
3720gcattggcgg ccttgctgtt cttctacggc aaggtgctgt gcacggatct
gccctggctt 3780caggagatcg gaagacctcg gccgtcgcgg cgcttgccgg
tggtgctgac cccggatgaa 3840gtggttcgca tcctcggttt tctggaaggc
gagcatcgtt tgttcgccca gcttctgtat 3900ggaacgggca tgcggatcag
tgagggtttg caactgcggg tcaaggatct ggatttcgat 3960cacggcacga
tcatcgtgcg ggagggcaag ggctccaagg atcgggcctt gatgttaccc
4020gagagcttgg cacccagcct gcgcgagcag gggaattaat tcccacgggt
tttgctgccc 4080gcaaacgggc tgttctggtg ttgctagttt gttatcagaa
tcgcagatcc ggcttcagcc 4140ggtttgccgg ctgaaagcgc tatttcttcc
agaattgcca tgattttttc cccacgggag 4200gcgtcactgg ctcccgtgtt
gtcggcagct ttgattcgat aagcagcatc gcctgtttca 4260ggctgtctat
gtgtgactgt tgagctgtaa caagttgtct caggtgttca atttcatgtt
4320ctagttgctt tgttttactg gtttcacctg ttctattagg tgttacatgc
tgttcatctg 4380ttacattgtc gatctgttca tggtgaacag ctttgaatgc
accaaaaact cgtaaaagct 4440ctgatgtatc tatctttttt acaccgtttt
catctgtgca tatggacagt tttccctttg 4500atatgtaacg gtgaacagtt
gttctacttt tgtttgttag tcttgatgct tcactgatag 4560atacaagagc
cataagaacc tcagatcctt ccgtatttag ccagtatgtt ctctagtgtg
4620gttcgttgtt tttgcgtgag ccatgagaac gaaccattga gatcatactt
actttgcatg 4680tcactcaaaa attttgcctc aaaactggtg agctgaattt
ttgcagttaa agcatcgtgt 4740agtgtttttc ttagtccgtt atgtaggtag
gaatctgatg taatggttgt tggtattttg 4800tcaccattca tttttatctg
gttgttctca agttcggtta cgagatccat ttgtctatct 4860agttcaactt
ggaaaatcaa cgtatcagtc gggcggcctc gcttatcaac caccaatttc
4920atattgctgt aagtgtttaa atctttactt attggtttca aaacccattg
gttaagcctt 4980ttaaactcat ggtagttatt ttcaagcatt aacatgaact
taaattcatc aaggctaatc 5040tctatatttg ccttgtgagt tttcttttgt
gttagttctt ttaataacca ctcataaatc 5100ctcatagagt atttgttttc
aaaagactta acatgttcca gattatattt tatgaatttt 5160tttaactgga
aaagataagg caatatctct tcactaaaaa ctaattctaa tttttcgctt
5220gagaacttgg catagtttgt ccactggaaa atctcaaagc ctttaaccaa
aggattcctg 5280atttccacag ttctcgtcat cagctctctg gttgctttag
ctaatacacc ataagcattt 5340tccctactga tgttcatcat ctgagcgtat
tggttataag tgaacgatac cgtccgttct 5400ttccttgtag ggttttcaat
cgtggggttg agtagtgcca cacagcataa aattagcttg 5460gtttcatgct
ccgttaagtc atagcgacta atcgctagtt catttgcttt gaaaacaact
5520aattcagaca tacatctcaa ttggtctagg tgattttaat cactatacca
attgagatgg 5580gctagtcaat gataattact agtccttttc ctttgagttg
tgggtatctg taaattctgc 5640tagacctttg ctggaaaact tgtaaattct
gctagaccct ctgtaaattc cgctagacct 5700ttgtgtgttt tttttgttta
tattcaagtg gttataattt atagaataaa gaaagaataa 5760aaaaagataa
aaagaataga tcccagccct gtgtataact cactacttta gtcagttccg
5820cagtattaca aaaggatgtc gcaaacgctg tttgctcctc tacaaaacag
accttaaaac 5880cctaaaggct taagtagcac cctcgcaagc tcgggcaaat
cgctgaatat tccttttgtc 5940tccgaccatc aggcacctga gtcgctgtct
ttttcgtgac attcagttcg ctgcgctcac 6000ggctctggca gtgaatgggg
gtaaatggca ctacaggcgc cttttatgga ttcatgcaag 6060gaaactaccc
ataatacaag aaaagcccgt cacgggcttc tcagggcgtt ttatggcggg
6120tctgctatgt ggtgctatct gactttttgc tgttcagcag ttcctgccct
ctgattttcc 6180agtctgacca cttcggatta tcccgtgaca ggtcattcag
actggctaat gcacccagta 6240aggcagcggt atcatcaaca ggctta
626681695DNAArtificial SequenceSynthetic Construct 8atgtgtgcaa
cctcctccca gtttactcag attaccgagc ataattctcg acgatctgct 60aactaccagc
cgaacctttg gaactttgag tttctccagt ctctcgaaaa tgacctgaag
120gtggaaaagc tcgaggagaa ggcgaccaaa ctcgaggagg aggtgcgatg
tatgatcaac 180agagttgaca cccaacccct gtctttgctg gagctgatcg
acgatgtgca gcggttgggt 240ttgacttata aattcgagaa ggacattatc
aaggcactgg agaacattgt gctcctcgac 300gagaacaaga agaacaagtc
tgatcttcac gctaccgctc tctctttccg acttcttcga 360caacacggct
tcgaggtgtc gcaggacgtc ttcgagagat ttaaggacaa ggagggagga
420tttagcggcg agctgaaggg agacgttcag ggtcttctct ccttgtacga
ggcgtcctac 480ctgggattcg agggagagaa cctcctggag gaagctcgta
cattttccat cactcacctt 540aagaataacc ttaaggaggg aattaacacc
aaggtggccg agcaggtttc tcacgccctg 600gagctcccct accaccaacg
gctccataga ctggaggctc gttggttcct ggacaaatat 660gagccaaagg
agcctcatca tcagttgctg ttggagttgg ccaagctgga cttcaatatg
720gttcagacgc tgcaccaaaa ggagttgcag gacctgtctc gatggtggac
cgagatggga 780ttggcctcga agctggattt tgtccgtgac cgacttatgg
aggtctattt ttgggccctt 840ggaatggcgc ctgaccccca gttcggagag
tgccggaagg cggtgacgaa gatgttcggt 900cttgtgacta tcatcgacga
cgtctacgat gtctacggca cactcgacga gttgcagctg 960ttcactgacg
ccgtcgagcg atgggatgtg aacgccatta atactctccc tgactatatg
1020aagctgtgct tcctggctct gtacaacact gtcaacgata cctcgtactc
tatcctcaag 1080gagaagggac acaacaatct ctcctacttg accaaatcct
ggcgagaact gtgcaaggct 1140tttctgcagg aggctaaatg gtccaataac
aagatcattc ctgctttttc taaatacctg 1200gaaaatgcct cggtgtcgag
ctctggcgtc gcccttctgg ccccttccta cttctccgtc 1260tgccagcagc
aggaggatat ttccgatcat gctcttagat cgctgaccga ttttcacggc
1320ctcgtgcgat cttcctgcgt gatttttcgg ttgtgtaatg accttgcgac
ctctgctgct 1380gagctggaac gaggcgagac tacaaattcc attatttctt
acatgcacga aaacgatgga 1440acatctgaag aacaggctag agaggaactg
cgaaagttga tcgacgccga gtggaagaag 1500atgaacagag agcgggtgtc
cgactctacc ctgcttccca aggccttcat ggagatcgcc 1560gtgaacatgg
ctcgagtttc ccattgtact taccagtacg gtgacggcct gggtcgtccg
1620gactacgcta cagagaaccg aatcaagctg ctgctcatcg accccttccc
tatcaaccaa 1680ttgatgtacg tgtaa 1695940DNAArtificial
SequenceSynthetic Construct 9gcttatggat cctctagact attacacgta
catcaattgg 401030DNAArtificial SequenceSynthetic Construct
10caccatgtgt gcaacctcct cccagtttac 30118191DNAArtificial
SequenceSynthetic Construct 11tcgaccggtg agaagaacag catcgggaca
agggaaggaa gaacaaagac aaagaaaaca 60aaagaaagca attgaaaaca aaacaaaaca
attttcattc cttctcttat cattcctttt 120cttttctttt ctctcattca
acgcactcca tcgtatccgt attcctctta ttttttctct 180ttctctatat
ccatttcttt ctctctaggt gtgtcctctc tctctcttca atttctctac
240tccgcattcc aacgcatcct tcccccaacc tcccatttcc tccttacggc
ccgatagcga 300tcgtctttcc ctcgctatca ctcgctaccg gcccctcctc
tgcaccgtaa cctcctacgt 360atttaccata tcataaagtt ttttccgacg
cttatcgctg accccctgtc gccctcctat 420tggcttccgg attatcttct
tgtccataag gtgatccatg cttcctgaag attcccgaaa 480tgtgtccact
ttggcgggga atcattccat ccacttcttt ctctctcgct ttcctcattc
540ggcgctcccc ttccgcgtct cattggtctt ccgctccgtt tttgctttgc
cgatgttact 600tggggagagg tgcgataatc ctttcgcaaa aactcggttt
gacgcctccc atggtataaa 660tagtgggtgg tggacaggtg ccttcgcttt
tctttaagca agagaatccc attgtcttga 720ctatcacgaa ttcacataca
ttatgaagat caccgctgtc attgcccttt tattctcact 780tgctgctgcc
tcacctattc cagttgccga tcctggtgtg gtttcagtta gcaagtcata
840tgctgatttc cttcgtgttt accaaagttg gaacactttt gctaatcctg
atagacccaa 900ccttaagaag agaaatgata cacctgcaag tggatatcaa
gttgaaaaag tcgtaatttt 960gtcacgtcac ggtgttaggg cccctacaaa
aatgactcaa accatgcgtg atgtcactcc 1020taatacatgg ccagaatggc
ccgttaaatt aggatatatt acaccaagag gtgaacactt 1080gatatcactt
atgggcggtt tttaccgtca aaaattccag caacaaggaa tcctttctca
1140gggctcctgt cctactccta actccatata tgtctgggct gacgtcgatc
agcgtacttt 1200aaaaactggt gaagcattcc ttgctggttt ggcaccacaa
tgtggcttga caattcatca 1260ccaacaaaat cttgagaaag ctgatcctct
ttttcatccc gttaaagctg gaacctgctc 1320tatggataaa actcaagttc
aacaagctgt tgagaaggag gcacaaactc ctatagataa 1380tttgaatcaa
cattacatcc cctttttagc tttaatgaat acaacattaa attttagtac
1440ttctgcctgg tgccaaaaac actctgctga taaatcctgt gacctaggtt
tatccatgcc 1500ttctaaattg tccataaaag ataatggtaa caaggtcgca
ttggatggag ctattggtct 1560atcctctact ttggccgaga tttttcttct
tgaatatgct caaggcatgc ctcaagctgc 1620ttggggtaac atccactcag
agcaagagtg ggcttccttg ctaaagttgc ataatgttca 1680attcgatttg
atggcccgaa caccttatat tgctcgacat aacggtactc ctttattgca
1740agctatatca aatgccctta atcccaacgc cactgaatca aaacttccag
atatttcacc 1800tgataacaaa atattgttca ttgcaggtca tgacacaaat
attgctaata tagccggcat 1860gttaaatatg cgttggacat taccaggtca
accagataat actcctccag gtggtgccct 1920agtatttgaa cgtcttgctg
ataaaagtgg aaaacaatat gtttctgtat ctatggttta 1980tcaaacacta
gaacaacttc gatcacagac tcccctttct ctaaatcagc ctgccggatc
2040tgttcaactt aaaattccag gttgcaatga tcaaacagcc gagggttact
gtcctctttc 2100cacttttaca agagttgttt cccaatctgt tgaacctgga
tgccaacttc aataatgagg 2160atccaagtaa gggaatgaga atgtgatcca
cttttaattc ctaatgaata catgcctata 2220gttcttttct tttgttcttt
atgtcgtttt tcgatggtac ggccgttgtc aatctcagtt 2280tgtgtgcttg
gttgcagctt ggtttcaaat ctgttcatct catgaatctt ttaccatttc
2340accacacgtt tataccattc tctcatagaa tcttcatcaa accatctcgg
ggttagagtg 2400gaaagaaagt cttgttcttt tatttccttt tttccatctt
caaggctttt cttttcttcc 2460tcctcctcgt tcatcttgag gtttgacgtg
tctgtttaga attttgagct gttgcagcat 2520cttatttttt gttttgcgaa
aacgaagcgc tttactctct tcatcagttg gacgattgta 2580cctttgaaaa
ccaactactt ttgcatgttt tgtatagaaa tcaatgatat tagaatccca
2640tcctttaatt tctttcaaag tagttgagct atagttaagt gtaagggccc
tactgcgaaa 2700gcatttgcca aggatgtttt cattaatcaa gaacgaaagt
taggggatcg aagacgatca 2760gataccgtcg tagtcttaac cataaactat
gccgactagg gatcgggcaa tgtttcattt 2820atcgacttgc tcggcacctt
acgagaaatc aaagtctttg ggttccgggg ggagtatggt 2880cgcaaggctg
aaacttaaag gaattgacgg aagggcacca caatggagtg gagcctgcgg
2940cttaatttga ctcaacacgg ggaaactcac caggtccaga catagtaagg
attgacagat 3000tgagagctct ttcttgattc tatgggtggt ggtgcatggc
cgttcttagt tggtggagtg 3060atttgtctgc ttaattgcga taacgaacga
gaccttaacc tgctaaatag ctggatcagc 3120cattttggct gatcattagc
ttcttagagg gactattggc ataaagccaa tggaagtttg 3180aggcaataac
aggtctgtga tgcccttaga tgttctgggc cgcacgcgcg ctacactgac
3240ggagccaacg agttgaaaaa aatcttttga ttttttatcc ttggccggaa
ggtctgggta 3300atcttgttaa actccgtcgt gctggggata gagcattgca
attattgcgg ccgctcctca 3360attcgatgtt gcagatttta caagttttta
aaatgtattt cattattact ttttatatgc 3420ctaataaaaa agccatagtt
taatctatag ataacttttt ttccagtgca ctaacggacg 3480ttacattccc
atacaaaact gcgtagttaa agctaaggaa aagttaatat catgttaatt
3540aaatacgcta tttacaataa gacattgaac tcatttatat cgttgaatat
gaataaccaa 3600tttcagcgaa tttttaacaa acatcgttca cctcgtttaa
ggatatcttg tgtatggggt 3660gttgacttgc tttatcgaat aattaccgta
cctgtaattg gcttgctgga tatagcggta 3720gtctaatatc tagcaaaaat
cttttgggtg aaaaggcttg caatttcacg acaccgaact 3780atttgtcatt
ttttaataag gaagttttcc ataaattcct gtaattctcg gttgatctaa
3840ttgaaaagag tagttttgca tcacgatgag gagggctttt gtagaaagaa
atacgaacga 3900aacgaaaatc agcgttgcca tcgctttgga caaagctccc
ttacctgaag agtcgaattt 3960tattgatgaa cttataactt ccaagcatgc
aaaccaaaag ggagaacaag taatccaagt 4020agacacggga attggattct
tggatcacat gtatcatgca ctggctaaac atgcaggctg 4080gagcttacga
ctttactcaa gaggtgattt aatcatcgat gatcatcaca ctgcagaaga
4140tactgctatt gcacttggta ttgcattcaa gcaggctatg ggtaactttg
ccggcgttaa 4200aagatttgga catgcttatt gtccacttga cgaagctctt
tctagaagcg tagttgactt 4260gtcgggacgg ccctatgctg ttatcgattt
gggattaaag cgtgaaaagg ttggggaatt 4320gtcctgtgaa atgatccctc
acttactata ttccttttcg gtagcagctg gaattacttt 4380gcatgttacc
tgcttatatg gtagtaatga ccatcatcgt gctgaaagcg cttttaaatc
4440tctggctgtt gccatgcgcg cggctactag tcttactgga agttctgaag
tcccaagcac 4500gaagggagtg ttgtaaagat gaattggatt atgtcaggaa
aagaacgaca attttgcatc 4560caaattgtct aaattttaga gttgcttgaa
aacaatagaa ccttacttgc tttataatta 4620cgttaattag aagcgttatc
tcgtgaagga atatagtacg tagccgtata aattgaattg 4680aatgttcagc
ttatagaata gagacacttt gctgttcaat gcgtcgtcac ttaccatact
4740cactttatta tacgacttta agtataaact ccgcggttat ggtaaaatta
atgatgcaca 4800aacgtccgat tccatatggg tacactacaa ttaaatactt
ttaagctgat cccccacaca 4860ccatagcttc aaaatgtttc tactcctttt
ttactcttcc agattttctc ggactccgcg 4920catcgccgta ccacttcaaa
acacccaagc acagcatact aaattttccc tctttcttcc 4980tctagggtgt
cgttaattac ccgtactaaa ggtttggaaa agaaaaaaga gaccgcctcg
5040tttctttttc ttcgtcgaaa aaggcaataa aaatttttat cacgtttctt
tttcttgaaa 5100tttttttttt tagttttttt ctctttcagt gacctccatt
gatatttaag ttaataaacg 5160gtcttcaatt tctcaagttt cagtttcatt
tttcttgttc tattacaact ttttttactt 5220cttgttcatt agaaagaaag
catagcaatc taatctaagg gcggtgttga caattaatca 5280tcggcatagt
atatcggcat agtataatac gacaaggtga ggaactaaac catggccaag
5340ttgaccagtg ccgttccggt gctcaccgcg cgcgacgtcg ccggagcggt
cgagttctgg 5400accgaccggc tcgggttctc ccgggacttc gtggaggacg
acttcgccgg tgtggtccgg 5460gacgacgtga ccctgttcat cagcgcggtc
caggaccagg tggtgccgga caacaccctg 5520gcctgggtgt gggtgcgcgg
cctggacgag ctgtacgccg agtggtcgga ggtcgtgtcc 5580acgaacttcc
gggacgcctc cgggccggcc atgaccgaga tcggcgagca gccgtggggg
5640cgggagttcg ccctgcgcga cccggccggc aactgcgtgc acttcgtggc
cgaggagcag 5700gactgacacg tccgacggcg gcccacgggt cccaggcctc
ggagatccgt cccccttttc 5760ctttgtcgat atcatgtaat tagttatgtc
acgcttacat tcacgccctc cccccacatc 5820cgctctaacc gaaaaggaag
gagttagaca acctgaagtc taggtcccta tttatttttt 5880tatagttatg
ttagtattaa gaacgttatt tatatttcaa atttttcttt tttttctgta
5940cagacgcgag cttcccagta aatgtgccat ctcgtaggca gaaaacggtt
cccccgtagg 6000gtctctctct tggcctcctt tctaggtcgg gctgattgct
cttgaagctc tctagggggg 6060ctcacaccat aggcagataa cgttccccac
cggctcgcct cgtaagcgca caaggactgc 6120tcccaaagat cctaggcggg
attttgccga tttcggccta aaggaaccgg aacacgtaga 6180aagccagtcc
gcagaaacgg tgctgacccc ggatgaatgt cagctactgg gctatctgga
6240caagggaaaa cgcaagcgca aagagaaagc aggtagcttg cagtgggctt
acatggcgat 6300agctagactg ggcggtttta tggacagcaa gcgaaccgga
attgccagct ggggcgccct 6360ctggtaaggt tgggaagccc tgcaaagtaa
actggatggc tttcttgccg ccaaggatct 6420gatggcgcag gggatcaaga
tctgatcaag agacaggatg aggatcgttt cgcatgattg 6480aacaagatgg
attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg
6540actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg
tcagcgcagg 6600ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc
cctgaatgaa ctgcaggacg 6660aggcagcgcg gctatcgtgg ctggccacga
cgggcgttcc ttgcgcagct gtgctcgacg 6720ttgtcactga agcgggaagg
gactggctgc tattgggcga agtgccgggg caggatctcc 6780tgtcatctcg
ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc
6840tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat
cgcatcgagc 6900gagcacgtac tcggatggaa gccggtcttg tcgatcagga
tgatctggac gaagagcatc 6960aggggctcgc gccagccgaa ctgttcgcca
ggctcaaggc gcgcatgccc gacggcgagg 7020atctcgtcgt gatccatggc
gatgcctgct tgccgaatat catggtggaa aatggccgct 7080tttctggatt
caacgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt
7140tggatacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc
ttcctcgtgc 7200tttacggtat cgccgctccc gattcgcagc gcatcgcctt
ctatcgcctt cttgacgagt 7260tcttctgaat tgaaaaaggt accaagttta
ctcatatata ctttagattg atttaaaact 7320tcatttttaa tttaaaagga
tctaggtgaa gatccttttt gataatctca tgaccaaaat 7380cccttaacgt
gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
7440ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
aaccaccgct 7500accagcggtg gtttgtttgc cggatcaaga gctaccaact
ctttttccga aggtaactgg 7560cttcagcaga gcgcagatac caaatactgt
ccttctagtg tagccgtagt taggccacca 7620cttcaagaac tctgtagcac
cgcctacata cctcgctctg ctaatcctgt taccagtggc 7680tgctgccagt
ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
7740taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
tggagcgaac 7800gacctacacc gaactgagat acctacagcg tgagcattga
gaaagcgcca cgcttcccga 7860agggagaaag gcggacaggt atccggtaag
cggcagggtc ggaacaggag agcgcacgag 7920ggagcttcca gggggaaacg
cctggtatct ttatagtcct gtcgggtttc gccacctctg 7980acttgagcgt
cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
8040caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
tgttctttcc 8100tgcgttatcc cctgattctg tggataaccg tattaccgcc
tttgagtgag ctgataccgc 8160tcgccgcagc cgaacgaccg agcgcagcga g
8191121724DNAArtificial SequenceSynthetic Construct 12gaattcaaaa
caaaatgtgt gcaacctcct cccagtttac tcagattacc gagcataatt 60ctcgacgatc
tgctaactac cagccgaacc tttggaactt tgagtttctc cagtctctcg
120aaaatgacct gaaggtggaa aagctcgagg agaaggcgac caaactcgag
gaggaggtgc 180gatgtatgat caacagagtt gacacccaac ccctgtcttt
gctggagctg atcgacgatg 240tgcagcggtt gggtttgact tataaattcg
agaaggacat tatcaaggca ctggagaaca 300ttgtgctcct cgacgagaac
aagaagaaca agtctgatct tcacgctacc gctctctctt 360tccgacttct
tcgacaacac ggcttcgagg tgtcgcagga cgtcttcgag agatttaagg
420acaaggaggg aggatttagc ggcgagctga agggagacgt tcagggtctt
ctctccttgt 480acgaggcgtc ctacctggga ttcgagggag agaacctcct
ggaggaagct cgtacatttt 540ccatcactca ccttaagaat aaccttaagg
agggaattaa caccaaggtg gccgagcagg 600tttctcacgc cctggagctc
ccctaccacc aacggctcca tagactggag gctcgttggt 660tcctggacaa
atatgagcca aaggagcctc atcatcagtt gctgttggag ttggccaagc
720tggacttcaa tatggttcag acgctgcacc aaaaggagtt gcaggacctg
tctcgatggt 780ggaccgagat gggattggcc tcgaagctgg attttgtccg
tgaccgactt atggaggtct 840atttttgggc ccttggaatg gcgcctgacc
cccagttcgg agagtgccgg aaggcggtga 900cgaagatgtt cggtcttgtg
actatcatcg acgacgtcta cgatgtctac ggcacactcg 960acgagttgca
gctgttcact gacgccgtcg agcgatggga tgtgaacgcc attaatactc
1020tccctgacta tatgaagctg tgcttcctgg ctctgtacaa cactgtcaac
gatacctcgt 1080actctatcct caaggagaag ggacacaaca atctctccta
cttgaccaaa tcctggcgag 1140aactgtgcaa ggcttttctg caggaggcta
aatggtccaa taacaagatc attcctgctt 1200tttctaaata cctggaaaat
gcctcggtgt cgagctctgg cgtcgccctt ctggcccctt 1260cctacttctc
cgtctgccag cagcaggagg atatttccga tcatgctctt agatcgctga
1320ccgattttca cggcctcgtg cgatcttcct gcgtgatttt tcggttgtgt
aatgaccttg 1380cgacctctgc tgctgagctg gaacgaggcg agactacaaa
ttccattatt tcttacatgc 1440acgaaaacga tggaacatct gaagaacagg
ctagagagga actgcgaaag ttgatcgacg 1500ccgagtggaa gaagatgaac
agagagcggg tgtccgactc taccctgctt cccaaggcct 1560tcatggagat
cgccgtgaac atggctcgag tttcccattg tacttaccag tacggtgacg
1620gcctgggtcg tccggactac gctacagaga accgaatcaa gctgctgctc
atcgacccct 1680tccctatcaa ccaattgatg tacgtgtaat agtctagagg atcc
1724131701DNAArtificial SequenceSynthetic Construct 13gaattcaaca
aaaatgtgct ctgtttccac tgagaacgtg tcctttactg agactgagac 60tgaagcacgt
agaagcgcca actacgaacc caactcctgg gattatgact ttctgctgtc
120ttctgacacc gacgagtcga tcgaggttta taaggataag gccaagaaac
ttgaggccga 180ggtcagacga gagattaaca acgagaaggc cgagttcctg
acccttcttg agctgatcga 240caacgttcaa cgacttggtc ttggttaccg
tttcgaatcc gatatccgac gtgcattgga 300tcgatttgtc tcgtccggag
gtttcgatgg tgtgactaag acgtcgctgc acgccacagc 360tctttccttc
agactgttgc ggcagcatgg atttgaggtt tcccaggaag ccttttctgg
420tttcaaggat cagaacggaa actttttgga gaatctcaag gaggacacca
aggccatcct 480gtcgttgtat gaggcctcgt tcctggctct tgagggcgag
aatattctgg atgaggctcg 540ggttttcgct atttcgcacc tgaaggagtt
gtcggaggaa aagatcggaa aggaactggc 600cgagcaggtc aaccatgcac
ttgaacttcc cctgcatcga cgtacccagc gactggaggc 660cgtgtggagc
atcgaggcgt acagaaaaaa ggaggatgct aatcaggttc tgctcgaact
720cgctatcctc gactataaca tgattcagag cgtgtaccag cgtgacttgc
gagagacaag 780ccggtggtgg cgacgggtgg gactggccac gaagctccac
tttgctaaag atcgattgat 840tgagtcgttc tactgggcag tgggtgtggc
ctttgagcct cagtactccg actgccgaaa 900ctccgttgca aagatgtttt
cttttgtcac tatcatcgac gacatctacg atgtttacgg 960cactctcgat
gaactcgaac tcttcacgga cgctgtcgag cgatgggatg tgaatgccat
1020taatgatctg ccagattata tgaagttgtg tttcttggcg ctctacaaca
caattaatga 1080aattgcctac gacaacctca aggacaaggg agagaacatt
ctgccctacc ttactaaagc 1140ctgggccgac ctgtgtaacg cctttttgca
ggaagccaag tggctctata acaaatctac 1200tcctacattt gatgactact
tcggcaacgc ttggaagtct tccagcggcc ctctccagtt 1260gatcttcgct
tactttgcag tggtccagaa catcaagaaa gaggagattg agaacctcca
1320gaagtatcac gacatcatct cccgaccttc gcacatcttt cgactgtgca
atgaccttgc 1380ctccgcatcc gctgagattg cccgaggaga aacagccaat
tctgtgtcgt gttacatgcg 1440tacaaagggc atctccgagg agctggctac
cgagtctgtg atgaacctga tcgatgaaac 1500ctgtaagaag atgaacaaag
agaaactggg cggttctctg ttcgccaaac catttgttga 1560aaccgcgatc
aatctggctc gtcagtctca ttgtacttac cataacggtg acgcgcacac
1620ttcgccggac gaattgaccc gtaagcgtgt gctttcggtg attaccgagc
cgatcctgcc 1680gttcgaaaga taataggatc c 17011428DNAArtificial
SequenceSynthetic Construct 14gatcaagctt aaccggaatt gccagctg
281533DNAArtificial SequenceSynthetic Construct 15gatccgatcg
tcagaagaac tcgtcaagaa ggc 331638DNAArtificial SequenceSynthetic
Construct 16catcaatgca tcgcccttag gaggtaaaaa aaaatgac
381726DNAArtificial SequenceSynthetic Construct 17ccttctgcag
gacgcgttgt tatagc 261860DNAArtificial SequenceSynthetic Construct
18gatcatgcat tcgcccttag gaggtaaaaa aacatgagtt ttgatattgc caaatacccg
601931DNAArtificial SequenceSynthetic Construct 19catgctgcag
ttatgccagc caggccttga t 31208804DNAArtificial SequenceSynthetic
Construct 20gctggtacca tatgggaatt cgaagctttc tagaacaaaa actcatctca
gaagaggatc 60tgaatagcgc cgtcgaccat catcatcatc atcattgagt ttaaacggtc
tccagcttgg 120ctgttttggc ggatgagaga agattttcag cctgatacag
attaaatcag aacgcagaag 180cggtctgata aaacagaatt tgcctggcgg
cagtagcgcg gtggtcccac ctgaccccat 240gccgaactca gaagtgaaac
gccgtagcgc cgatggtagt gtggggtctc cccatgcgag 300agtagggaac
tgccaggcat caaataaaac gaaaggctca gtcgaaagac tgggcctttc
360gttttatctg ttgtttgtcg gtgaacgctc tcctgagtag gacaaatccg
ccgggagcgg 420atttgaacgt tgcgaagcaa cggcccggag ggtggcgggc
aggacgcccg ccataaactg 480ccaggcatca aattaagcag aaggccatcc
tgacggatgg cctttttgcg tttctacaaa 540ctctttttgt ttatttttct
aaatacattc aaatatgtat ccgcttaacc ggaattgcca 600gctggggcgc
cctctggtaa ggttgggaag ccctgcaaag taaactggat ggctttctcg
660ccgccaagga tctgatggcg caggggatca agctctgatc aagagacagg
atgaggatcg 720tttcgcatga ttgaacaaga tggattgcac gcaggttctc
cggccgcttg ggtggagagg 780ctattcggct atgactgggc acaacagaca
atcggctgct ctgatgccgc cgtgttccgg 840ctgtcagcgc aggggcgccc
ggttcttttt gtcaagaccg acctgtccgg tgccctgaat 900gaactgcaag
acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca
960gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg
cgaagtgccg 1020gggcaggatc tcctgtcatc tcaccttgct cctgccgaga
aagtatccat catggctgat 1080gcaatgcggc ggctgcatac gcttgatccg
gctacctgcc cattcgacca ccaagcgaaa 1140catcgcatcg agcgagcacg
tactcggatg gaagccggtc ttgtcgatca ggatgatctg 1200gacgaagagc
atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgagcatg
1260cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa
tatcatggtg 1320gaaaatggcc gcttttctgg attcatcgac tgtggccggc
tgggtgtggc ggaccgctat 1380caggacatag cgttggctac ccgtgatatt
gctgaagagc ttggcggcga atgggctgac 1440cgcttcctcg tgctttacgg
tatcgccgct cccgattcgc agcgcatcgc cttctatcgc 1500cttcttgacg
agttcttctg acatgaccaa aatcccttaa cgtgagtttt cgttccactg
1560agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
ttctgcgcgt 1620aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
gtggtttgtt tgccggatca 1680agagctacca actctttttc cgaaggtaac
tggcttcagc agagcgcaga taccaaatac 1740tgtccttcta gtgtagccgt
agttaggcca ccacttcaag aactctgtag caccgcctac 1800atacctcgct
ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
1860taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
gctgaacggg 1920gggttcgtgc acacagccca gcttggagcg aacgacctac
accgaactga gatacctaca 1980gcgtgagcta tgagaaagcg ccacgcttcc
cgaagggaga aaggcggaca ggtatccggt 2040aagcggcagg gtcggaacag
gagagcgcac gagggagctt ccagggggaa acgcctggta 2100tctttatagt
cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
2160gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
ggttcctggc 2220cttttgctgg ccttttgctc acatgttctt tcctgcgtta
tcccctgatt ctgtggataa 2280ccgtattacc gcctttgagt gagctgatac
cgctcgccgc agccgaacga ccgagcgcag 2340cgagtcagtg agcgaggaag
cggaagagcg cctgatgcgg tattttctcc ttacgcatct 2400gtgcggtatt
tcacaccgca tatggtgcac tctcagtaca atctgctctg atgccgcata
2460gttaagccag tatacactcc gctatcgcta cgtgactggg tcatggctgc
gccccgacac 2520ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc
tcccggcatc cgcttacaga 2580caagctgtga ccgtctccgg gagctgcatg
tgtcagaggt tttcaccgtc atcaccgaaa 2640cgcgcgaggc agcagatcaa
ttcgcgcgcg aaggcgaagc ggcatgcatt tacgttgaca 2700ccatcgaatg
gtgcaaaacc tttcgcggta tggcatgata gcgcccggaa gagagtcaat
2760tcagggtggt gaatgtgaaa ccagtaacgt tatacgatgt cgcagagtat
gccggtgtct 2820cttatcagac cgtttcccgc gtggtgaacc aggccagcca
cgtttctgcg aaaacgcggg 2880aaaaagtgga agcggcgatg gcggagctga
attacattcc caaccgcgtg gcacaacaac 2940tggcgggcaa acagtcgttg
ctgattggcg ttgccacctc cagtctggcc ctgcacgcgc 3000cgtcgcaaat
tgtcgcggcg attaaatctc gcgccgatca actgggtgcc agcgtggtgg
3060tgtcgatggt agaacgaagc ggcgtcgaag cctgtaaagc ggcggtgcac
aatcttctcg 3120cgcaacgcgt cagtgggctg atcattaact atccgctgga
tgaccaggat gccattgctg 3180tggaagctgc ctgcactaat gttccggcgt
tatttcttga tgtctctgac cagacaccca 3240tcaacagtat tattttctcc
catgaagacg gtacgcgact gggcgtggag catctggtcg 3300cattgggtca
ccagcaaatc gcgctgttag cgggcccatt aagttctgtc tcggcgcgtc
3360tgcgtctggc tggctggcat aaatatctca ctcgcaatca aattcagccg
atagcggaac 3420gggaaggcga ctggagtgcc atgtccggtt ttcaacaaac
catgcaaatg ctgaatgagg 3480gcatcgttcc cactgcgatg ctggttgcca
acgatcagat ggcgctgggc gcaatgcgcg 3540ccattaccga gtccgggctg
cgcgttggtg cggatatctc ggtagtggga tacgacgata 3600ccgaagacag
ctcatgttat atcccgccgt caaccaccat caaacaggat tttcgcctgc
3660tggggcaaac cagcgtggac cgcttgctgc aactctctca gggccaggcg
gtgaagggca 3720atcagctgtt gcccgtctca ctggtgaaaa gaaaaaccac
cctggcgccc aatacgcaaa 3780ccgcctctcc ccgcgcgttg gccgattcat
taatgcagct ggcacgacag gtttcccgac 3840tggaaagcgg gcagtgagcg
caacgcaatt aatgtgagtt agcgcgaatt gatctggttt 3900gacagcttat
catcgactgc acggtgcacc aatgcttctg gcgtcaggca gccatcggaa
3960gctgtggtat ggctgtgcag gtcgtaaatc actgcataat tcgtgtcgct
caaggcgcac 4020tcccgttctg gataatgttt tttgcgccga catcataacg
gttctggcaa atattctgaa 4080atgagctgtt gacaattaat catccggctc
gtataatgtg tggaattgtg agcggataac 4140aatttcacac aggaaacagc
gccgctgaga aaaagcgaag cggcactgct ctttaacaat 4200ttatcagaca
atctgtgtgg gcactcgacc ggaattatcg attaacttta ttattaaaaa
4260ttaaagaggt atatattaat gtatcgatta aataaggagg aataaaccat
gtgtgcgacc 4320tcttctcaat ttactcagat taccgagcat aattcccgtc
gttccgcaaa ctatcagcca 4380aacctgtgga atttcgaatt cctgcaatcc
ctggagaacg acctgaaagt ggaaaagctg 4440gaggagaaag cgaccaaact
ggaggaagaa gttcgctgca tgatcaaccg tgtagacacc 4500cagccgctgt
ccctgctgga gctgatcgac gatgtgcagc gcctgggtct gacctacaaa
4560tttgaaaaag acatcattaa agccctggaa aacatcgtac tgctggacga
aaacaaaaag 4620aacaaatctg acctgcacgc aaccgctctg tctttccgtc
tgctgcgtca gcacggtttc 4680gaggtttctc aggatgtttt tgagcgtttc
aaggataaag aaggtggttt cagcggtgaa 4740ctgaaaggtg acgtccaagg
cctgctgagc ctgtatgaag cgtcttacct gggtttcgag 4800ggtgagaacc
tgctggagga ggcgcgtacc ttttccatca cccacctgaa gaacaacctg
4860aaagaaggca ttaataccaa ggttgcagaa caagtgagcc acgccctgga
actgccatat 4920caccagcgtc tgcaccgtct ggaggcacgt tggttcctgg
ataaatacga accgaaagaa 4980ccgcatcacc agctgctgct ggagctggcg
aagctggatt ttaacatggt acagaccctg 5040caccagaaag agctgcaaga
tctgtcccgc tggtggaccg agatgggcct ggctagcaaa 5100ctggattttg
tacgcgaccg cctgatggaa gtttatttct gggcactggg tatggcgcca
5160gacccgcagt ttggtgaatg tcgcaaagct gttactaaaa tgtttggtct
ggtgacgatc 5220atcgatgacg tgtatgacgt ttatggcact ctggacgaac
tgcaactgtt caccgatgct 5280gtagagcgct gggacgttaa cgctattaac
accctgccgg actatatgaa actgtgtttc 5340ctggcactgt acaacaccgt
taacgacacg tcctattcta ttctgaaaga gaaaggtcat 5400aacaacctgt
cctatctgac gaaaagctgg cgtgaactgt gcaaagcctt tctgcaagag
5460gcgaaatggt ccaacaacaa aattatcccg gctttctcca agtacctgga
aaacgccagc 5520gtttcctcct ccggtgtagc gctgctggcg ccgtcttact
tttccgtatg ccagcagcag 5580gaagacatct ccgaccacgc gctgcgttcc
ctgaccgact tccatggtct ggtgcgttct 5640agctgcgtta tcttccgcct
gtgcaacgat ctggccacct ctgcggcgga gctggaacgt 5700ggcgagacta
ccaattctat cattagctac atgcacgaaa acgatggtac cagcgaggaa
5760caggcccgcg aagaactgcg taaactgatc gacgccgaat ggaaaaagat
gaatcgtgaa 5820cgcgttagcg actccaccct gctgcctaaa gcgttcatgg
aaatcgcagt taacatggca 5880cgtgtttccc actgcaccta ccagtatggc
gatggtctgg gtcgcccaga ctacgcgact 5940gaaaaccgca tcaaactgct
gctgattgac cctttcccga ttaaccagct gatgtatgtc 6000taactgcatc
gcccttagga ggtaaaaaaa aatgactgcc gacaacaata gtatgcccca
6060tggtgcagta tctagttacg ccaaattagt gcaaaaccaa acacctgaag
acattttgga 6120agagtttcct gaaattattc cattacaaca aagacctaat
acccgatcta gtgagacgtc 6180aaatgacgaa agcggagaaa catgtttttc
tggtcatgat gaggagcaaa ttaagttaat 6240gaatgaaaat tgtattgttt
tggattggga cgataatgct attggtgccg gtaccaagaa 6300agtttgtcat
ttaatggaaa atattgaaaa gggtttacta catcgtgcat tctccgtctt
6360tattttcaat gaacaaggtg aattactttt acaacaaaga gccactgaaa
aaataacttt 6420ccctgatctt tggactaaca catgctgctc tcatccacta
tgtattgatg acgaattagg 6480tttgaagggt aagctagacg ataagattaa
gggcgctatt actgcggcgg tgagaaaact 6540agatcatgaa ttaggtattc
cagaagatga aactaagaca aggggtaagt ttcacttttt 6600aaacagaatc
cattacatgg caccaagcaa tgaaccatgg ggtgaacatg aaattgatta
6660catcctattt tataagatca acgctaaaga aaacttgact gtcaacccaa
acgtcaatga 6720agttagagac ttcaaatggg tttcaccaaa tgatttgaaa
actatgtttg ctgacccaag 6780ttacaagttt acgccttggt ttaagattat
ttgcgagaat tacttattca actggtggga 6840gcaattagat gacctttctg
aagtggaaaa tgacaggcaa attcatagaa tgctataaca 6900acgcgtcctg
cattcgccct taggaggtaa aaaaacatga gttttgatat tgccaaatac
6960ccgaccctgg cactggtcga ctccacccag gagttacgac tgttgccgaa
agagagttta 7020ccgaaactct gcgacgaact gcgccgctat ttactcgaca
gcgtgagccg ttccagcggg 7080cacttcgcct ccgggctggg cacggtcgaa
ctgaccgtgg cgctgcacta tgtctacaac 7140accccgtttg accaattgat
ttgggatgtg gggcatcagg cttatccgca taaaattttg 7200accggacgcc
gcgacaaaat cggcaccatc cgtcagaaag gcggtctgca cccgttcccg
7260tggcgcggcg aaagcgaata tgacgtatta agcgtcgggc attcatcaac
ctccatcagt 7320gccggaattg gtattgcggt tgctgccgaa aaagaaggca
aaaatcgccg caccgtctgt 7380gtcattggcg atggcgcgat taccgcaggc
atggcgtttg aagcgatgaa tcacgcgggc 7440gatatccgtc ctgatatgct
ggtgattctc aacgacaatg aaatgtcgat ttccgaaaat 7500gtcggcgcgc
tcaacaacca tctggcacag ctgctttccg gtaagcttta ctcttcactg
7560cgcgaaggcg ggaaaaaagt tttctctggc gtgccgccaa ttaaagagct
gctcaaacgc 7620accgaagaac atattaaagg catggtagtg cctggcacgt
tgtttgaaga gctgggcttt 7680aactacatcg gcccggtgga cggtcacgat
gtgctggggc ttatcaccac gctaaagaac 7740atgcgcgacc tgaaaggccc
gcagttcctg catatcatga ccaaaaaagg tcgtggttat 7800gaaccggcag
aaaaagaccc gatcactttc cacgccgtgc ctaaatttga tccctccagc
7860ggttgtttgc cgaaaagtag cggcggtttg ccgagctatt caaaaatctt
tggcgactgg 7920ttgtgcgaaa cggcagcgaa agacaacaag ctgatggcga
ttactccggc gatgcgtgaa 7980ggttccggca tggtcgagtt ttcacgtaaa
ttcccggatc gctacttcga cgtggcaatt 8040gccgagcaac acgcggtgac
ctttgctgcg ggtctggcga ttggtgggta caaacccatt 8100gtcgcgattt
actccacttt cctgcaacgc gcctatgatc aggtgctgca tgacgtggcg
8160attcaaaagc ttccggtcct gttcgccatc gaccgcgcgg gcattgttgg
tgctgacggt 8220caaacccatc agggtgcttt tgatctctct tacctgcgct
gcataccgga aatggtcatt 8280atgaccccga gcgatgaaaa cgaatgtcgc
cagatgctct ataccggcta tcactataac 8340gatggcccgt cagcggtgcg
ctacccgcgt ggcaacgcgg tcggcgtgga actgacgccg 8400ctggaaaaac
taccaattgg caaaggcatt gtgaagcgtc gtggcgagaa actggcgatc
8460cttaactttg gtacgctgat gccagaagcg gcgaaagtcg ccgaatcgct
gaacgccacg 8520ctggtcgata tgcgttttgt gaaaccgctt gatgaagcgt
taattctgga aatggccgcc 8580agccatgaag cgctggtcac cgtagaagaa
aacgccatta tgggcggcgc aggcagcggc 8640gtgaacgaag tgctgatggc
ccatcgtaaa ccagtacccg tgctgaacat tggcctgccg 8700gacttcttta
ttccgcaagg aactcaggaa gaaatgcgcg ccgaactcgg cctcgatgcc
8760gctggtatgg aagccaaaat caaggcctgg ctggcataac tgca
8804214339DNAArtificial SequenceSynthetic Construct 21tagaaaaact
catcgagcat caaatgaaac tgcaatttat tcatatcagg attatcaata 60ccatattttt
gaaaaagccg tttctgtaat gaaggagaaa actcaccgag gcagttccat
120aggatggcaa gatcctggta tcggtctgcg attccgactc gtccaacatc
aatacaacct 180attaatttcc cctcgtcaaa aataaggtta tcaagtgaga
aatcaccatg agtgacgact 240gaatccggtg agaatggcaa aagtttatgc
atttctttcc agacttgttc aacaggccag 300ccattacgct cgtcatcaaa
atcactcgca tcaaccaaac cgttattcat tcgtgattgc 360gcctgagcga
ggcgaaatac gcgatcgctg ttaaaaggac aattacaaac aggaatcgag
420tgcaaccggc gcaggaacac tgccagcgca tcaacaatat tttcacctga
atcaggatat 480tcttctaata cctggaacgc tgtttttccg gggatcgcag
tggtgagtaa ccatgcatca 540tcaggagtac ggataaaatg cttgatggtc
ggaagtggca taaattccgt cagccagttt 600agtctgacca tctcatctgt
aacatcattg gcaacgctac ctttgccatg tttcagaaac 660aactctggcg
catcgggctt cccatacaag cgatagattg tcgcacctga ttgcccgaca
720ttatcgcgag cccatttata cccatataaa tcagcatcca tgttggaatt
taatcgcggc 780ctcgacgttt cccgttgaat atggctcata ttcttccttt
ttcaatatta ttgaagcatt 840tatcagggtt attgtctcat gagcggatac
atatttgaat gtatttagaa aaataaacaa 900ataggggtca gtgttacaac
caattaacca attctgaaca ttatcgcgag cccatttata 960cctgaatatg
gctcataaca ccccttgttt gcctggcggc agtagcgcgg tggtcccacc
1020tgaccccatg ccgaactcag aagtgaaacg ccgtagcgcc gatggtagtg
tggggactcc 1080ccatgcgaga gtagggaact gccaggcatc aaataaaacg
aaaggctcag tcgaaagact 1140gggcctttcg cccgggctaa ttagggggtg
tcgcccttta gtcgctgaac atgtgctctg 1200tttctaccga gaacgtttcc
ttcactgaga cggaaaccga ggcacgtcgt agcgcgaact 1260acgagccgaa
tagctgggac tacgatttcc tgctgtcttc cgatactgac gaatctattg
1320aggtgtacaa agacaaagca aagaaactgg aggctgaagt gcgccgcgaa
attaacaacg 1380agaaagctga attcctgact ctgctggagc tgatcgataa
cgtacagcgc ctgggtctgg 1440gttaccgctt cgaatctgat atccgtcgcg
cactggatcg tttcgtaagc agcggcggtt 1500tcgatggcgt gaccaaaacg
agcctgcacg ctaccgcgct gtccttccgt ctgctgcgtc 1560agcacggctt
cgaagtttct caggaagcat tctccggttt caaagatcaa aacggtaact
1620tcctggaaaa cctgaaagaa gacactaagg cgatcctgag cctgtatgag
gcaagctttc 1680tggccctgga gggtgagaac atcctggatg aggcgcgcgt
attcgccatc tcccatctga 1740aagagctgtc tgaagagaaa atcggtaagg
aactggcaga gcaggttaat cacgcactgg 1800aactgccgct gcatcgtcgt
acccagcgtc tggaggcggt ttggtccatc gaagcgtacc 1860gcaaaaagga
ggatgctaac caggttctgc tggaactggc catcctggac tacaacatga
1920tccagtccgt ttaccagcgt gatctgcgtg aaacctcccg ttggtggcgc
cgtgtgggcc 1980tggcgaccaa actgcacttc gctaaggacc gcctgattga
gtctttttac tgggcagtcg 2040gcgttgcgtt cgaacctcag tattctgact
gccgtaacag cgttgcgaaa atgttcagct 2100tcgttactat tatcgacgac
atctacgacg tttacggtac tctggacgag ctggaactgt 2160ttaccgacgc
tgtcgaacgt tgggatgtta acgccatcaa cgatctgcct gactacatga
2220aactgtgctt cctggcactg tataacacga tcaacgaaat tgcatacgac
aacctgaaag 2280acaaaggtga aaacatcctg ccgtacctga ctaaagcgtg
ggcggatctg tgtaacgctt 2340ttctgcaaga agcgaaatgg ctgtataaca
aatccactcc gacctttgac gattatttcg 2400gcaatgcctg gaaatccagc
tctggcccgc tgcaactgat cttcgcttat tttgcggttg 2460tccaaaacat
caaaaaggag gaaattgaaa acctgcaaaa ataccacgat atcattagcc
2520gtccttctca tatctttcgc ctgtgcaacg acctggcaag cgcgtccgca
gagatcgcac 2580gtggcgaaac cgctaactct gtttcctgct acatgcgcac
caagggcatt tccgaagagc 2640tggcaaccga gagcgtaatg aatctgatcg
acgaaacctg taagaaaatg aacaaagaaa 2700aactgggtgg ctccctgttc
gctaaaccgt tcgtagagac tgctattaac ctggcacgtc 2760agagccactg
cacctaccac aatggtgacg cacatactag cccggatgaa ctgactcgta
2820aacgtgtact gtctgttatc accgaaccga ttctgccgtt cgaacgttaa
ctgcagcgtc 2880aatcgaaagg gcgacacaaa atttattcta aatgcataat
aaatactgat aacatcttat 2940agtttgtatt atattttgta ttatcgttga
catgtataat tttgatatca aaaactgatt 3000ttccctttat tattttcgag
atttattttc ttaattctct ttaacaaact agaaatattg 3060tatatacaaa
aaatcataaa taatagatga atagtttaat tataggtgtt catcaatcga
3120aaaagcaacg tatcttattt aaagtgcgtt gcttttttct catttataag
gttaaataat 3180tctcatatat caagcaaagt gacaggcgcc cttaaatatt
ctgacaaatg ctctttccct 3240aaactccccc cataaaaaaa cccgccgaag
cgggttttta cgttatttgc ggattaacga 3300ttactcgtta tcagaaccgc
ccagggggcc cgagcttaag actggccgtc gttttacaac 3360acagaaagag
tttgtagaaa cgcaaaaagg ccatccgtca ggggccttct gcttagtttg
3420atgcctggca gttccctact ctcgccttcc gcttcctcgc tcactgactc
gctgcgctcg 3480gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
cggtaatacg gttatccaca 3540gaatcagggg ataacgcagg aaagaacatg
tgagcaaaag gccagcaaaa ggccaggaac 3600cgtaaaaagg ccgcgttgct
ggcgtttttc cataggctcc gcccccctga cgagcatcac 3660aaaaatcgac
gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
3720tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
taccggatac 3780ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
atagctcacg ctgtaggtat 3840ctcagttcgg tgtaggtcgt tcgctccaag
ctgggctgtg tgcacgaacc ccccgttcag 3900cccgaccgct gcgccttatc
cggtaactat cgtcttgagt ccaacccggt aagacacgac 3960ttatcgccac
tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
4020gctacagagt tcttgaagtg gtgggctaac tacggctaca ctagaagaac
agtatttggt 4080atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
ttggtagctc ttgatccggc 4140aaacaaacca ccgctggtag cggtggtttt
tttgtttgca agcagcagat tacgcgcaga 4200aaaaaaggat ctcaagaaga
tcctttgatc ttttctacgg ggtctgacgc tcagtggaac 4260gacgcgcgcg
taactcacgt taagggattt tggtcatgag cttgcgccgt cccgtcaagt
4320cagcgtaatg ctctgcttt 4339226065DNAArtificial SequenceSynthetic
Construct 22gtttgacagc ttatcatcga ctgcacggtg caccaatgct tctggcgtca
ggcagccatc 60ggaagctgtg gtatggctgt gcaggtcgta aatcactgca taattcgtgt
cgctcaaggc 120gcactcccgt tctggataat gttttttgcg ccgacatcat
aacggttctg gcaaatattc 180tgaaatgagc tgttgacaat taatcatccg
gctcgtataa tgtgtggaat tgtgagcgga 240taacaatttc acacaggaaa
cagcgccgct gagaaaaagc gaagcggcac tgctctttaa 300caatttatca
gacaatctgt gtgggcactc gaccggaatt atcgattaac tttattatta
360aaaattaaag aggtatatat taatgtatcg attaaataag gaggaataaa
ccatgtgctc 420tgtttctacc gagaacgttt ccttcactga gacggaaacc
gaggcacgtc gtagcgcgaa 480ctacgagccg aatagctggg actacgattt
cctgctgtct tccgatactg acgaatctat 540tgaggtgtac aaagacaaag
caaagaaact ggaggctgaa gtgcgccgcg aaattaacaa 600cgagaaagct
gaattcctga ctctgctgga gctgatcgat aacgtacagc gcctgggtct
660gggttaccgc ttcgaatctg atatccgtcg cgcactggat cgtttcgtaa
gcagcggcgg 720tttcgatggc gtgaccaaaa cgagcctgca cgctaccgcg
ctgtccttcc gtctgctgcg 780tcagcacggc ttcgaagttt ctcaggaagc
attctccggt ttcaaagatc aaaacggtaa 840cttcctggaa aacctgaaag
aagacactaa ggcgatcctg agcctgtatg aggcaagctt 900tctggccctg
gagggtgaga acatcctgga tgaggcgcgc gtattcgcca tctcccatct
960gaaagagctg tctgaagaga aaatcggtaa ggaactggca gagcaggtta
atcacgcact 1020ggaactgccg ctgcatcgtc gtacccagcg tctggaggcg
gtttggtcca tcgaagcgta 1080ccgcaaaaag gaggatgcta accaggttct
gctggaactg gccatcctgg actacaacat 1140gatccagtcc gtttaccagc
gtgatctgcg tgaaacctcc cgttggtggc gccgtgtggg 1200cctggcgacc
aaactgcact tcgctaagga ccgcctgatt gagtcttttt actgggcagt
1260cggcgttgcg ttcgaacctc agtattctga ctgccgtaac agcgttgcga
aaatgttcag 1320cttcgttact attatcgacg acatctacga cgtttacggt
actctggacg agctggaact 1380gtttaccgac gctgtcgaac gttgggatgt
taacgccatc aacgatctgc ctgactacat 1440gaaactgtgc ttcctggcac
tgtataacac gatcaacgaa attgcatacg acaacctgaa 1500agacaaaggt
gaaaacatcc tgccgtacct gactaaagcg tgggcggatc tgtgtaacgc
1560ttttctgcaa gaagcgaaat ggctgtataa caaatccact ccgacctttg
acgattattt 1620cggcaatgcc tggaaatcca gctctggccc gctgcaactg
atcttcgctt attttgcggt 1680tgtccaaaac atcaaaaagg aggaaattga
aaacctgcaa aaataccacg atatcattag 1740ccgtccttct catatctttc
gcctgtgcaa cgacctggca agcgcgtccg cagagatcgc 1800acgtggcgaa
accgctaact ctgtttcctg ctacatgcgc accaagggca tttccgaaga
1860gctggcaacc gagagcgtaa tgaatctgat cgacgaaacc tgtaagaaaa
tgaacaaaga 1920aaaactgggt ggctccctgt tcgctaaacc gttcgtagag
actgctatta acctggcacg 1980tcagagccac tgcacctacc acaatggtga
cgcacatact agcccggatg aactgactcg 2040taaacgtgta ctgtctgtta
tcaccgaacc gattctgccg ttcgaacgtt aactgcagct 2100ggtaccatat
gggaattcga agctttctag aacaaaaact catctcagaa gaggatctga
2160atagcgccgt cgaccatcat catcatcatc attgagttta aacggtctcc
agcttggctg 2220ttttggcgga tgagagaaga ttttcagcct gatacagatt
aaatcagaac gcagaagcgg 2280tctgataaaa cagaatttgc ctggcggcag
tagcgcggtg gtcccacctg accccatgcc 2340gaactcagaa gtgaaacgcc
gtagcgccga tggtagtgtg gggtctcccc atgcgagagt 2400agggaactgc
caggcatcaa ataaaacgaa aggctcagtc gaaagactgg gcctttcgtt
2460ttatctgttg tttgtcggtg aacgctctcc tgagtaggac aaatccgccg
ggagcggatt 2520tgaacgttgc gaagcaacgg cccggagggt ggcgggcagg
acgcccgcca taaactgcca 2580ggcatcaaat taagcagaag gccatcctga
cggatggcct ttttgcgttt ctacaaactc 2640tttttgttta tttttctaaa
tacattcaaa tatgtatccg ctcatgagac aataaccctg 2700ataaatgctt
caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc
2760ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
aaacgctggt 2820gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
ggttacatcg aactggatct 2880caacagcggt aagatccttg agagttttcg
ccccgaagaa cgttttccaa tgatgagcac 2940ttttaaagtt ctgctatgtg
gcgcggtatt atcccgtgtt gacgccgggc aagagcaact 3000cggtcgccgc
atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa
3060gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
ccatgagtga 3120taacactgcg gccaacttac ttctgacaac gatcggagga
ccgaaggagc taaccgcttt 3180tttgcacaac atgggggatc atgtaactcg
ccttgatcgt tgggaaccgg agctgaatga 3240agccatacca aacgacgagc
gtgacaccac gatgcctgta gcaatggcaa caacgttgcg 3300caaactatta
actggcgaac tacttactct agcttcccgg caacaattaa tagactggat
3360ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
gctggtttat 3420tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
atcattgcag cactggggcc 3480agatggtaag ccctcccgta tcgtagttat
ctacacgacg gggagtcagg caactatgga 3540tgaacgaaat agacagatcg
ctgagatagg tgcctcactg attaagcatt ggtaactgtc 3600agaccaagtt
tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag
3660gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
gtgagttttc 3720gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
tcttcttgag atcctttttt 3780tctgcgcgta atctgctgct tgcaaacaaa
aaaaccaccg ctaccagcgg tggtttgttt 3840gccggatcaa gagctaccaa
ctctttttcc gaaggtaact ggcttcagca gagcgcagat 3900accaaatact
gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
3960accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
gtggcgataa 4020gtcgtgtctt accgggttgg actcaagacg atagttaccg
gataaggcgc agcggtcggg 4080ctgaacgggg ggttcgtgca cacagcccag
cttggagcga acgacctaca ccgaactgag 4140atacctacag cgtgagctat
gagaaagcgc cacgcttccc gaagggagaa aggcggacag 4200gtatccggta
agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
4260cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
gtcgattttt 4320gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
agcaacgcgg cctttttacg 4380gttcctggcc ttttgctggc cttttgctca
catgttcttt cctgcgttat cccctgattc 4440tgtggataac cgtattaccg
cctttgagtg agctgatacc gctcgccgca gccgaacgac 4500cgagcgcagc
gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct
4560tacgcatctg tgcggtattt cacaccgcat atggtgcact ctcagtacaa
tctgctctga 4620tgccgcatag ttaagccagt atacactccg ctatcgctac
gtgactgggt catggctgcg 4680ccccgacacc cgccaacacc cgctgacgcg
ccctgacggg cttgtctgct cccggcatcc 4740gcttacagac aagctgtgac
cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca 4800tcaccgaaac
gcgcgaggca gcagatcaat tcgcgcgcga aggcgaagcg gcatgcattt
4860acgttgacac catcgaatgg tgcaaaacct ttcgcggtat ggcatgatag
cgcccggaag 4920agagtcaatt cagggtggtg aatgtgaaac cagtaacgtt
atacgatgtc gcagagtatg 4980ccggtgtctc ttatcagacc gtttcccgcg
tggtgaacca ggccagccac gtttctgcga 5040aaacgcggga aaaagtggaa
gcggcgatgg cggagctgaa ttacattccc aaccgcgtgg 5100cacaacaact
ggcgggcaaa cagtcgttgc tgattggcgt tgccacctcc agtctggccc
5160tgcacgcgcc gtcgcaaatt gtcgcggcga ttaaatctcg cgccgatcaa
ctgggtgcca 5220gcgtggtggt gtcgatggta gaacgaagcg gcgtcgaagc
ctgtaaagcg gcggtgcaca 5280atcttctcgc gcaacgcgtc agtgggctga
tcattaacta tccgctggat gaccaggatg 5340ccattgctgt ggaagctgcc
tgcactaatg ttccggcgtt atttcttgat gtctctgacc 5400agacacccat
caacagtatt attttctccc atgaagacgg tacgcgactg ggcgtggagc
5460atctggtcgc attgggtcac cagcaaatcg cgctgttagc gggcccatta
agttctgtct 5520cggcgcgtct gcgtctggct ggctggcata aatatctcac
tcgcaatcaa attcagccga 5580tagcggaacg ggaaggcgac tggagtgcca
tgtccggttt tcaacaaacc atgcaaatgc 5640tgaatgaggg catcgttccc
actgcgatgc tggttgccaa cgatcagatg gcgctgggcg 5700caatgcgcgc
cattaccgag tccgggctgc gcgttggtgc ggatatctcg gtagtgggat
5760acgacgatac cgaagacagc tcatgttata tcccgccgtc aaccaccatc
aaacaggatt 5820ttcgcctgct ggggcaaacc agcgtggacc gcttgctgca
actctctcag ggccaggcgg 5880tgaagggcaa tcagctgttg cccgtctcac
tggtgaaaag aaaaaccacc ctggcgccca 5940atacgcaaac cgcctctccc
cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg 6000tttcccgact
ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gcgcgaattg 6060atctg
6065236912DNAArtificial SequenceSynthetic Construct 23ttgtctgctc
ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg 60tcagaggttt
tcaccgtcat caccgaaacg cgcgaggcag cagatcaatt cgcgcgcgaa
120ggcgaagcgg catgcattta cgttgacacc atcgaatggt gcaaaacctt
tcgcggtatg 180gcatgatagc gcccggaaga gagtcaattc agggtggtga
atgtgaaacc agtaacgtta 240tacgatgtcg cagagtatgc cggtgtctct
tatcagaccg tttcccgcgt ggtgaaccag 300gccagccacg tttctgcgaa
aacgcgggaa aaagtggaag cggcgatggc ggagctgaat 360tacattccca
accgcgtggc acaacaactg gcgggcaaac agtcgttgct gattggcgtt
420gccacctcca gtctggccct gcacgcgccg tcgcaaattg tcgcggcgat
taaatctcgc 480gccgatcaac tgggtgccag cgtggtggtg tcgatggtag
aacgaagcgg cgtcgaagcc 540tgtaaagcgg cggtgcacaa tcttctcgcg
caacgcgtca gtgggctgat cattaactat 600ccgctggatg accaggatgc
cattgctgtg gaagctgcct gcactaatgt tccggcgtta 660tttcttgatg
tctctgacca gacacccatc aacagtatta ttttctccca tgaagacggt
720acgcgactgg gcgtggagca tctggtcgca ttgggtcacc agcaaatcgc
gctgttagcg 780ggcccattaa gttctgtctc ggcgcgtctg cgtctggctg
gctggcataa atatctcact 840cgcaatcaaa ttcagccgat agcggaacgg
gaaggcgact ggagtgccat gtccggtttt 900caacaaacca tgcaaatgct
gaatgagggc atcgttccca ctgcgatgct ggttgccaac 960gatcagatgg
cgctgggcgc aatgcgcgcc attaccgagt ccgggctgcg cgttggtgcg
1020gatatctcgg tagtgggata cgacgatacc gaagacagct catgttatat
cccgccgtca 1080accaccatca aacaggattt tcgcctgctg gggcaaacca
gcgtggaccg cttgctgcaa 1140ctctctcagg gccaggcggt gaagggcaat
cagctgttgc ccgtctcact ggtgaaaaga 1200aaaaccaccc tggcgcccaa
tacgcaaacc gcctctcccc gcgcgttggc cgattcatta 1260atgcagctgg
cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa
1320tgtgagttag cgcgaattga tctggtttga cagcttatca tcgactgcac
ggtgcaccaa 1380tgcttctggc gtcaggcagc catcggaagc tgtggtatgg
ctgtgcaggt cgtaaatcac 1440tgcataattc gtgtcgctca aggcgcactc
ccgttctgga taatgttttt tgcgccgaca 1500tcataacggt tctggcaaat
attctgaaat gagctgttga caattaatca tccggctcgt 1560ataatgtgtg
gaattgtgag cggataacaa tttcacacag gaaacagcgc cgctgagaaa
1620aagcgaagcg gcactgctct ttaacaattt atcagacaat ctgtgtgggc
actcgaccgg 1680aattatcgat taactttatt attaaaaatt aaagaggtat
atattaatgt atcgattaaa 1740taaggaggaa taaaccatgt gtgcgacctc
ttctcaattt actcagatta ccgagcataa 1800ttcccgtcgt tccgcaaact
atcagccaaa cctgtggaat ttcgaattcc tgcaatccct 1860ggagaacgac
ctgaaagtgg aaaagctgga ggagaaagcg accaaactgg aggaagaagt
1920tcgctgcatg atcaaccgtg tagacaccca gccgctgtcc ctgctggagc
tgatcgacga 1980tgtgcagcgc ctgggtctga cctacaaatt tgaaaaagac
atcattaaag ccctggaaaa 2040catcgtactg ctggacgaaa acaaaaagaa
caaatctgac ctgcacgcaa ccgctctgtc 2100tttccgtctg ctgcgtcagc
acggtttcga ggtttctcag gatgtttttg agcgtttcaa 2160ggataaagaa
ggtggtttca gcggtgaact gaaaggtgac gtccaaggcc tgctgagcct
2220gtatgaagcg tcttacctgg gtttcgaggg tgagaacctg ctggaggagg
cgcgtacctt 2280ttccatcacc cacctgaaga acaacctgaa agaaggcatt
aataccaagg ttgcagaaca 2340agtgagccac gccctggaac tgccatatca
ccagcgtctg caccgtctgg aggcacgttg 2400gttcctggat aaatacgaac
cgaaagaacc gcatcaccag ctgctgctgg agctggcgaa 2460gctggatttt
aacatggtac agaccctgca ccagaaagag ctgcaagatc tgtcccgctg
2520gtggaccgag atgggcctgg ctagcaaact ggattttgta cgcgaccgcc
tgatggaagt 2580ttatttctgg gcactgggta tggcgccaga cccgcagttt
ggtgaatgtc gcaaagctgt 2640tactaaaatg tttggtctgg tgacgatcat
cgatgacgtg tatgacgttt atggcactct 2700ggacgaactg caactgttca
ccgatgctgt agagcgctgg gacgttaacg ctattaacac 2760cctgccggac
tatatgaaac tgtgtttcct ggcactgtac aacaccgtta acgacacgtc
2820ctattctatt ctgaaagaga aaggtcataa caacctgtcc tatctgacga
aaagctggcg 2880tgaactgtgc aaagcctttc tgcaagaggc gaaatggtcc
aacaacaaaa ttatcccggc 2940tttctccaag tacctggaaa acgccagcgt
ttcctcctcc ggtgtagcgc tgctggcgcc 3000gtcttacttt tccgtatgcc
agcagcagga agacatctcc gaccacgcgc tgcgttccct 3060gaccgacttc
catggtctgg tgcgttctag ctgcgttatc ttccgcctgt gcaacgatct
3120ggccacctct gcggcggagc tggaacgtgg cgagactacc aattctatca
ttagctacat 3180gcacgaaaac gatggtacca gcgaggaaca ggcccgcgaa
gaactgcgta aactgatcga 3240cgccgaatgg aaaaagatga atcgtgaacg
cgttagcgac tccaccctgc tgcctaaagc 3300gttcatggaa atcgcagtta
acatggcacg tgtttcccac tgcacctacc agtatggcga 3360tggtctgggt
cgcccagact acgcgactga aaaccgcatc aaactgctgc tgattgaccc
3420tttcccgatt aaccagctga tgtatgtcta actgcatcgc ccttaggagg
taaaaaaaaa 3480tgactgccga caacaatagt atgccccatg gtgcagtatc
tagttacgcc aaattagtgc 3540aaaaccaaac acctgaagac attttggaag
agtttcctga aattattcca ttacaacaaa 3600gacctaatac ccgatctagt
gagacgtcaa atgacgaaag cggagaaaca tgtttttctg 3660gtcatgatga
ggagcaaatt aagttaatga atgaaaattg tattgttttg gattgggacg
3720ataatgctat tggtgccggt accaagaaag tttgtcattt aatggaaaat
attgaaaagg 3780gtttactaca tcgtgcattc tccgtcttta ttttcaatga
acaaggtgaa ttacttttac 3840aacaaagagc cactgaaaaa ataactttcc
ctgatctttg gactaacaca tgctgctctc 3900atccactatg tattgatgac
gaattaggtt tgaagggtaa gctagacgat aagattaagg 3960gcgctattac
tgcggcggtg agaaaactag atcatgaatt aggtattcca gaagatgaaa
4020ctaagacaag gggtaagttt cactttttaa acagaatcca ttacatggca
ccaagcaatg 4080aaccatgggg tgaacatgaa attgattaca tcctatttta
taagatcaac gctaaagaaa 4140acttgactgt caacccaaac gtcaatgaag
ttagagactt caaatgggtt tcaccaaatg 4200atttgaaaac tatgtttgct
gacccaagtt acaagtttac gccttggttt aagattattt 4260gcgagaatta
cttattcaac tggtgggagc aattagatga cctttctgaa gtggaaaatg
4320acaggcaaat tcatagaatg ctataacaac gcgtcctgca gctggtacca
tatgggaatt 4380cgaagctttc tagaacaaaa actcatctca gaagaggatc
tgaatagcgc cgtcgaccat 4440catcatcatc atcattgagt ttaaacggtc
tccagcttgg ctgttttggc ggatgagaga 4500agattttcag cctgatacag
attaaatcag aacgcagaag cggtctgata aaacagaatt 4560tgcctggcgg
cagtagcgcg gtggtcccac ctgaccccat gccgaactca gaagtgaaac
4620gccgtagcgc cgatggtagt gtggggtctc cccatgcgag agtagggaac
tgccaggcat 4680caaataaaac gaaaggctca gtcgaaagac tgggcctttc
gttttatctg ttgtttgtcg 4740gtgaacgctc tcctgagtag gacaaatccg
ccgggagcgg atttgaacgt tgcgaagcaa 4800cggcccggag ggtggcgggc
aggacgcccg ccataaactg ccaggcatca aattaagcag 4860aaggccatcc
tgacggatgg cctttttgcg tttctacaaa ctctttttgt ttatttttct
4920aaatacattc aaatatgtat ccgcttaacc ggaattgcca gctggggcgc
cctctggtaa 4980ggttgggaag ccctgcaaag taaactggat ggctttctcg
ccgccaagga tctgatggcg 5040caggggatca agctctgatc aagagacagg
atgaggatcg tttcgcatga ttgaacaaga 5100tggattgcac gcaggttctc
cggccgcttg ggtggagagg ctattcggct atgactgggc 5160acaacagaca
atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc
5220ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcaag
acgaggcagc 5280gcggctatcg tggctggcca cgacgggcgt tccttgcgca
gctgtgctcg acgttgtcac 5340tgaagcggga agggactggc tgctattggg
cgaagtgccg gggcaggatc tcctgtcatc 5400tcaccttgct cctgccgaga
aagtatccat catggctgat gcaatgcggc ggctgcatac 5460gcttgatccg
gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg
5520tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc
atcaggggct 5580cgcgccagcc gaactgttcg ccaggctcaa ggcgagcatg
cccgacggcg aggatctcgt 5640cgtgacccat ggcgatgcct gcttgccgaa
tatcatggtg gaaaatggcc gcttttctgg 5700attcatcgac tgtggccggc
tgggtgtggc ggaccgctat caggacatag cgttggctac 5760ccgtgatatt
gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg
5820tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg
agttcttctg 5880acatgaccaa aatcccttaa cgtgagtttt cgttccactg
agcgtcagac cccgtagaaa 5940agatcaaagg atcttcttga gatccttttt
ttctgcgcgt aatctgctgc ttgcaaacaa 6000aaaaaccacc gctaccagcg
gtggtttgtt tgccggatca agagctacca actctttttc 6060cgaaggtaac
tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt
6120agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
ctgctaatcc 6180tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
taccgggttg gactcaagac 6240gatagttacc ggataaggcg cagcggtcgg
gctgaacggg gggttcgtgc acacagccca 6300gcttggagcg aacgacctac
accgaactga gatacctaca gcgtgagcta tgagaaagcg 6360ccacgcttcc
cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
6420gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
cctgtcgggt 6480ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
gtcagggggg cggagcctat 6540ggaaaaacgc cagcaacgcg gcctttttac
ggttcctggc cttttgctgg ccttttgctc 6600acatgttctt tcctgcgtta
tcccctgatt ctgtggataa ccgtattacc gcctttgagt 6660gagctgatac
cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
6720cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
tcacaccgca 6780tatggtgcac tctcagtaca atctgctctg atgccgcata
gttaagccag tatacactcc 6840gctatcgcta cgtgactggg tcatggctgc
gccccgacac ccgccaacac ccgctgacgc 6900gccctgacgg gc
6912247902DNAArtificial SequenceSynthetic Construct 24ttgtctgctc
ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg 60tcagaggttt
tcaccgtcat caccgaaacg cgcgaggcag cagatcaatt cgcgcgcgaa
120ggcgaagcgg catgcattta cgttgacacc atcgaatggt gcaaaacctt
tcgcggtatg 180gcatgatagc gcccggaaga gagtcaattc agggtggtga
atgtgaaacc agtaacgtta 240tacgatgtcg cagagtatgc cggtgtctct
tatcagaccg tttcccgcgt ggtgaaccag 300gccagccacg tttctgcgaa
aacgcgggaa aaagtggaag cggcgatggc ggagctgaat 360tacattccca
accgcgtggc acaacaactg gcgggcaaac agtcgttgct gattggcgtt
420gccacctcca gtctggccct gcacgcgccg tcgcaaattg tcgcggcgat
taaatctcgc 480gccgatcaac tgggtgccag cgtggtggtg tcgatggtag
aacgaagcgg cgtcgaagcc 540tgtaaagcgg cggtgcacaa tcttctcgcg
caacgcgtca gtgggctgat cattaactat 600ccgctggatg accaggatgc
cattgctgtg gaagctgcct gcactaatgt tccggcgtta 660tttcttgatg
tctctgacca gacacccatc aacagtatta ttttctccca tgaagacggt
720acgcgactgg gcgtggagca tctggtcgca ttgggtcacc agcaaatcgc
gctgttagcg 780ggcccattaa gttctgtctc ggcgcgtctg cgtctggctg
gctggcataa atatctcact 840cgcaatcaaa ttcagccgat agcggaacgg
gaaggcgact ggagtgccat gtccggtttt 900caacaaacca tgcaaatgct
gaatgagggc atcgttccca ctgcgatgct ggttgccaac 960gatcagatgg
cgctgggcgc aatgcgcgcc attaccgagt ccgggctgcg cgttggtgcg
1020gatatctcgg tagtgggata cgacgatacc gaagacagct catgttatat
cccgccgtca 1080accaccatca aacaggattt tcgcctgctg gggcaaacca
gcgtggaccg cttgctgcaa 1140ctctctcagg gccaggcggt gaagggcaat
cagctgttgc ccgtctcact ggtgaaaaga 1200aaaaccaccc tggcgcccaa
tacgcaaacc gcctctcccc gcgcgttggc cgattcatta 1260atgcagctgg
cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa
1320tgtgagttag cgcgaattga tctggtttga cagcttatca tcgactgcac
ggtgcaccaa 1380tgcttctggc gtcaggcagc catcggaagc tgtggtatgg
ctgtgcaggt cgtaaatcac 1440tgcataattc gtgtcgctca aggcgcactc
ccgttctgga taatgttttt tgcgccgaca 1500tcataacggt tctggcaaat
attctgaaat gagctgttga caattaatca tccggctcgt 1560ataatgtgtg
gaattgtgag cggataacaa tttcacacag gaaacagcgc cgctgagaaa
1620aagcgaagcg gcactgctct ttaacaattt atcagacaat ctgtgtgggc
actcgaccgg 1680aattatcgat taactttatt attaaaaatt aaagaggtat
atattaatgt atcgattaaa 1740taaggaggaa taaaccatgt gtgcgacctc
ttctcaattt actcagatta ccgagcataa 1800ttcccgtcgt tccgcaaact
atcagccaaa cctgtggaat ttcgaattcc tgcaatccct 1860ggagaacgac
ctgaaagtgg aaaagctgga ggagaaagcg accaaactgg aggaagaagt
1920tcgctgcatg atcaaccgtg tagacaccca gccgctgtcc ctgctggagc
tgatcgacga 1980tgtgcagcgc ctgggtctga cctacaaatt tgaaaaagac
atcattaaag ccctggaaaa 2040catcgtactg ctggacgaaa acaaaaagaa
caaatctgac ctgcacgcaa ccgctctgtc 2100tttccgtctg ctgcgtcagc
acggtttcga ggtttctcag gatgtttttg agcgtttcaa 2160ggataaagaa
ggtggtttca gcggtgaact gaaaggtgac gtccaaggcc tgctgagcct
2220gtatgaagcg tcttacctgg gtttcgaggg tgagaacctg ctggaggagg
cgcgtacctt 2280ttccatcacc cacctgaaga acaacctgaa agaaggcatt
aataccaagg ttgcagaaca 2340agtgagccac gccctggaac tgccatatca
ccagcgtctg caccgtctgg aggcacgttg 2400gttcctggat aaatacgaac
cgaaagaacc gcatcaccag ctgctgctgg agctggcgaa 2460gctggatttt
aacatggtac agaccctgca ccagaaagag ctgcaagatc tgtcccgctg
2520gtggaccgag atgggcctgg ctagcaaact ggattttgta cgcgaccgcc
tgatggaagt 2580ttatttctgg gcactgggta tggcgccaga cccgcagttt
ggtgaatgtc gcaaagctgt 2640tactaaaatg tttggtctgg tgacgatcat
cgatgacgtg tatgacgttt atggcactct 2700ggacgaactg caactgttca
ccgatgctgt agagcgctgg gacgttaacg ctattaacac 2760cctgccggac
tatatgaaac tgtgtttcct ggcactgtac aacaccgtta acgacacgtc
2820ctattctatt ctgaaagaga aaggtcataa caacctgtcc tatctgacga
aaagctggcg 2880tgaactgtgc aaagcctttc tgcaagaggc gaaatggtcc
aacaacaaaa ttatcccggc 2940tttctccaag tacctggaaa acgccagcgt
ttcctcctcc ggtgtagcgc tgctggcgcc 3000gtcttacttt tccgtatgcc
agcagcagga agacatctcc gaccacgcgc tgcgttccct 3060gaccgacttc
catggtctgg tgcgttctag ctgcgttatc ttccgcctgt gcaacgatct
3120ggccacctct gcggcggagc tggaacgtgg cgagactacc aattctatca
ttagctacat 3180gcacgaaaac gatggtacca gcgaggaaca ggcccgcgaa
gaactgcgta aactgatcga 3240cgccgaatgg aaaaagatga atcgtgaacg
cgttagcgac tccaccctgc tgcctaaagc 3300gttcatggaa atcgcagtta
acatggcacg tgtttcccac tgcacctacc agtatggcga 3360tggtctgggt
cgcccagact acgcgactga aaaccgcatc aaactgctgc tgattgaccc
3420tttcccgatt aaccagctga tgtatgtcta actgcattcg cccttaggag
gtaaaaaaac 3480atgagttttg atattgccaa atacccgacc ctggcactgg
tcgactccac ccaggagtta 3540cgactgttgc cgaaagagag tttaccgaaa
ctctgcgacg aactgcgccg ctatttactc 3600gacagcgtga gccgttccag
cgggcacttc gcctccgggc tgggcacggt cgaactgacc 3660gtggcgctgc
actatgtcta caacaccccg tttgaccaat tgatttggga tgtggggcat
3720caggcttatc cgcataaaat tttgaccgga cgccgcgaca aaatcggcac
catccgtcag 3780aaaggcggtc tgcacccgtt cccgtggcgc ggcgaaagcg
aatatgacgt attaagcgtc 3840gggcattcat caacctccat cagtgccgga
attggtattg cggttgctgc cgaaaaagaa 3900ggcaaaaatc gccgcaccgt
ctgtgtcatt ggcgatggcg cgattaccgc aggcatggcg 3960tttgaagcga
tgaatcacgc gggcgatatc cgtcctgata tgctggtgat tctcaacgac
4020aatgaaatgt cgatttccga aaatgtcggc gcgctcaaca accatctggc
acagctgctt 4080tccggtaagc tttactcttc actgcgcgaa ggcgggaaaa
aagttttctc tggcgtgccg 4140ccaattaaag agctgctcaa acgcaccgaa
gaacatatta aaggcatggt agtgcctggc 4200acgttgtttg aagagctggg
ctttaactac atcggcccgg tggacggtca cgatgtgctg 4260gggcttatca
ccacgctaaa gaacatgcgc gacctgaaag gcccgcagtt cctgcatatc
4320atgaccaaaa aaggtcgtgg ttatgaaccg gcagaaaaag acccgatcac
tttccacgcc 4380gtgcctaaat ttgatccctc cagcggttgt ttgccgaaaa
gtagcggcgg tttgccgagc 4440tattcaaaaa tctttggcga ctggttgtgc
gaaacggcag cgaaagacaa caagctgatg 4500gcgattactc cggcgatgcg
tgaaggttcc ggcatggtcg agttttcacg taaattcccg 4560gatcgctact
tcgacgtggc aattgccgag caacacgcgg tgacctttgc tgcgggtctg
4620gcgattggtg ggtacaaacc cattgtcgcg atttactcca ctttcctgca
acgcgcctat 4680gatcaggtgc tgcatgacgt ggcgattcaa aagcttccgg
tcctgttcgc catcgaccgc 4740gcgggcattg ttggtgctga cggtcaaacc
catcagggtg cttttgatct ctcttacctg 4800cgctgcatac cggaaatggt
cattatgacc ccgagcgatg aaaacgaatg tcgccagatg 4860ctctataccg
gctatcacta taacgatggc ccgtcagcgg tgcgctaccc gcgtggcaac
4920gcggtcggcg tggaactgac gccgctggaa aaactaccaa ttggcaaagg
cattgtgaag 4980cgtcgtggcg agaaactggc gatccttaac tttggtacgc
tgatgccaga agcggcgaaa 5040gtcgccgaat cgctgaacgc cacgctggtc
gatatgcgtt ttgtgaaacc gcttgatgaa 5100gcgttaattc tggaaatggc
cgccagccat gaagcgctgg tcaccgtaga agaaaacgcc 5160attatgggcg
gcgcaggcag cggcgtgaac gaagtgctga tggcccatcg taaaccagta
5220cccgtgctga acattggcct gccggacttc tttattccgc aaggaactca
ggaagaaatg 5280cgcgccgaac tcggcctcga tgccgctggt atggaagcca
aaatcaaggc ctggctggca 5340taactgcagc tggtaccata tgggaattcg
aagctttcta gaacaaaaac tcatctcaga 5400agaggatctg aatagcgccg
tcgaccatca tcatcatcat cattgagttt aaacggtctc 5460cagcttggct
gttttggcgg atgagagaag attttcagcc tgatacagat taaatcagaa
5520cgcagaagcg gtctgataaa acagaatttg cctggcggca gtagcgcggt
ggtcccacct 5580gaccccatgc cgaactcaga agtgaaacgc cgtagcgccg
atggtagtgt ggggtctccc 5640catgcgagag tagggaactg ccaggcatca
aataaaacga aaggctcagt cgaaagactg 5700ggcctttcgt tttatctgtt
gtttgtcggt gaacgctctc ctgagtagga caaatccgcc 5760gggagcggat
ttgaacgttg cgaagcaacg gcccggaggg tggcgggcag gacgcccgcc
5820ataaactgcc aggcatcaaa ttaagcagaa ggccatcctg acggatggcc
tttttgcgtt 5880tctacaaact ctttttgttt atttttctaa atacattcaa
atatgtatcc gcttaaccgg 5940aattgccagc tggggcgccc tctggtaagg
ttgggaagcc ctgcaaagta aactggatgg 6000ctttctcgcc gccaaggatc
tgatggcgca ggggatcaag ctctgatcaa gagacaggat 6060gaggatcgtt
tcgcatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg
6120tggagaggct attcggctat gactgggcac aacagacaat cggctgctct
gatgccgccg 6180tgttccggct gtcagcgcag gggcgcccgg ttctttttgt
caagaccgac ctgtccggtg 6240ccctgaatga actgcaagac gaggcagcgc
ggctatcgtg gctggccacg acgggcgttc 6300cttgcgcagc tgtgctcgac
gttgtcactg aagcgggaag ggactggctg ctattgggcg 6360aagtgccggg
gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca
6420tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca
ttcgaccacc 6480aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga
agccggtctt gtcgatcagg 6540atgatctgga cgaagagcat caggggctcg
cgccagccga actgttcgcc aggctcaagg 6600cgagcatgcc cgacggcgag
gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata 6660tcatggtgga
aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg
6720accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt
ggcggcgaat 6780gggctgaccg cttcctcgtg ctttacggta tcgccgctcc
cgattcgcag cgcatcgcct 6840tctatcgcct tcttgacgag ttcttctgac
gcatgaccaa aatcccttaa cgtgagtttt 6900cgttccactg agcgtcagac
cccgtagaaa agatcaaagg atcttcttga gatccttttt 6960ttctgcgcgt
aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
7020tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
agagcgcaga 7080taccaaatac tgtccttcta gtgtagccgt agttaggcca
ccacttcaag aactctgtag 7140caccgcctac atacctcgct ctgctaatcc
tgttaccagt ggctgctgcc agtggcgata 7200agtcgtgtct taccgggttg
gactcaagac gatagttacc ggataaggcg cagcggtcgg 7260gctgaacggg
gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
7320gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
aaggcggaca 7380ggtatccggt aagcggcagg gtcggaacag gagagcgcac
gagggagctt ccagggggaa 7440acgcctggta tctttatagt cctgtcgggt
ttcgccacct ctgacttgag cgtcgatttt 7500tgtgatgctc gtcagggggg
cggagcctat ggaaaaacgc cagcaacgcg gcctttttac 7560ggttcctggc
cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
7620ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
agccgaacga 7680ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg
cctgatgcgg tattttctcc 7740ttacgcatct gtgcggtatt tcacaccgca
tatggtgcac tctcagtaca atctgctctg 7800atgccgcata gttaagccag
tatacactcc gctatcgcta cgtgactggg tcatggctgc 7860gccccgacac
ccgccaacac ccgctgacgc gccctgacgg gc 7902256783DNAArtificial
SequenceSynthetic Construct 25ctggcgtaat agcgaagagg cccgcaccga
tcgcccttcc caacagttgc gcagcctgaa 60tggcgaatgg cgcctgatgc ggtattttct
ccttacgcat ctgtgcggta tttcacaccg 120catatggtgc actctcagta
caatctgctc tgatgccgca tagttaagcc agccccgaca 180cccgccaaca
cccgctgacg agcttagtaa agccctcgct agattttaat gcggatgttg
240cgattacttc gccaactatt gcgataacaa gaaaaagcca gcctttcatg
atatatctcc 300caatttgtgt agggcttatt atgcacgctt aaaaataata
aaagcagact tgacctgata 360gtttggctgt gagcaattat gtgcttagtg
catctaacgc ttgagttaag ccgcgccgcg 420aagcggcgtc ggcttgaacg
aattgttaga cattatttgc cgactacctt ggtgatctcg 480cctttcacgt
agtggacaaa ttcttccaac tgatctgcgc gcgaggccaa gcgatcttct
540tcttgtccaa gataagcctg tctagcttca agtatgacgg gctgatactg
ggccggcagg 600cgctccattg cccagtcggc agcgacatcc ttcggcgcga
ttttgccggt tactgcgctg 660taccaaatgc gggacaacgt aagcactaca
tttcgctcat cgccagccca gtcgggcggc 720gagttccata gcgttaaggt
ttcatttagc gcctcaaata gatcctgttc aggaaccgga 780tcaaagagtt
cctccgccgc tggacctacc aaggcaacgc tatgttctct tgcttttgtc
840agcaagatag ccagatcaat gtcgatcgtg gctggctcga agatacctgc
aagaatgtca 900ttgcgctgcc attctccaaa ttgcagttcg cgcttagctg
gataacgcca cggaatgatg 960tcgtcgtgca caacaatggt gacttctaca
gcgcggagaa tctcgctctc tccaggggaa 1020gccgaagttt ccaaaaggtc
gttgatcaaa gctcgccgcg ttgtttcatc aagccttacg 1080gtcaccgtaa
ccagcaaatc aatatcactg tgtggcttca ggccgccatc cactgcggag
1140ccgtacaaat gtacggccag caacgtcggt tcgagatggc gctcgatgac
gccaactacc 1200tctgatagtt gagtcgatac ttcggcgatc accgcttccc
tcatgatgtt taactttgtt 1260ttagggcgac tgccctgctg cgtaacatcg
ttgctgctcc ataacatcaa acatcgaccc 1320acggcgtaac gcgcttgctg
cttggatgcc cgaggcatag actgtacccc aaaaaaacag 1380tcataacaag
ccatgaaaac cgccactgcg ccgttaccac cgctgcgttc ggtcaaggtt
1440ctggaccagt tgcgtgagcg catacgctac ttgcattaca gcttacgaac
cgaacaggct 1500tatgtccact gggttcgtgc cttcatccgt ttccacggtg
tgcgtcaccc ggcaaccttg 1560ggcagcagcg aagtcgaggc atttctgtcc
tggctggcga acgagcgcaa ggtttcggtc 1620tccacgcatc gtcaggcatt
ggcggccttg ctgttcttct acggcaaggt gctgtgcacg 1680gatctgccct
ggcttcagga gatcggaaga cctcggccgt cgcggcgctt gccggtggtg
1740ctgaccccgg atgaagtggt tcgcatcctc ggttttctgg aaggcgagca
tcgtttgttc 1800gcccagcttc tgtatggaac gggcatgcgg atcagtgagg
gtttgcaact gcgggtcaag 1860gatctggatt tcgatcacgg cacgatcatc
gtgcgggagg gcaagggctc caaggatcgg 1920gccttgatgt tacccgagag
cttggcaccc agcctgcgcg agcaggggaa ttaattccca 1980cgggttttgc
tgcccgcaaa cgggctgttc tggtgttgct agtttgttat cagaatcgca
2040gatccggctt cagccggttt gccggctgaa agcgctattt cttccagaat
tgccatgatt 2100ttttccccac gggaggcgtc actggctccc gtgttgtcgg
cagctttgat tcgataagca 2160gcatcgcctg tttcaggctg tctatgtgtg
actgttgagc tgtaacaagt tgtctcaggt 2220gttcaatttc atgttctagt
tgctttgttt tactggtttc acctgttcta ttaggtgtta 2280catgctgttc
atctgttaca ttgtcgatct gttcatggtg aacagctttg aatgcaccaa
2340aaactcgtaa aagctctgat gtatctatct tttttacacc gttttcatct
gtgcatatgg 2400acagttttcc ctttgatatg taacggtgaa cagttgttct
acttttgttt gttagtcttg 2460atgcttcact gatagataca agagccataa
gaacctcaga tccttccgta tttagccagt 2520atgttctcta gtgtggttcg
ttgtttttgc gtgagccatg agaacgaacc attgagatca 2580tacttacttt
gcatgtcact caaaaatttt gcctcaaaac tggtgagctg aatttttgca
2640gttaaagcat cgtgtagtgt ttttcttagt ccgttatgta ggtaggaatc
tgatgtaatg 2700gttgttggta ttttgtcacc attcattttt atctggttgt
tctcaagttc ggttacgaga 2760tccatttgtc tatctagttc aacttggaaa
atcaacgtat cagtcgggcg gcctcgctta 2820tcaaccacca atttcatatt
gctgtaagtg tttaaatctt tacttattgg tttcaaaacc 2880cattggttaa
gccttttaaa ctcatggtag ttattttcaa gcattaacat gaacttaaat
2940tcatcaaggc taatctctat atttgccttg tgagttttct tttgtgttag
ttcttttaat 3000aaccactcat aaatcctcat agagtatttg ttttcaaaag
acttaacatg ttccagatta 3060tattttatga atttttttaa ctggaaaaga
taaggcaata tctcttcact aaaaactaat 3120tctaattttt cgcttgagaa
cttggcatag tttgtccact ggaaaatctc aaagccttta 3180accaaaggat
tcctgatttc cacagttctc gtcatcagct ctctggttgc tttagctaat
3240acaccataag cattttccct actgatgttc atcatctgag cgtattggtt
ataagtgaac 3300gataccgtcc gttctttcct tgtagggttt tcaatcgtgg
ggttgagtag tgccacacag 3360cataaaatta gcttggtttc atgctccgtt
aagtcatagc gactaatcgc tagttcattt 3420gctttgaaaa caactaattc
agacatacat ctcaattggt ctaggtgatt ttaatcacta 3480taccaattga
gatgggctag tcaatgataa ttactagtcc ttttcctttg agttgtgggt
3540atctgtaaat tctgctagac ctttgctgga aaacttgtaa attctgctag
accctctgta 3600aattccgcta gacctttgtg tgtttttttt gtttatattc
aagtggttat aatttataga 3660ataaagaaag aataaaaaaa gataaaaaga
atagatccca gccctgtgta taactcacta 3720ctttagtcag ttccgcagta
ttacaaaagg atgtcgcaaa cgctgtttgc tcctctacaa 3780aacagacctt
aaaaccctaa aggcttaagt agcaccctcg caagctcggg caaatcgctg
3840aatattcctt ttgtctccga ccatcaggca cctgagtcgc tgtctttttc
gtgacattca 3900gttcgctgcg ctcacggctc tggcagtgaa tgggggtaaa
tggcactaca ggcgcctttt 3960atggattcat gcaaggaaac tacccataat
acaagaaaag cccgtcacgg gcttctcagg 4020gcgttttatg gcgggtctgc
tatgtggtgc tatctgactt tttgctgttc agcagttcct 4080gccctctgat
tttccagtct gaccacttcg gattatcccg tgacaggtca ttcagactgg
4140ctaatgcacc cagtaaggca gcggtatcat caacaggctt acccgtctta
ctgtcgggaa 4200ttcgcgttgg ccgattcatt aatgcagatt ctgaaatgag
ctgttgacaa ttaatcatcc 4260ggctcgtata atgtgtggaa ttgtgagcgg
ataacaattt cacacaggaa acagcgccgc 4320tgagaaaaag
cgaagcggca ctgctcttta acaatttatc agacaatctg tgtgggcact
4380cgaccggaat tatcgattaa ctttattatt aaaaattaaa gaggtatata
ttaatgtatc 4440gattaaataa ggaggaataa accatgtgtg cgacctcttc
tcaatttact cagattaccg 4500agcataattc ccgtcgttcc gcaaactatc
agccaaacct gtggaatttc gaattcctgc 4560aatccctgga gaacgacctg
aaagtggaaa agctggagga gaaagcgacc aaactggagg 4620aagaagttcg
ctgcatgatc aaccgtgtag acacccagcc gctgtccctg ctggagctga
4680tcgacgatgt gcagcgcctg ggtctgacct acaaatttga aaaagacatc
attaaagccc 4740tggaaaacat cgtactgctg gacgaaaaca aaaagaacaa
atctgacctg cacgcaaccg 4800ctctgtcttt ccgtctgctg cgtcagcacg
gtttcgaggt ttctcaggat gtttttgagc 4860gtttcaagga taaagaaggt
ggtttcagcg gtgaactgaa aggtgacgtc caaggcctgc 4920tgagcctgta
tgaagcgtct tacctgggtt tcgagggtga gaacctgctg gaggaggcgc
4980gtaccttttc catcacccac ctgaagaaca acctgaaaga aggcattaat
accaaggttg 5040cagaacaagt gagccacgcc ctggaactgc catatcacca
gcgtctgcac cgtctggagg 5100cacgttggtt cctggataaa tacgaaccga
aagaaccgca tcaccagctg ctgctggagc 5160tggcgaagct ggattttaac
atggtacaga ccctgcacca gaaagagctg caagatctgt 5220cccgctggtg
gaccgagatg ggcctggcta gcaaactgga ttttgtacgc gaccgcctga
5280tggaagttta tttctgggca ctgggtatgg cgccagaccc gcagtttggt
gaatgtcgca 5340aagctgttac taaaatgttt ggtctggtga cgatcatcga
tgacgtgtat gacgtttatg 5400gcactctgga cgaactgcaa ctgttcaccg
atgctgtaga gcgctgggac gttaacgcta 5460ttaacaccct gccggactat
atgaaactgt gtttcctggc actgtacaac accgttaacg 5520acacgtccta
ttctattctg aaagagaaag gtcataacaa cctgtcctat ctgacgaaaa
5580gctggcgtga actgtgcaaa gcctttctgc aagaggcgaa atggtccaac
aacaaaatta 5640tcccggcttt ctccaagtac ctggaaaacg ccagcgtttc
ctcctccggt gtagcgctgc 5700tggcgccgtc ttacttttcc gtatgccagc
agcaggaaga catctccgac cacgcgctgc 5760gttccctgac cgacttccat
ggtctggtgc gttctagctg cgttatcttc cgcctgtgca 5820acgatctggc
cacctctgcg gcggagctgg aacgtggcga gactaccaat tctatcatta
5880gctacatgca cgaaaacgat ggtaccagcg aggaacaggc ccgcgaagaa
ctgcgtaaac 5940tgatcgacgc cgaatggaaa aagatgaatc gtgaacgcgt
tagcgactcc accctgctgc 6000ctaaagcgtt catggaaatc gcagttaaca
tggcacgtgt ttcccactgc acctaccagt 6060atggcgatgg tctgggtcgc
ccagactacg cgactgaaaa ccgcatcaaa ctgctgctga 6120ttgacccttt
cccgattaac cagctgatgt atgtctaact gcagctggta ccatatggga
6180attcgaagct ttctagaaca aaaactcatc tcagaagagg atctgaatag
cgccgtcgac 6240catcatcatc atcatcattg agtttaaacg gtctccagct
tggctgtttt ggcggatgag 6300agaagatttt cagcctgata cagattaaat
cagaacgcag aagcggtctg ataaaacaga 6360atttgcctgg cggcagtagc
gcggtggtcc cacctgaccc catgccgaac tcagaagtga 6420aacgccgtag
cgccgatggt agtgtggggt ctccccatgc gagagtaggg aactgccagg
6480catcaaataa aacgaaaggc tcagtcgaaa gactgggcct ttcgttttat
ctgttgtttg 6540tcggtgaacg ctctcctgag taggacaaat ccgccgggag
cggatttgaa cgttgcgaag 6600caacggcccg gagggtggcg ggcaggacgc
ccgccataaa ctgccaggca tcaaattaag 6660cagaaggcca tcctgacgga
tggccttttt gcgtttctac aaactctttt tgtttatttt 6720tctaaataca
ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat 6780aat
6783266783DNAArtificial SequenceSynthetic Construct 26cccgtcttac
tgtcgggaat tcgcgttggc cgattcatta atgcagatta ttgaagcatt 60tatcagggtt
attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
120aaagagtttg tagaaacgca aaaaggccat ccgtcaggat ggccttctgc
ttaatttgat 180gcctggcagt ttatggcggg cgtcctgccc gccaccctcc
gggccgttgc ttcgcaacgt 240tcaaatccgc tcccggcgga tttgtcctac
tcaggagagc gttcaccgac aaacaacaga 300taaaacgaaa ggcccagtct
ttcgactgag cctttcgttt tatttgatgc ctggcagttc 360cctactctcg
catggggaga ccccacacta ccatcggcgc tacggcgttt cacttctgag
420ttcggcatgg ggtcaggtgg gaccaccgcg ctactgccgc caggcaaatt
ctgttttatc 480agaccgcttc tgcgttctga tttaatctgt atcaggctga
aaatcttctc tcatccgcca 540aaacagccaa gctggagacc gtttaaactc
aatgatgatg atgatgatgg tcgacggcgc 600tattcagatc ctcttctgag
atgagttttt gttctagaaa gcttcgaatt cccatatggt 660accagctgca
gttagacata catcagctgg ttaatcggga aagggtcaat cagcagcagt
720ttgatgcggt tttcagtcgc gtagtctggg cgacccagac catcgccata
ctggtaggtg 780cagtgggaaa cacgtgccat gttaactgcg atttccatga
acgctttagg cagcagggtg 840gagtcgctaa cgcgttcacg attcatcttt
ttccattcgg cgtcgatcag tttacgcagt 900tcttcgcggg cctgttcctc
gctggtacca tcgttttcgt gcatgtagct aatgatagaa 960ttggtagtct
cgccacgttc cagctccgcc gcagaggtgg ccagatcgtt gcacaggcgg
1020aagataacgc agctagaacg caccagacca tggaagtcgg tcagggaacg
cagcgcgtgg 1080tcggagatgt cttcctgctg ctggcatacg gaaaagtaag
acggcgccag cagcgctaca 1140ccggaggagg aaacgctggc gttttccagg
tacttggaga aagccgggat aattttgttg 1200ttggaccatt tcgcctcttg
cagaaaggct ttgcacagtt cacgccagct tttcgtcaga 1260taggacaggt
tgttatgacc tttctctttc agaatagaat aggacgtgtc gttaacggtg
1320ttgtacagtg ccaggaaaca cagtttcata tagtccggca gggtgttaat
agcgttaacg 1380tcccagcgct ctacagcatc ggtgaacagt tgcagttcgt
ccagagtgcc ataaacgtca 1440tacacgtcat cgatgatcgt caccagacca
aacattttag taacagcttt gcgacattca 1500ccaaactgcg ggtctggcgc
catacccagt gcccagaaat aaacttccat caggcggtcg 1560cgtacaaaat
ccagtttgct agccaggccc atctcggtcc accagcggga cagatcttgc
1620agctctttct ggtgcagggt ctgtaccatg ttaaaatcca gcttcgccag
ctccagcagc 1680agctggtgat gcggttcttt cggttcgtat ttatccagga
accaacgtgc ctccagacgg 1740tgcagacgct ggtgatatgg cagttccagg
gcgtggctca cttgttctgc aaccttggta 1800ttaatgcctt ctttcaggtt
gttcttcagg tgggtgatgg aaaaggtacg cgcctcctcc 1860agcaggttct
caccctcgaa acccaggtaa gacgcttcat acaggctcag caggccttgg
1920acgtcacctt tcagttcacc gctgaaacca ccttctttat ccttgaaacg
ctcaaaaaca 1980tcctgagaaa cctcgaaacc gtgctgacgc agcagacgga
aagacagagc ggttgcgtgc 2040aggtcagatt tgttcttttt gttttcgtcc
agcagtacga tgttttccag ggctttaatg 2100atgtcttttt caaatttgta
ggtcagaccc aggcgctgca catcgtcgat cagctccagc 2160agggacagcg
gctgggtgtc tacacggttg atcatgcagc gaacttcttc ctccagtttg
2220gtcgctttct cctccagctt ttccactttc aggtcgttct ccagggattg
caggaattcg 2280aaattccaca ggtttggctg atagtttgcg gaacgacggg
aattatgctc ggtaatctga 2340gtaaattgag aagaggtcgc acacatggtt
tattcctcct tatttaatcg atacattaat 2400atatacctct ttaattttta
ataataaagt taatcgataa ttccggtcga gtgcccacac 2460agattgtctg
ataaattgtt aaagagcagt gccgcttcgc tttttctcag cggcgctgtt
2520tcctgtgtga aattgttatc cgctcacaat tccacacatt atacgagccg
gatgattaat 2580tgtcaacagc tcatttcaga atctggcgta atagcgaaga
ggcccgcacc gatcgccctt 2640cccaacagtt gcgcagcctg aatggcgaat
ggcgcctgat gcggtatttt ctccttacgc 2700atctgtgcgg tatttcacac
cgcatatggt gcactctcag tacaatctgc tctgatgccg 2760catagttaag
ccagccccga cacccgccaa cacccgctga cgagcttagt aaagccctcg
2820ctagatttta atgcggatgt tgcgattact tcgccaacta ttgcgataac
aagaaaaagc 2880cagcctttca tgatatatct cccaatttgt gtagggctta
ttatgcacgc ttaaaaataa 2940taaaagcaga cttgacctga tagtttggct
gtgagcaatt atgtgcttag tgcatctaac 3000gcttgagtta agccgcgccg
cgaagcggcg tcggcttgaa cgaattgtta gacattattt 3060gccgactacc
ttggtgatct cgcctttcac gtagtggaca aattcttcca actgatctgc
3120gcgcgaggcc aagcgatctt cttcttgtcc aagataagcc tgtctagctt
caagtatgac 3180gggctgatac tgggccggca ggcgctccat tgcccagtcg
gcagcgacat ccttcggcgc 3240gattttgccg gttactgcgc tgtaccaaat
gcgggacaac gtaagcacta catttcgctc 3300atcgccagcc cagtcgggcg
gcgagttcca tagcgttaag gtttcattta gcgcctcaaa 3360tagatcctgt
tcaggaaccg gatcaaagag ttcctccgcc gctggaccta ccaaggcaac
3420gctatgttct cttgcttttg tcagcaagat agccagatca atgtcgatcg
tggctggctc 3480gaagatacct gcaagaatgt cattgcgctg ccattctcca
aattgcagtt cgcgcttagc 3540tggataacgc cacggaatga tgtcgtcgtg
cacaacaatg gtgacttcta cagcgcggag 3600aatctcgctc tctccagggg
aagccgaagt ttccaaaagg tcgttgatca aagctcgccg 3660cgttgtttca
tcaagcctta cggtcaccgt aaccagcaaa tcaatatcac tgtgtggctt
3720caggccgcca tccactgcgg agccgtacaa atgtacggcc agcaacgtcg
gttcgagatg 3780gcgctcgatg acgccaacta cctctgatag ttgagtcgat
acttcggcga tcaccgcttc 3840cctcatgatg tttaactttg ttttagggcg
actgccctgc tgcgtaacat cgttgctgct 3900ccataacatc aaacatcgac
ccacggcgta acgcgcttgc tgcttggatg cccgaggcat 3960agactgtacc
ccaaaaaaac agtcataaca agccatgaaa accgccactg cgccgttacc
4020accgctgcgt tcggtcaagg ttctggacca gttgcgtgag cgcatacgct
acttgcatta 4080cagcttacga accgaacagg cttatgtcca ctgggttcgt
gccttcatcc gtttccacgg 4140tgtgcgtcac ccggcaacct tgggcagcag
cgaagtcgag gcatttctgt cctggctggc 4200gaacgagcgc aaggtttcgg
tctccacgca tcgtcaggca ttggcggcct tgctgttctt 4260ctacggcaag
gtgctgtgca cggatctgcc ctggcttcag gagatcggaa gacctcggcc
4320gtcgcggcgc ttgccggtgg tgctgacccc ggatgaagtg gttcgcatcc
tcggttttct 4380ggaaggcgag catcgtttgt tcgcccagct tctgtatgga
acgggcatgc ggatcagtga 4440gggtttgcaa ctgcgggtca aggatctgga
tttcgatcac ggcacgatca tcgtgcggga 4500gggcaagggc tccaaggatc
gggccttgat gttacccgag agcttggcac ccagcctgcg 4560cgagcagggg
aattaattcc cacgggtttt gctgcccgca aacgggctgt tctggtgttg
4620ctagtttgtt atcagaatcg cagatccggc ttcagccggt ttgccggctg
aaagcgctat 4680ttcttccaga attgccatga ttttttcccc acgggaggcg
tcactggctc ccgtgttgtc 4740ggcagctttg attcgataag cagcatcgcc
tgtttcaggc tgtctatgtg tgactgttga 4800gctgtaacaa gttgtctcag
gtgttcaatt tcatgttcta gttgctttgt tttactggtt 4860tcacctgttc
tattaggtgt tacatgctgt tcatctgtta cattgtcgat ctgttcatgg
4920tgaacagctt tgaatgcacc aaaaactcgt aaaagctctg atgtatctat
cttttttaca 4980ccgttttcat ctgtgcatat ggacagtttt ccctttgata
tgtaacggtg aacagttgtt 5040ctacttttgt ttgttagtct tgatgcttca
ctgatagata caagagccat aagaacctca 5100gatccttccg tatttagcca
gtatgttctc tagtgtggtt cgttgttttt gcgtgagcca 5160tgagaacgaa
ccattgagat catacttact ttgcatgtca ctcaaaaatt ttgcctcaaa
5220actggtgagc tgaatttttg cagttaaagc atcgtgtagt gtttttctta
gtccgttatg 5280taggtaggaa tctgatgtaa tggttgttgg tattttgtca
ccattcattt ttatctggtt 5340gttctcaagt tcggttacga gatccatttg
tctatctagt tcaacttgga aaatcaacgt 5400atcagtcggg cggcctcgct
tatcaaccac caatttcata ttgctgtaag tgtttaaatc 5460tttacttatt
ggtttcaaaa cccattggtt aagcctttta aactcatggt agttattttc
5520aagcattaac atgaacttaa attcatcaag gctaatctct atatttgcct
tgtgagtttt 5580cttttgtgtt agttctttta ataaccactc ataaatcctc
atagagtatt tgttttcaaa 5640agacttaaca tgttccagat tatattttat
gaattttttt aactggaaaa gataaggcaa 5700tatctcttca ctaaaaacta
attctaattt ttcgcttgag aacttggcat agtttgtcca 5760ctggaaaatc
tcaaagcctt taaccaaagg attcctgatt tccacagttc tcgtcatcag
5820ctctctggtt gctttagcta atacaccata agcattttcc ctactgatgt
tcatcatctg 5880agcgtattgg ttataagtga acgataccgt ccgttctttc
cttgtagggt tttcaatcgt 5940ggggttgagt agtgccacac agcataaaat
tagcttggtt tcatgctccg ttaagtcata 6000gcgactaatc gctagttcat
ttgctttgaa aacaactaat tcagacatac atctcaattg 6060gtctaggtga
ttttaatcac tataccaatt gagatgggct agtcaatgat aattactagt
6120ccttttcctt tgagttgtgg gtatctgtaa attctgctag acctttgctg
gaaaacttgt 6180aaattctgct agaccctctg taaattccgc tagacctttg
tgtgtttttt ttgtttatat 6240tcaagtggtt ataatttata gaataaagaa
agaataaaaa aagataaaaa gaatagatcc 6300cagccctgtg tataactcac
tactttagtc agttccgcag tattacaaaa ggatgtcgca 6360aacgctgttt
gctcctctac aaaacagacc ttaaaaccct aaaggcttaa gtagcaccct
6420cgcaagctcg ggcaaatcgc tgaatattcc ttttgtctcc gaccatcagg
cacctgagtc 6480gctgtctttt tcgtgacatt cagttcgctg cgctcacggc
tctggcagtg aatgggggta 6540aatggcacta caggcgcctt ttatggattc
atgcaaggaa actacccata atacaagaaa 6600agcccgtcac gggcttctca
gggcgtttta tggcgggtct gctatgtggt gctatctgac 6660tttttgctgt
tcagcagttc ctgccctctg attttccagt ctgaccactt cggattatcc
6720cgtgacaggt cattcagact ggctaatgca cccagtaagg cagcggtatc
atcaacaggc 6780tta 6783277687DNAArtificial SequenceSynthetic
Construct 27ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc
gcagcctgaa 60tggcgaatgg cgcctgatgc ggtattttct ccttacgcat ctgtgcggta
tttcacaccg 120catatggtgc actctcagta caatctgctc tgatgccgca
tagttaagcc agccccgaca 180cccgccaaca cccgctgacg agcttagtaa
agccctcgct agattttaat gcggatgttg 240cgattacttc gccaactatt
gcgataacaa gaaaaagcca gcctttcatg atatatctcc 300caatttgtgt
agggcttatt atgcacgctt aaaaataata aaagcagact tgacctgata
360gtttggctgt gagcaattat gtgcttagtg catctaacgc ttgagttaag
ccgcgccgcg 420aagcggcgtc ggcttgaacg aattgttaga cattatttgc
cgactacctt ggtgatctcg 480cctttcacgt agtggacaaa ttcttccaac
tgatctgcgc gcgaggccaa gcgatcttct 540tcttgtccaa gataagcctg
tctagcttca agtatgacgg gctgatactg ggccggcagg 600cgctccattg
cccagtcggc agcgacatcc ttcggcgcga ttttgccggt tactgcgctg
660taccaaatgc gggacaacgt aagcactaca tttcgctcat cgccagccca
gtcgggcggc 720gagttccata gcgttaaggt ttcatttagc gcctcaaata
gatcctgttc aggaaccgga 780tcaaagagtt cctccgccgc tggacctacc
aaggcaacgc tatgttctct tgcttttgtc 840agcaagatag ccagatcaat
gtcgatcgtg gctggctcga agatacctgc aagaatgtca 900ttgcgctgcc
attctccaaa ttgcagttcg cgcttagctg gataacgcca cggaatgatg
960tcgtcgtgca caacaatggt gacttctaca gcgcggagaa tctcgctctc
tccaggggaa 1020gccgaagttt ccaaaaggtc gttgatcaaa gctcgccgcg
ttgtttcatc aagccttacg 1080gtcaccgtaa ccagcaaatc aatatcactg
tgtggcttca ggccgccatc cactgcggag 1140ccgtacaaat gtacggccag
caacgtcggt tcgagatggc gctcgatgac gccaactacc 1200tctgatagtt
gagtcgatac ttcggcgatc accgcttccc tcatgatgtt taactttgtt
1260ttagggcgac tgccctgctg cgtaacatcg ttgctgctcc ataacatcaa
acatcgaccc 1320acggcgtaac gcgcttgctg cttggatgcc cgaggcatag
actgtacccc aaaaaaacag 1380tcataacaag ccatgaaaac cgccactgcg
ccgttaccac cgctgcgttc ggtcaaggtt 1440ctggaccagt tgcgtgagcg
catacgctac ttgcattaca gcttacgaac cgaacaggct 1500tatgtccact
gggttcgtgc cttcatccgt ttccacggtg tgcgtcaccc ggcaaccttg
1560ggcagcagcg aagtcgaggc atttctgtcc tggctggcga acgagcgcaa
ggtttcggtc 1620tccacgcatc gtcaggcatt ggcggccttg ctgttcttct
acggcaaggt gctgtgcacg 1680gatctgccct ggcttcagga gatcggaaga
cctcggccgt cgcggcgctt gccggtggtg 1740ctgaccccgg atgaagtggt
tcgcatcctc ggttttctgg aaggcgagca tcgtttgttc 1800gcccagcttc
tgtatggaac gggcatgcgg atcagtgagg gtttgcaact gcgggtcaag
1860gatctggatt tcgatcacgg cacgatcatc gtgcgggagg gcaagggctc
caaggatcgg 1920gccttgatgt tacccgagag cttggcaccc agcctgcgcg
agcaggggaa ttaattccca 1980cgggttttgc tgcccgcaaa cgggctgttc
tggtgttgct agtttgttat cagaatcgca 2040gatccggctt cagccggttt
gccggctgaa agcgctattt cttccagaat tgccatgatt 2100ttttccccac
gggaggcgtc actggctccc gtgttgtcgg cagctttgat tcgataagca
2160gcatcgcctg tttcaggctg tctatgtgtg actgttgagc tgtaacaagt
tgtctcaggt 2220gttcaatttc atgttctagt tgctttgttt tactggtttc
acctgttcta ttaggtgtta 2280catgctgttc atctgttaca ttgtcgatct
gttcatggtg aacagctttg aatgcaccaa 2340aaactcgtaa aagctctgat
gtatctatct tttttacacc gttttcatct gtgcatatgg 2400acagttttcc
ctttgatatg taacggtgaa cagttgttct acttttgttt gttagtcttg
2460atgcttcact gatagataca agagccataa gaacctcaga tccttccgta
tttagccagt 2520atgttctcta gtgtggttcg ttgtttttgc gtgagccatg
agaacgaacc attgagatca 2580tacttacttt gcatgtcact caaaaatttt
gcctcaaaac tggtgagctg aatttttgca 2640gttaaagcat cgtgtagtgt
ttttcttagt ccgttatgta ggtaggaatc tgatgtaatg 2700gttgttggta
ttttgtcacc attcattttt atctggttgt tctcaagttc ggttacgaga
2760tccatttgtc tatctagttc aacttggaaa atcaacgtat cagtcgggcg
gcctcgctta 2820tcaaccacca atttcatatt gctgtaagtg tttaaatctt
tacttattgg tttcaaaacc 2880cattggttaa gccttttaaa ctcatggtag
ttattttcaa gcattaacat gaacttaaat 2940tcatcaaggc taatctctat
atttgccttg tgagttttct tttgtgttag ttcttttaat 3000aaccactcat
aaatcctcat agagtatttg ttttcaaaag acttaacatg ttccagatta
3060tattttatga atttttttaa ctggaaaaga taaggcaata tctcttcact
aaaaactaat 3120tctaattttt cgcttgagaa cttggcatag tttgtccact
ggaaaatctc aaagccttta 3180accaaaggat tcctgatttc cacagttctc
gtcatcagct ctctggttgc tttagctaat 3240acaccataag cattttccct
actgatgttc atcatctgag cgtattggtt ataagtgaac 3300gataccgtcc
gttctttcct tgtagggttt tcaatcgtgg ggttgagtag tgccacacag
3360cataaaatta gcttggtttc atgctccgtt aagtcatagc gactaatcgc
tagttcattt 3420gctttgaaaa caactaattc agacatacat ctcaattggt
ctaggtgatt ttaatcacta 3480taccaattga gatgggctag tcaatgataa
ttactagtcc ttttcctttg agttgtgggt 3540atctgtaaat tctgctagac
ctttgctgga aaacttgtaa attctgctag accctctgta 3600aattccgcta
gacctttgtg tgtttttttt gtttatattc aagtggttat aatttataga
3660ataaagaaag aataaaaaaa gataaaaaga atagatccca gccctgtgta
taactcacta 3720ctttagtcag ttccgcagta ttacaaaagg atgtcgcaaa
cgctgtttgc tcctctacaa 3780aacagacctt aaaaccctaa aggcttaagt
agcaccctcg caagctcggg caaatcgctg 3840aatattcctt ttgtctccga
ccatcaggca cctgagtcgc tgtctttttc gtgacattca 3900gttcgctgcg
ctcacggctc tggcagtgaa tgggggtaaa tggcactaca ggcgcctttt
3960atggattcat gcaaggaaac tacccataat acaagaaaag cccgtcacgg
gcttctcagg 4020gcgttttatg gcgggtctgc tatgtggtgc tatctgactt
tttgctgttc agcagttcct 4080gccctctgat tttccagtct gaccacttcg
gattatcccg tgacaggtca ttcagactgg 4140ctaatgcacc cagtaaggca
gcggtatcat caacaggctt acccgtctta ctgtcgggaa 4200ttcgcgttgg
ccgattcatt aatgcagatt ctgaaatgag ctgttgacaa ttaatcatcc
4260ggctcgtata atgtgtggaa ttgtgagcgg ataacaattt cacacaggaa
acagcgccgc 4320tgagaaaaag cgaagcggca ctgctcttta acaatttatc
agacaatctg tgtgggcact 4380cgaccggaat tatcgattaa ctttattatt
aaaaattaaa gaggtatata ttaatgtatc 4440gattaaataa ggaggaataa
accatgtgtg cgacctcttc tcaatttact cagattaccg 4500agcataattc
ccgtcgttcc gcaaactatc agccaaacct gtggaatttc gaattcctgc
4560aatccctgga gaacgacctg aaagtggaaa agctggagga gaaagcgacc
aaactggagg 4620aagaagttcg ctgcatgatc aaccgtgtag acacccagcc
gctgtccctg ctggagctga 4680tcgacgatgt gcagcgcctg ggtctgacct
acaaatttga aaaagacatc attaaagccc 4740tggaaaacat cgtactgctg
gacgaaaaca aaaagaacaa atctgacctg cacgcaaccg 4800ctctgtcttt
ccgtctgctg cgtcagcacg gtttcgaggt ttctcaggat gtttttgagc
4860gtttcaagga taaagaaggt ggtttcagcg gtgaactgaa aggtgacgtc
caaggcctgc 4920tgagcctgta tgaagcgtct tacctgggtt tcgagggtga
gaacctgctg gaggaggcgc 4980gtaccttttc catcacccac ctgaagaaca
acctgaaaga aggcattaat accaaggttg 5040cagaacaagt gagccacgcc
ctggaactgc catatcacca gcgtctgcac cgtctggagg 5100cacgttggtt
cctggataaa tacgaaccga aagaaccgca tcaccagctg ctgctggagc
5160tggcgaagct ggattttaac atggtacaga ccctgcacca gaaagagctg
caagatctgt 5220cccgctggtg gaccgagatg ggcctggcta gcaaactgga
ttttgtacgc gaccgcctga 5280tggaagttta tttctgggca ctgggtatgg
cgccagaccc gcagtttggt gaatgtcgca 5340aagctgttac taaaatgttt
ggtctggtga cgatcatcga tgacgtgtat gacgtttatg 5400gcactctgga
cgaactgcaa ctgttcaccg atgctgtaga gcgctgggac gttaacgcta
5460ttaacaccct gccggactat atgaaactgt gtttcctggc actgtacaac
accgttaacg 5520acacgtccta ttctattctg aaagagaaag gtcataacaa
cctgtcctat ctgacgaaaa 5580gctggcgtga
actgtgcaaa gcctttctgc aagaggcgaa atggtccaac aacaaaatta
5640tcccggcttt ctccaagtac ctggaaaacg ccagcgtttc ctcctccggt
gtagcgctgc 5700tggcgccgtc ttacttttcc gtatgccagc agcaggaaga
catctccgac cacgcgctgc 5760gttccctgac cgacttccat ggtctggtgc
gttctagctg cgttatcttc cgcctgtgca 5820acgatctggc cacctctgcg
gcggagctgg aacgtggcga gactaccaat tctatcatta 5880gctacatgca
cgaaaacgat ggtaccagcg aggaacaggc ccgcgaagaa ctgcgtaaac
5940tgatcgacgc cgaatggaaa aagatgaatc gtgaacgcgt tagcgactcc
accctgctgc 6000ctaaagcgtt catggaaatc gcagttaaca tggcacgtgt
ttcccactgc acctaccagt 6060atggcgatgg tctgggtcgc ccagactacg
cgactgaaaa ccgcatcaaa ctgctgctga 6120ttgacccttt cccgattaac
cagctgatgt atgtctaact gcatcgccct taggaggtaa 6180aaaaaaatga
ctgccgacaa caatagtatg ccccatggtg cagtatctag ttacgccaaa
6240ttagtgcaaa accaaacacc tgaagacatt ttggaagagt ttcctgaaat
tattccatta 6300caacaaagac ctaatacccg atctagtgag acgtcaaatg
acgaaagcgg agaaacatgt 6360ttttctggtc atgatgagga gcaaattaag
ttaatgaatg aaaattgtat tgttttggat 6420tgggacgata atgctattgg
tgccggtacc aagaaagttt gtcatttaat ggaaaatatt 6480gaaaagggtt
tactacatcg tgcattctcc gtctttattt tcaatgaaca aggtgaatta
6540cttttacaac aaagagccac tgaaaaaata actttccctg atctttggac
taacacatgc 6600tgctctcatc cactatgtat tgatgacgaa ttaggtttga
agggtaagct agacgataag 6660attaagggcg ctattactgc ggcggtgaga
aaactagatc atgaattagg tattccagaa 6720gatgaaacta agacaagggg
taagtttcac tttttaaaca gaatccatta catggcacca 6780agcaatgaac
catggggtga acatgaaatt gattacatcc tattttataa gatcaacgct
6840aaagaaaact tgactgtcaa cccaaacgtc aatgaagtta gagacttcaa
atgggtttca 6900ccaaatgatt tgaaaactat gtttgctgac ccaagttaca
agtttacgcc ttggtttaag 6960attatttgcg agaattactt attcaactgg
tgggagcaat tagatgacct ttctgaagtg 7020gaaaatgaca ggcaaattca
tagaatgcta taacgacgcg tcctgcagct ggtaccatat 7080gggaattcga
agctttctag aacgaaaact catctcagaa gaggatctga atagcgccgt
7140cgaccatcat catcatcatc attgagttta aacggtctcc agcttggctg
ttttggcgga 7200tgagagaaga ttttcagcct gatacagatt aaatcagaac
gcagaagcgg tctgataaaa 7260cagaatttgc ctggcggcag tagcgcggtg
gtcccacctg accccatgcc gaactcagaa 7320gtgaaacgcc gtagcgccga
tggtagtgtg gggtctcccc atgcgagagt agggaactgc 7380caggcatcaa
ataaaacgaa aggctcagtc gaaagactgg gcctttcgtt ttatctgttg
7440tttgtcggtg aacgctctcc tgagtaggac aaatccgccg ggagcggatt
tgaacgttgc 7500gaagcaacgg cccggagggt ggcgggcagg acgcccgcca
taaactgcca ggcatcaaat 7560taagcagaag gccatcctga cggatggcct
ttttgcgttt ctacaaactc tttttgttta 7620tttttctaaa tacattcaaa
tatgtatccg ctcatgagac aataaccctg ataaatgctt 7680caataat
7687288675DNAArtificial SequenceSynthetic Construct 28cccgtcttac
tgtcgggaat tcgcgttggc cgattcatta atgcagatta ttgaagcatt 60tatcagggtt
attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
120aaagagtttg tagaaacgca aaaaggccat ccgtcaggat ggccttctgc
ttaatttgat 180gcctggcagt ttatggcggg cgtcctgccc gccaccctcc
gggccgttgc ttcgcaacgt 240tcaaatccgc tcccggcgga tttgtcctac
tcaggagagc gttcaccgac aaacaacaga 300taaaacgaaa ggcccagtct
ttcgactgag cctttcgttt tatttgatgc ctggcagttc 360cctactctcg
catggggaga ccccacacta ccatcggcgc tacggcgttt cacttctgag
420ttcggcatgg ggtcaggtgg gaccaccgcg ctactgccgc caggcaaatt
ctgttttatc 480agaccgcttc tgcgttctga tttaatctgt atcaggctga
aaatcttctc tcatccgcca 540aaacagccaa gctggagacc gtttaaactc
aatgatgatg atgatgatgg tcgacggcgc 600tattcagatc ctcttctgag
atgagttttt gttctagaaa gcttcgaatt cccatatggt 660accagctgca
gttatgccag ccaggccttg attttggctt ccataccagc ggcatcgagg
720ccgagttcgg cgcgcatttc ttcctgagtt ccttgcggaa taaagaagtc
cggcaggcca 780atgttcagca cgggtactgg tttacgatgg gccatcagca
cttcgttcac gccgctgcct 840gcgccgccca taatggcgtt ttcttctacg
gtgaccagcg cttcatggct ggcggccatt 900tccagaatta acgcttcatc
aagcggtttc acaaaacgca tatcgaccag cgtggcgttc 960agcgattcgg
cgactttcgc cgcttctggc atcagcgtac caaagttaag gatcgccagt
1020ttctcgccac gacgcttcac aatgcctttg ccaattggta gtttttccag
cggcgtcagt 1080tccacgccga ccgcgttgcc acgcgggtag cgcaccgctg
acgggccatc gttatagtga 1140tagccggtat agagcatctg gcgacattcg
ttttcatcgc tcggggtcat aatgaccatt 1200tccggtatgc agcgcaggta
agagagatca aaagcaccct gatgggtttg accgtcagca 1260ccaacaatgc
ccgcgcggtc gatggcgaac aggaccggaa gcttttgaat cgccacgtca
1320tgcagcacct gatcataggc gcgttgcagg aaagtggagt aaatcgcgac
aatgggtttg 1380tacccaccaa tcgccagacc cgcagcaaag gtcaccgcgt
gttgctcggc aattgccacg 1440tcgaagtagc gatccgggaa tttacgtgaa
aactcgacca tgccggaacc ttcacgcatc 1500gccggagtaa tcgccatcag
cttgttgtct ttcgctgccg tttcgcacaa ccagtcgcca 1560aagatttttg
aatagctcgg caaaccgccg ctacttttcg gcaaacaacc gctggaggga
1620tcaaatttag gcacggcgtg gaaagtgatc gggtcttttt ctgccggttc
ataaccacga 1680ccttttttgg tcatgatatg caggaactgc gggcctttca
ggtcgcgcat gttctttagc 1740gtggtgataa gccccagcac atcgtgaccg
tccaccgggc cgatgtagtt aaagcccagc 1800tcttcaaaca acgtgccagg
cactaccatg cctttaatat gttcttcggt gcgtttgagc 1860agctctttaa
ttggcggcac gccagagaaa acttttttcc cgccttcgcg cagtgaagag
1920taaagcttac cggaaagcag ctgtgccaga tggttgttga gcgcgccgac
attttcggaa 1980atcgacattt cattgtcgtt gagaatcacc agcatatcag
gacggatatc gcccgcgtga 2040ttcatcgctt caaacgccat gcctgcggta
atcgcgccat cgccaatgac acagacggtg 2100cggcgatttt tgccttcttt
ttcggcagca accgcaatac caattccggc actgatggag 2160gttgatgaat
gcccgacgct taatacgtca tattcgcttt cgccgcgcca cgggaacggg
2220tgcagaccgc ctttctgacg gatggtgccg attttgtcgc ggcgtccggt
caaaatttta 2280tgcggataag cctgatgccc cacatcccaa atcaattggt
caaacggggt gttgtagaca 2340tagtgcagcg ccacggtcag ttcgaccgtg
cccagcccgg aggcgaagtg cccgctggaa 2400cggctcacgc tgtcgagtaa
atagcggcgc agttcgtcgc agagtttcgg taaactctct 2460ttcggcaaca
gtcgtaactc ctgggtggag tcgaccagtg ccagggtcgg gtatttggca
2520atatcaaaac tcatgttttt ttacctccta agggcgaatg cagttagaca
tacatcagct 2580ggttaatcgg gaaagggtca atcagcagca gtttgatgcg
gttttcagtc gcgtagtctg 2640ggcgacccag accatcgcca tactggtagg
tgcagtggga aacacgtgcc atgttaactg 2700cgatttccat gaacgcttta
ggcagcaggg tggagtcgct aacgcgttca cgattcatct 2760ttttccattc
ggcgtcgatc agtttacgca gttcttcgcg ggcctgttcc tcgctggtac
2820catcgttttc gtgcatgtag ctaatgatag aattggtagt ctcgccacgt
tccagctccg 2880ccgcagaggt ggccagatcg ttgcacaggc ggaagataac
gcagctagaa cgcaccagac 2940catggaagtc ggtcagggaa cgcagcgcgt
ggtcggagat gtcttcctgc tgctggcata 3000cggaaaagta agacggcgcc
agcagcgcta caccggagga ggaaacgctg gcgttttcca 3060ggtacttgga
gaaagccggg ataattttgt tgttggacca tttcgcctct tgcagaaagg
3120ctttgcacag ttcacgccag cttttcgtca gataggacag gttgttatga
cctttctctt 3180tcagaataga ataggacgtg tcgttaacgg tgttgtacag
tgccaggaaa cacagtttca 3240tatagtccgg cagggtgtta atagcgttaa
cgtcccagcg ctctacagca tcggtgaaca 3300gttgcagttc gtccagagtg
ccataaacgt catacacgtc atcgatgatc gtcaccagac 3360caaacatttt
agtaacagct ttgcgacatt caccaaactg cgggtctggc gccataccca
3420gtgcccagaa ataaacttcc atcaggcggt cgcgtacaaa atccagtttg
ctagccaggc 3480ccatctcggt ccaccagcgg gacagatctt gcagctcttt
ctggtgcagg gtctgtacca 3540tgttaaaatc cagcttcgcc agctccagca
gcagctggtg atgcggttct ttcggttcgt 3600atttatccag gaaccaacgt
gcctccagac ggtgcagacg ctggtgatat ggcagttcca 3660gggcgtggct
cacttgttct gcaaccttgg tattaatgcc ttctttcagg ttgttcttca
3720ggtgggtgat ggaaaaggta cgcgcctcct ccagcaggtt ctcaccctcg
aaacccaggt 3780aagacgcttc atacaggctc agcaggcctt ggacgtcacc
tttcagttca ccgctgaaac 3840caccttcttt atccttgaaa cgctcaaaaa
catcctgaga aacctcgaaa ccgtgctgac 3900gcagcagacg gaaagacaga
gcggttgcgt gcaggtcaga tttgttcttt ttgttttcgt 3960ccagcagtac
gatgttttcc agggctttaa tgatgtcttt ttcaaatttg taggtcagac
4020ccaggcgctg cacatcgtcg atcagctcca gcagggacag cggctgggtg
tctacacggt 4080tgatcatgca gcgaacttct tcctccagtt tggtcgcttt
ctcctccagc ttttccactt 4140tcaggtcgtt ctccagggat tgcaggaatt
cgaaattcca caggtttggc tgatagtttg 4200cggaacgacg ggaattatgc
tcggtaatct gagtaaattg agaagaggtc gcacacatgg 4260tttattcctc
cttatttaat cgatacatta atatatacct ctttaatttt taataataaa
4320gttaatcgat aattccggtc gagtgcccac acagattgtc tgataaattg
ttaaagagca 4380gtgccgcttc gctttttctc agcggcgctg tttcctgtgt
gaaattgtta tccgctcaca 4440attccacaca ttatacgagc cggatgatta
attgtcaaca gctcatttca gaatctggcg 4500taatagcgaa gaggcccgca
ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga 4560atggcgcctg
atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatatg
4620gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc
gacacccgcc 4680aacacccgct gacgagctta gtaaagccct cgctagattt
taatgcggat gttgcgatta 4740cttcgccaac tattgcgata acaagaaaaa
gccagccttt catgatatat ctcccaattt 4800gtgtagggct tattatgcac
gcttaaaaat aataaaagca gacttgacct gatagtttgg 4860ctgtgagcaa
ttatgtgctt agtgcatcta acgcttgagt taagccgcgc cgcgaagcgg
4920cgtcggcttg aacgaattgt tagacattat ttgccgacta ccttggtgat
ctcgcctttc 4980acgtagtgga caaattcttc caactgatct gcgcgcgagg
ccaagcgatc ttcttcttgt 5040ccaagataag cctgtctagc ttcaagtatg
acgggctgat actgggccgg caggcgctcc 5100attgcccagt cggcagcgac
atccttcggc gcgattttgc cggttactgc gctgtaccaa 5160atgcgggaca
acgtaagcac tacatttcgc tcatcgccag cccagtcggg cggcgagttc
5220catagcgtta aggtttcatt tagcgcctca aatagatcct gttcaggaac
cggatcaaag 5280agttcctccg ccgctggacc taccaaggca acgctatgtt
ctcttgcttt tgtcagcaag 5340atagccagat caatgtcgat cgtggctggc
tcgaagatac ctgcaagaat gtcattgcgc 5400tgccattctc caaattgcag
ttcgcgctta gctggataac gccacggaat gatgtcgtcg 5460tgcacaacaa
tggtgacttc tacagcgcgg agaatctcgc tctctccagg ggaagccgaa
5520gtttccaaaa ggtcgttgat caaagctcgc cgcgttgttt catcaagcct
tacggtcacc 5580gtaaccagca aatcaatatc actgtgtggc ttcaggccgc
catccactgc ggagccgtac 5640aaatgtacgg ccagcaacgt cggttcgaga
tggcgctcga tgacgccaac tacctctgat 5700agttgagtcg atacttcggc
gatcaccgct tccctcatga tgtttaactt tgttttaggg 5760cgactgccct
gctgcgtaac atcgttgctg ctccataaca tcaaacatcg acccacggcg
5820taacgcgctt gctgcttgga tgcccgaggc atagactgta ccccaaaaaa
acagtcataa 5880caagccatga aaaccgccac tgcgccgtta ccaccgctgc
gttcggtcaa ggttctggac 5940cagttgcgtg agcgcatacg ctacttgcat
tacagcttac gaaccgaaca ggcttatgtc 6000cactgggttc gtgccttcat
ccgtttccac ggtgtgcgtc acccggcaac cttgggcagc 6060agcgaagtcg
aggcatttct gtcctggctg gcgaacgagc gcaaggtttc ggtctccacg
6120catcgtcagg cattggcggc cttgctgttc ttctacggca aggtgctgtg
cacggatctg 6180ccctggcttc aggagatcgg aagacctcgg ccgtcgcggc
gcttgccggt ggtgctgacc 6240ccggatgaag tggttcgcat cctcggtttt
ctggaaggcg agcatcgttt gttcgcccag 6300cttctgtatg gaacgggcat
gcggatcagt gagggtttgc aactgcgggt caaggatctg 6360gatttcgatc
acggcacgat catcgtgcgg gagggcaagg gctccaagga tcgggccttg
6420atgttacccg agagcttggc acccagcctg cgcgagcagg ggaattaatt
cccacgggtt 6480ttgctgcccg caaacgggct gttctggtgt tgctagtttg
ttatcagaat cgcagatccg 6540gcttcagccg gtttgccggc tgaaagcgct
atttcttcca gaattgccat gattttttcc 6600ccacgggagg cgtcactggc
tcccgtgttg tcggcagctt tgattcgata agcagcatcg 6660cctgtttcag
gctgtctatg tgtgactgtt gagctgtaac aagttgtctc aggtgttcaa
6720tttcatgttc tagttgcttt gttttactgg tttcacctgt tctattaggt
gttacatgct 6780gttcatctgt tacattgtcg atctgttcat ggtgaacagc
tttgaatgca ccaaaaactc 6840gtaaaagctc tgatgtatct atctttttta
caccgttttc atctgtgcat atggacagtt 6900ttccctttga tatgtaacgg
tgaacagttg ttctactttt gtttgttagt cttgatgctt 6960cactgataga
tacaagagcc ataagaacct cagatccttc cgtatttagc cagtatgttc
7020tctagtgtgg ttcgttgttt ttgcgtgagc catgagaacg aaccattgag
atcatactta 7080ctttgcatgt cactcaaaaa ttttgcctca aaactggtga
gctgaatttt tgcagttaaa 7140gcatcgtgta gtgtttttct tagtccgtta
tgtaggtagg aatctgatgt aatggttgtt 7200ggtattttgt caccattcat
ttttatctgg ttgttctcaa gttcggttac gagatccatt 7260tgtctatcta
gttcaacttg gaaaatcaac gtatcagtcg ggcggcctcg cttatcaacc
7320accaatttca tattgctgta agtgtttaaa tctttactta ttggtttcaa
aacccattgg 7380ttaagccttt taaactcatg gtagttattt tcaagcatta
acatgaactt aaattcatca 7440aggctaatct ctatatttgc cttgtgagtt
ttcttttgtg ttagttcttt taataaccac 7500tcataaatcc tcatagagta
tttgttttca aaagacttaa catgttccag attatatttt 7560atgaattttt
ttaactggaa aagataaggc aatatctctt cactaaaaac taattctaat
7620ttttcgcttg agaacttggc atagtttgtc cactggaaaa tctcaaagcc
tttaaccaaa 7680ggattcctga tttccacagt tctcgtcatc agctctctgg
ttgctttagc taatacacca 7740taagcatttt ccctactgat gttcatcatc
tgagcgtatt ggttataagt gaacgatacc 7800gtccgttctt tccttgtagg
gttttcaatc gtggggttga gtagtgccac acagcataaa 7860attagcttgg
tttcatgctc cgttaagtca tagcgactaa tcgctagttc atttgctttg
7920aaaacaacta attcagacat acatctcaat tggtctaggt gattttaatc
actataccaa 7980ttgagatggg ctagtcaatg ataattacta gtccttttcc
tttgagttgt gggtatctgt 8040aaattctgct agacctttgc tggaaaactt
gtaaattctg ctagaccctc tgtaaattcc 8100gctagacctt tgtgtgtttt
ttttgtttat attcaagtgg ttataattta tagaataaag 8160aaagaataaa
aaaagataaa aagaatagat cccagccctg tgtataactc actactttag
8220tcagttccgc agtattacaa aaggatgtcg caaacgctgt ttgctcctct
acaaaacaga 8280ccttaaaacc ctaaaggctt aagtagcacc ctcgcaagct
cgggcaaatc gctgaatatt 8340ccttttgtct ccgaccatca ggcacctgag
tcgctgtctt tttcgtgaca ttcagttcgc 8400tgcgctcacg gctctggcag
tgaatggggg taaatggcac tacaggcgcc ttttatggat 8460tcatgcaagg
aaactaccca taatacaaga aaagcccgtc acgggcttct cagggcgttt
8520tatggcgggt ctgctatgtg gtgctatctg actttttgct gttcagcagt
tcctgccctc 8580tgattttcca gtctgaccac ttcggattat cccgtgacag
gtcattcaga ctggctaatg 8640cacccagtaa ggcagcggta tcatcaacag gctta
86752931DNAArtificial SequenceSynthetic Construct 29gacatcaatt
gctccatttt cttctgctat c 313041DNAArtificial SequenceSynthetic
Construct 30attgagaaga ggtcgcacac actctttacc ctctcctttt a
413141DNAArtificial SequenceSynthetic Construct 31taaaaggaga
gggtaaagag tgtgtgcgac ctcttctcaa t 413241DNAArtificial
SequenceSynthetic Construct 32ccaaggccgg ttttttttag acatacatca
gctggttaat c 413341DNAArtificial SequenceSynthetic Construct
33gattaaccag ctgatgtatg tctaaaaaaa accggccttg g 413432DNAArtificial
SequenceSynthetic Construct 34gacatgacgg atccgattac gaatgccgtc tc
323531DNAArtificial SequenceSynthetic Construct 35gacatcaatt
gctccatttt cttctgctat c 313627DNAArtificial SequenceSynthetic
Construct 36gacatgaatt cctccatttt cttctgc 273720DNAArtificial
SequenceSynthetic Construct 37aggagagggt aaagagtgag
203822DNAArtificial SequenceSynthetic Construct 38cttttccatc
acccacctga ag 223928DNAArtificial SequenceSynthetic Construct
39ggcgaaatgg tccaacaaca aaattatc 284051DNAArtificial
SequenceSynthetic Construct 40ggtgaattca gtctactggg gattcccaaa
tctatatata ctgcaggtga c 514123DNAArtificial SequenceSynthetic
Construct 41gcaggtggga aactatgcac tcc 234236DNAArtificial
SequenceSynthetic Construct 42cctgaattct gttggattgg aggattggat
agtggg 364330DNAArtificial SequenceSynthetic Construct 43ggtgtcgacg
tacggtcgag cttattgacc 304432DNAArtificial SequenceSynthetic
Construct 44ggtgggcccg cattttgcca cctacaagcc ag 324540DNAArtificial
SequenceSynthetic Construct 45ggtgaattct agaggatccc aacgctgttg
cctacaacgg 404636DNAArtificial SequenceSynthetic Construct
46ggtgcggccg ctgtctggac ctggtgagtt tccccg 364737DNAArtificial
SequenceSynthetic Construct 47ggtgggccca ttaaatcagt tatcgtttat
ttgatag 374835DNAArtificial SequenceSynthetic Construct
48ggtgaccagc aagtccatgg gtggtttgat catgg 354935DNAArtificial
SequenceSynthetic Construct 49ggtgcggccg cctttggagt acgactccaa
ctatg 355031DNAArtificial SequenceSynthetic Construct 50gcggccgcag
actaaattta tttcagtctc c 31515356DNAArtificial SequenceSynthetic
Construct 51tctataatca cggcagaaaa gtccacattg attatttgca cggcgtcaca
ctttgctatg 60ccatagcatt tttatccata agattagcgg atcctacctg acgcttttta
tcgcaactct 120ctactgtttc tccatacccg tttttttggg ctagcgaatt
cgagctcggt acccggggat 180cctctagagt cgacctgcag gcatgcaagc
ttggctgttt tggcggatga gagaagattt 240tcagcctgat acagattaaa
tcagaacgca gaagcggtct gataaaacag aatttgcctg 300gcggcagtag
cgcggtggtc ccacctgacc ccatgccgaa ctcagaagtg aaacgccgta
360gcgccgatgg tagtgtgggg tctccccatg cgagagtagg gaactgccag
gcatcaaata 420aaacgaaagg ctcagtcgaa agactgggcc tttcgtttta
tctgttgttt gtcggtgaac 480gctctcctga gtaggacaaa tccgccggga
gcggatttga acgttgcgaa gcaacggccc 540ggagggtggc gggcaggacg
cccgccataa actgccaggc atcaaattaa gcagaaggcc 600atcctgacgg
atggcctttt tgcgtttcta caaactcttt tgtttatttt tctaaataca
660ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
aatattgaaa 720aaggaagagt atgagtattc aacatttccg tgtcgccctt
attccctttt ttgcggcatt 780ttgccttcct gtttttgctc acccagaaac
gctggtgaaa gtaaaagatg ctgaagatca 840gttgggtgca gcaaactatt
aactggcgaa ctacttactc tagcttcccg gcaacaatta 900atagactgga
tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct
960ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg
tatcattgca 1020gcactggggc cagatggtaa gccctcccgt atcgtagtta
tctacacgac ggggagtcag 1080gcaactatgg atgaacgaaa tagacagatc
gctgagatag gtgcctcact gattaagcat 1140tggtaactgt cagaccaagt
ttactcatat atactttaga ttgatttacg cgccctgtag 1200cggcgcatta
agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
1260cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
tcgccggctt 1320tccccgtcaa gctctaaatc gggggctccc tttagggttc
cgatttagtg ctttacggca 1380cctcgacccc aaaaaacttg atttgggtga
tggttcacgt agtgggccat cgccctgata 1440gacggttttt cgccctttga
cgttggagtc cacgttcttt aatagtggac tcttgttcca 1500aacttgaaca
acactcaacc ctatctcggg ctattctttt gatttataag ggattttgcc
1560gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg
cgaattttaa 1620caaaatatta acgtttacaa tttaaaagga tctaggtgaa
gatccttttt gataatctca 1680tgaccaaaat cccttaacgt gagttttcgt
tccactgagc gtcagacccc gtagaaaaga 1740tcaaaggatc ttcttgagat
cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa 1800aaccaccgct
accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
1860aggtaactgg cttcagcaga gcgcagatac caaatactgt
ccttctagtg tagccgtagt 1920taggccacca cttcaagaac tctgtagcac
cgcctacata cctcgctctg ctaatcctgt 1980taccagtcag gcatttgaga
agcacacggt cacactgctt ccggtagtca ataaaccggt 2040aaaccagcaa
tagacataag cggctattta acgaccctgc cctgaaccga cgaccgggtc
2100gaatttgctt tcgaatttct gccattcatc cgcttattat cacttattca
ggcgtagcac 2160caggcgttta agggcaccaa taactgcctt aaaaaaatta
cgccccgccc tgccactcat 2220cgcagtactg ttgtaattca ttaagcattc
tgccgacatg gaagccatca cagacggcat 2280gatgaacctg aatcgccagc
ggcatcagca ccttgtcgcc ttgcgtataa tatttgccca 2340tggtgaaaac
gggggcgaag aagttgtcca tattggccac gtttaaatca aaactggtga
2400aactcaccca gggattggct gagacgaaaa acatattctc aataaaccct
ttagggaaat 2460aggccaggtt ttcaccgtaa cacgccacat cttgcgaata
tatgtgtaga aactgccgga 2520aatcgtcgtg gtattcactc cagagcgatg
aaaacgtttc agtttgctca tggaaaacgg 2580tgtaacaagg gtgaacacta
tcccatatca ccagctcacc gtctttcatt gccatacgga 2640attccggatg
agcattcatc aggcgggcaa gaatgtgaat aaaggccgga taaaacttgt
2700gcttattttt ctttacggtc tttaaaaagg ccgtaatatc cagctgaacg
gtctggttat 2760aggtacattg agcaactgac tgaaatgcct caaaatgttc
tttacgatgc cattgggata 2820tatcaacggt ggtatatcca gtgatttttt
tctccatttt agcttcctta gctcctgaaa 2880atctcgataa ctcaaaaaat
acgcccggta gtgatcttat ttcattatgg tgaaagttgg 2940aacctcttac
gtgccgatca acgtctcatt ttcgccaaaa gttggcccag ggcttcccgg
3000tatcaacagg gacaccagga tttatttatt ctgcgaagtg atcttccgtc
acaggtattt 3060attcggcgca aagtgcgtcg ggtgatgctg ccaacttact
gatttagtgt atgatggtgt 3120ttttgaggtg ctccagtggc ttctgtttct
atcagctgtc cctcctgttc agctactgac 3180ggggtggtgc gtaacggcaa
aagcaccgcc ggacatcagc gctagcggag tgtatactgg 3240cttactatgt
tggcactgat gagggtgtca gtgaagtgct tcatgtggca ggagaaaaaa
3300ggctgcaccg gtgcgtcagc agaatatgtg atacaggata tattccgctt
cctcgctcac 3360tgactcgcta cgctcggtcg ttcgactgcg gcgagcggaa
atggcttacg aacggggcgg 3420agatttcctg gaagatgcca ggaagatact
taacagggaa gtgagagggc cgcggcaaag 3480ccgtttttcc ataggctccg
cccccctgac aagcatcacg aaatctgacg ctcaaatcag 3540tggtggcgaa
acccgacagg actataaaga taccaggcgt ttccccctgg cggctccctc
3600gtgcgctctc ctgttcctgc ctttcggttt accggtgtca ttccgctgtt
atggccgcgt 3660ttgtctcatt ccacgcctga cactcagttc cgggtaggca
gttcgctcca agctggactg 3720tatgcacgaa ccccccgttc agtccgaccg
ctgcgcctta tccggtaact atcgtcttga 3780gtccaacccg gaaagacatg
caaaagcacc actggcagca gccactggta attgatttag 3840aggagttagt
cttgaagtca tgcgccggtt aaggctaaac tgaaaggaca agttttggtg
3900actgcgctcc tccaagccag ttacctcggt tcaaagagtt ggtagctcag
agaaccttcg 3960aaaaaccgcc ctgcaaggcg gttttttcgt tttcagagca
agagattacg cgcagaccaa 4020aacgatctca agaagatcat cttattaatc
agataaaata tttgctcatg agcccgaagt 4080ggcgagcccg atcttcccca
tcggtgatgt cggcgatata ggcgccagca accgcacctg 4140tggcgccggt
gatgccggcc acgatgcgtc cggcgtagag gatctgctca tgtttgacag
4200cttatcatcg atgcataatg tgcctgtcaa atggacgaag cagggattct
gcaaacccta 4260tgctactccg tcaagccgtc aattgtctga ttcgttacca
attatgacaa cttgacggct 4320acatcattca ctttttcttc acaaccggca
cggaactcgc tcgggctggc cccggtgcat 4380tttttaaata cccgcgagaa
atagagttga tcgtcaaaac caacattgcg accgacggtg 4440gcgataggca
tccgggtggt gctcaaaagc agcttcgcct ggctgatacg ttggtcctcg
4500cgccagctta agacgctaat ccctaactgc tggcggaaaa gatgtgacag
acgcgacggc 4560gacaagcaaa catgctgtgc gacgctggcg atatcaaaat
tgctgtctgc caggtgatcg 4620ctgatgtact gacaagcctc gcgtacccga
ttatccatcg gtggatggag cgactcgtta 4680atcgcttcca tgcgccgcag
taacaattgc tcaagcagat ttatcgccag cagctccgaa 4740tagcgccctt
ccccttgccc ggcgttaatg atttgcccaa acaggtcgct gaaatgcggc
4800tggtgcgctt catccgggcg aaagaacccc gtattggcaa atattgacgg
ccagttaagc 4860cattcatgcc agtaggcgcg cggacgaaag taaacccact
ggtgatacca ttcgcgagcc 4920tccggatgac gaccgtagtg atgaatctct
cctggcggga acagcaaaat atcacccggt 4980cggcaaacaa attctcgtcc
ctgatttttc accaccccct gaccgcgaat ggtgagattg 5040agaatataac
ctttcattcc cagcggtcgg tcgataaaaa aatcgagata accgttggcc
5100tcaatcggcg ttaaacccgc caccagatgg gcattaaacg agtatcccgg
cagcagggga 5160tcattttgcg cttcagccat acttttcata ctcccgccat
tcagagaaga aaccaattgt 5220ccatattgca tcagacattg ccgtcactgc
gtcttttact ggctcttctc gctaaccaaa 5280ccggtaaccc cgcttattaa
aagcattctg taacaaagcg ggaccaaagc catgacaaaa 5340acgcgtaaca aaagtg
5356527271DNAArtificial SequenceSynthetic Construct 52gcaaactatt
aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga 60tggaggcgga
taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta
120ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca
gcactggggc 180cagatggtaa gccctcccgt atcgtagtta tctacacgac
ggggagtcag gcaactatgg 240atgaacgaaa tagacagatc gctgagatag
gtgcctcact gattaagcat tggtaactgt 300cagaccaagt ttactcatat
atactttaga ttgatttacg cgccctgtag cggcgcatta 360agcgcggcgg
gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg
420cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
tccccgtcaa 480gctctaaatc gggggctccc tttagggttc cgatttagtg
ctttacggca cctcgacccc 540aaaaaacttg atttgggtga tggttcacgt
agtgggccat cgccctgata gacggttttt 600cgccctttga cgttggagtc
cacgttcttt aatagtggac tcttgttcca aacttgaaca 660acactcaacc
ctatctcggg ctattctttt gatttataag ggattttgcc gatttcggcc
720tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa
caaaatatta 780acgtttacaa tttaaaagga tctaggtgaa gatccttttt
gataatctca tgaccaaaat 840cccttaacgt gagttttcgt tccactgagc
gtcagacccc gtagaaaaga tcaaaggatc 900ttcttgagat cctttttttc
tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct 960accagcggtg
gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
1020cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt
taggccacca 1080cttcaagaac tctgtagcac cgcctacata cctcgctctg
ctaatcctgt taccagtcag 1140gcatttgaga agcacacggt cacactgctt
ccggtagtca ataaaccggt aaaccagcaa 1200tagacataag cggctattta
acgaccctgc cctgaaccga cgaccgggtc gaatttgctt 1260tcgaatttct
gccattcatc cgcttattat cacttattca ggcgtagcac caggcgttta
1320agggcaccaa taactgcctt aaaaaaatta cgccccgccc tgccactcat
cgcagtactg 1380ttgtaattca ttaagcattc tgccgacatg gaagccatca
cagacggcat gatgaacctg 1440aatcgccagc ggcatcagca ccttgtcgcc
ttgcgtataa tatttgccca tggtgaaaac 1500gggggcgaag aagttgtcca
tattggccac gtttaaatca aaactggtga aactcaccca 1560gggattggct
gagacgaaaa acatattctc aataaaccct ttagggaaat aggccaggtt
1620ttcaccgtaa cacgccacat cttgcgaata tatgtgtaga aactgccgga
aatcgtcgtg 1680gtattcactc cagagcgatg aaaacgtttc agtttgctca
tggaaaacgg tgtaacaagg 1740gtgaacacta tcccatatca ccagctcacc
gtctttcatt gccatacgga attccggatg 1800agcattcatc aggcgggcaa
gaatgtgaat aaaggccgga taaaacttgt gcttattttt 1860ctttacggtc
tttaaaaagg ccgtaatatc cagctgaacg gtctggttat aggtacattg
1920agcaactgac tgaaatgcct caaaatgttc tttacgatgc cattgggata
tatcaacggt 1980ggtatatcca gtgatttttt tctccatttt agcttcctta
gctcctgaaa atctcgataa 2040ctcaaaaaat acgcccggta gtgatcttat
ttcattatgg tgaaagttgg aacctcttac 2100gtgccgatca acgtctcatt
ttcgccaaaa gttggcccag ggcttcccgg tatcaacagg 2160gacaccagga
tttatttatt ctgcgaagtg atcttccgtc acaggtattt attcggcgca
2220aagtgcgtcg ggtgatgctg ccaacttact gatttagtgt atgatggtgt
ttttgaggtg 2280ctccagtggc ttctgtttct atcagctgtc cctcctgttc
agctactgac ggggtggtgc 2340gtaacggcaa aagcaccgcc ggacatcagc
gctagcggag tgtatactgg cttactatgt 2400tggcactgat gagggtgtca
gtgaagtgct tcatgtggca ggagaaaaaa ggctgcaccg 2460gtgcgtcagc
agaatatgtg atacaggata tattccgctt cctcgctcac tgactcgcta
2520cgctcggtcg ttcgactgcg gcgagcggaa atggcttacg aacggggcgg
agatttcctg 2580gaagatgcca ggaagatact taacagggaa gtgagagggc
cgcggcaaag ccgtttttcc 2640ataggctccg cccccctgac aagcatcacg
aaatctgacg ctcaaatcag tggtggcgaa 2700acccgacagg actataaaga
taccaggcgt ttccccctgg cggctccctc gtgcgctctc 2760ctgttcctgc
ctttcggttt accggtgtca ttccgctgtt atggccgcgt ttgtctcatt
2820ccacgcctga cactcagttc cgggtaggca gttcgctcca agctggactg
tatgcacgaa 2880ccccccgttc agtccgaccg ctgcgcctta tccggtaact
atcgtcttga gtccaacccg 2940gaaagacatg caaaagcacc actggcagca
gccactggta attgatttag aggagttagt 3000cttgaagtca tgcgccggtt
aaggctaaac tgaaaggaca agttttggtg actgcgctcc 3060tccaagccag
ttacctcggt tcaaagagtt ggtagctcag agaaccttcg aaaaaccgcc
3120ctgcaaggcg gttttttcgt tttcagagca agagattacg cgcagaccaa
aacgatctca 3180agaagatcat cttattaatc agataaaata tttgctcatg
agcccgaagt ggcgagcccg 3240atcttcccca tcggtgatgt cggcgatata
ggcgccagca accgcacctg tggcgccggt 3300gatgccggcc acgatgcgtc
cggcgtagag gatctgctca tgtttgacag cttatcatcg 3360atgcataatg
tgcctgtcaa atggacgaag cagggattct gcaaacccta tgctactccg
3420tcaagccgtc aattgtctga ttcgttacca attatgacaa cttgacggct
acatcattca 3480ctttttcttc acaaccggca cggaactcgc tcgggctggc
cccggtgcat tttttaaata 3540cccgcgagaa atagagttga tcgtcaaaac
caacattgcg accgacggtg gcgataggca 3600tccgggtggt gctcaaaagc
agcttcgcct ggctgatacg ttggtcctcg cgccagctta 3660agacgctaat
ccctaactgc tggcggaaaa gatgtgacag acgcgacggc gacaagcaaa
3720catgctgtgc gacgctggcg atatcaaaat tgctgtctgc caggtgatcg
ctgatgtact 3780gacaagcctc gcgtacccga ttatccatcg gtggatggag
cgactcgtta atcgcttcca 3840tgcgccgcag taacaattgc tcaagcagat
ttatcgccag cagctccgaa tagcgccctt 3900ccccttgccc ggcgttaatg
atttgcccaa acaggtcgct gaaatgcggc tggtgcgctt 3960catccgggcg
aaagaacccc gtattggcaa atattgacgg ccagttaagc cattcatgcc
4020agtaggcgcg cggacgaaag taaacccact ggtgatacca ttcgcgagcc
tccggatgac 4080gaccgtagtg atgaatctct cctggcggga acagcaaaat
atcacccggt cggcaaacaa 4140attctcgtcc ctgatttttc accaccccct
gaccgcgaat ggtgagattg agaatataac 4200ctttcattcc cagcggtcgg
tcgataaaaa aatcgagata accgttggcc tcaatcggcg 4260ttaaacccgc
caccagatgg gcattaaacg agtatcccgg cagcagggga tcattttgcg
4320cttcagccat acttttcata ctcccgccat tcagagaaga aaccaattgt
ccatattgca 4380tcagacattg ccgtcactgc gtcttttact ggctcttctc
gctaaccaaa ccggtaaccc 4440cgcttattaa aagcattctg taacaaagcg
ggaccaaagc catgacaaaa acgcgtaaca 4500aaagtgtcta taatcacggc
agaaaagtcc acattgatta tttgcacggc gtcacacttt 4560gctatgccat
agcattttta tccataagat tagcggatcc tacctgacgc tttttatcgc
4620aactctctac tgtttctcca tacccgtttt tttgggctag cgaattcgag
ctcggtaccc 4680cccattctga aatgagctgt tgacaattaa tcatccggct
cgtataatgt gtggaattgt 4740gagcggataa caatttcaca caggaaacag
cgccgctgag aaaaagcgaa gcggcactgc 4800tctttaacaa tttatcagac
aatctgtgtg ggcactcgac cggaattatc gattaacttt 4860attattaaaa
attaaagagg tatatattaa tgtatcgatt aaataaggag gaataaacca
4920tgtgtgcgac ctcttctcaa tttactcaga ttaccgagca taattcccgt
cgttccgcaa 4980actatcagcc aaacctgtgg aatttcgaat tcctgcaatc
cctggagaac gacctgaaag 5040tggaaaagct ggaggagaaa gcgaccaaac
tggaggaaga agttcgctgc atgatcaacc 5100gtgtagacac ccagccgctg
tccctgctgg agctgatcga cgatgtgcag cgcctgggtc 5160tgacctacaa
atttgaaaaa gacatcatta aagccctgga aaacatcgta ctgctggacg
5220aaaacaaaaa gaacaaatct gacctgcacg caaccgctct gtctttccgt
ctgctgcgtc 5280agcacggttt cgaggtttct caggatgttt ttgagcgttt
caaggataaa gaaggtggtt 5340tcagcggtga actgaaaggt gacgtccaag
gcctgctgag cctgtatgaa gcgtcttacc 5400tgggtttcga gggtgagaac
ctgctggagg aggcgcgtac cttttccatc acccacctga 5460agaacaacct
gaaagaaggc attaatacca aggttgcaga acaagtgagc cacgccctgg
5520aactgccata tcaccagcgt ctgcaccgtc tggaggcacg ttggttcctg
gataaatacg 5580aaccgaaaga accgcatcac cagctgctgc tggagctggc
gaagctggat tttaacatgg 5640tacagaccct gcaccagaaa gagctgcaag
atctgtcccg ctggtggacc gagatgggcc 5700tggctagcaa actggatttt
gtacgcgacc gcctgatgga agtttatttc tgggcactgg 5760gtatggcgcc
agacccgcag tttggtgaat gtcgcaaagc tgttactaaa atgtttggtc
5820tggtgacgat catcgatgac gtgtatgacg tttatggcac tctggacgaa
ctgcaactgt 5880tcaccgatgc tgtagagcgc tgggacgtta acgctattaa
caccctgccg gactatatga 5940aactgtgttt cctggcactg tacaacaccg
ttaacgacac gtcctattct attctgaaag 6000agaaaggtca taacaacctg
tcctatctga cgaaaagctg gcgtgaactg tgcaaagcct 6060ttctgcaaga
ggcgaaatgg tccaacaaca aaattatccc ggctttctcc aagtacctgg
6120aaaacgccag cgtttcctcc tccggtgtag cgctgctggc gccgtcttac
ttttccgtat 6180gccagcagca ggaagacatc tccgaccacg cgctgcgttc
cctgaccgac ttccatggtc 6240tggtgcgttc tagctgcgtt atcttccgcc
tgtgcaacga tctggccacc tctgcggcgg 6300agctggaacg tggcgagact
accaattcta tcattagcta catgcacgaa aacgatggta 6360ccagcgagga
acaggcccgc gaagaactgc gtaaactgat cgacgccgaa tggaaaaaga
6420tgaatcgtga acgcgttagc gactccaccc tgctgcctaa agcgttcatg
gaaatcgcag 6480ttaacatggc acgtgtttcc cactgcacct accagtatgg
cgatggtctg ggtcgcccag 6540actacgcgac tgaaaaccgc atcaaactgc
tgctgattga ccctttcccg attaaccagc 6600tgatgtatgt ctaactgcag
ggcatgcaag cttggctgtt ttggcggatg agagaagatt 6660ttcagcctga
tacagattaa atcagaacgc agaagcggtc tgataaaaca gaatttgcct
6720ggcggcagta gcgcggtggt cccacctgac cccatgccga actcagaagt
gaaacgccgt 6780agcgccgatg gtagtgtggg gtctccccat gcgagagtag
ggaactgcca ggcatcaaat 6840aaaacgaaag gctcagtcga aagactgggc
ctttcgtttt atctgttgtt tgtcggtgaa 6900cgctctcctg agtaggacaa
atccgccggg agcggatttg aacgttgcga agcaacggcc 6960cggagggtgg
cgggcaggac gcccgccata aactgccagg catcaaatta agcagaaggc
7020catcctgacg gatggccttt ttgcgtttct acaaactctt ttgtttattt
ttctaaatac 7080attcaaatat gtatccgctc atgagacaat aaccctgata
aatgcttcaa taatattgaa 7140aaaggaagag tatgagtatt caacatttcc
gtgtcgccct tattcccttt tttgcggcat 7200tttgccttcc tgtttttgct
cacccagaaa cgctggtgaa agtaaaagat gctgaagatc 7260agttgggtgc a
7271537833DNAArtificial SequenceSynthetic Construct 53gcaaactatt
aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga 60tggaggcgga
taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta
120ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca
gcactggggc 180cagatggtaa gccctcccgt atcgtagtta tctacacgac
ggggagtcag gcaactatgg 240atgaacgaaa tagacagatc gctgagatag
gtgcctcact gattaagcat tggtaactgt 300cagaccaagt ttactcatat
atactttaga ttgatttacg cgccctgtag cggcgcatta 360agcgcggcgg
gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg
420cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
tccccgtcaa 480gctctaaatc gggggctccc tttagggttc cgatttagtg
ctttacggca cctcgacccc 540aaaaaacttg atttgggtga tggttcacgt
agtgggccat cgccctgata gacggttttt 600cgccctttga cgttggagtc
cacgttcttt aatagtggac tcttgttcca aacttgaaca 660acactcaacc
ctatctcggg ctattctttt gatttataag ggattttgcc gatttcggcc
720tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa
caaaatatta 780acgtttacaa tttaaaagga tctaggtgaa gatccttttt
gataatctca tgaccaaaat 840cccttaacgt gagttttcgt tccactgagc
gtcagacccc gtagaaaaga tcaaaggatc 900ttcttgagat cctttttttc
tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct 960accagcggtg
gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
1020cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt
taggccacca 1080cttcaagaac tctgtagcac cgcctacata cctcgctctg
ctaatcctgt taccagtcag 1140gcatttgaga agcacacggt cacactgctt
ccggtagtca ataaaccggt aaaccagcaa 1200tagacataag cggctattta
acgaccctgc cctgaaccga cgaccgggtc gaatttgctt 1260tcgaatttct
gccattcatc cgcttattat cacttattca ggcgtagcac caggcgttta
1320agggcaccaa taactgcctt aaaaaaatta cgccccgccc tgccactcat
cgcagtactg 1380ttgtaattca ttaagcattc tgccgacatg gaagccatca
cagacggcat gatgaacctg 1440aatcgccagc ggcatcagca ccttgtcgcc
ttgcgtataa tatttgccca tggtgaaaac 1500gggggcgaag aagttgtcca
tattggccac gtttaaatca aaactggtga aactcaccca 1560gggattggct
gagacgaaaa acatattctc aataaaccct ttagggaaat aggccaggtt
1620ttcaccgtaa cacgccacat cttgcgaata tatgtgtaga aactgccgga
aatcgtcgtg 1680gtattcactc cagagcgatg aaaacgtttc agtttgctca
tggaaaacgg tgtaacaagg 1740gtgaacacta tcccatatca ccagctcacc
gtctttcatt gccatacgga attccggatg 1800agcattcatc aggcgggcaa
gaatgtgaat aaaggccgga taaaacttgt gcttattttt 1860ctttacggtc
tttaaaaagg ccgtaatatc cagctgaacg gtctggttat aggtacattg
1920agcaactgac tgaaatgcct caaaatgttc tttacgatgc cattgggata
tatcaacggt 1980ggtatatcca gtgatttttt tctccatttt agcttcctta
gctcctgaaa atctcgataa 2040ctcaaaaaat acgcccggta gtgatcttat
ttcattatgg tgaaagttgg aacctcttac 2100gtgccgatca acgtctcatt
ttcgccaaaa gttggcccag ggcttcccgg tatcaacagg 2160gacaccagga
tttatttatt ctgcgaagtg atcttccgtc acaggtattt attcggcgca
2220aagtgcgtcg ggtgatgctg ccaacttact gatttagtgt atgatggtgt
ttttgaggtg 2280ctccagtggc ttctgtttct atcagctgtc cctcctgttc
agctactgac ggggtggtgc 2340gtaacggcaa aagcaccgcc ggacatcagc
gctagcggag tgtatactgg cttactatgt 2400tggcactgat gagggtgtca
gtgaagtgct tcatgtggca ggagaaaaaa ggctgcaccg 2460gtgcgtcagc
agaatatgtg atacaggata tattccgctt cctcgctcac tgactcgcta
2520cgctcggtcg ttcgactgcg gcgagcggaa atggcttacg aacggggcgg
agatttcctg 2580gaagatgcca ggaagatact taacagggaa gtgagagggc
cgcggcaaag ccgtttttcc 2640ataggctccg cccccctgac aagcatcacg
aaatctgacg ctcaaatcag tggtggcgaa 2700acccgacagg actataaaga
taccaggcgt ttccccctgg cggctccctc gtgcgctctc 2760ctgttcctgc
ctttcggttt accggtgtca ttccgctgtt atggccgcgt ttgtctcatt
2820ccacgcctga cactcagttc cgggtaggca gttcgctcca agctggactg
tatgcacgaa 2880ccccccgttc agtccgaccg ctgcgcctta tccggtaact
atcgtcttga gtccaacccg 2940gaaagacatg caaaagcacc actggcagca
gccactggta attgatttag aggagttagt 3000cttgaagtca tgcgccggtt
aaggctaaac tgaaaggaca agttttggtg actgcgctcc 3060tccaagccag
ttacctcggt tcaaagagtt ggtagctcag agaaccttcg aaaaaccgcc
3120ctgcaaggcg gttttttcgt tttcagagca agagattacg cgcagaccaa
aacgatctca 3180agaagatcat cttattaatc agataaaata tttgctcatg
agcccgaagt ggcgagcccg 3240atcttcccca tcggtgatgt cggcgatata
ggcgccagca accgcacctg tggcgccggt 3300gatgccggcc acgatgcgtc
cggcgtagag gatctgctca tgtttgacag cttatcatcg 3360atgcataatg
tgcctgtcaa atggacgaag cagggattct gcaaacccta tgctactccg
3420tcaagccgtc aattgtctga ttcgttacca attatgacaa cttgacggct
acatcattca 3480ctttttcttc acaaccggca cggaactcgc tcgggctggc
cccggtgcat tttttaaata 3540cccgcgagaa atagagttga tcgtcaaaac
caacattgcg accgacggtg gcgataggca 3600tccgggtggt gctcaaaagc
agcttcgcct ggctgatacg ttggtcctcg cgccagctta 3660agacgctaat
ccctaactgc tggcggaaaa gatgtgacag acgcgacggc gacaagcaaa
3720catgctgtgc gacgctggcg atatcaaaat tgctgtctgc caggtgatcg
ctgatgtact 3780gacaagcctc gcgtacccga ttatccatcg gtggatggag
cgactcgtta atcgcttcca 3840tgcgccgcag taacaattgc tcaagcagat
ttatcgccag cagctccgaa tagcgccctt 3900ccccttgccc ggcgttaatg
atttgcccaa acaggtcgct gaaatgcggc tggtgcgctt 3960catccgggcg
aaagaacccc gtattggcaa atattgacgg ccagttaagc cattcatgcc
4020agtaggcgcg cggacgaaag taaacccact ggtgatacca ttcgcgagcc
tccggatgac 4080gaccgtagtg atgaatctct cctggcggga acagcaaaat
atcacccggt cggcaaacaa 4140attctcgtcc
ctgatttttc accaccccct gaccgcgaat ggtgagattg agaatataac
4200ctttcattcc cagcggtcgg tcgataaaaa aatcgagata accgttggcc
tcaatcggcg 4260ttaaacccgc caccagatgg gcattaaacg agtatcccgg
cagcagggga tcattttgcg 4320cttcagccat acttttcata ctcccgccat
tcagagaaga aaccaattgt ccatattgca 4380tcagacattg ccgtcactgc
gtcttttact ggctcttctc gctaaccaaa ccggtaaccc 4440cgcttattaa
aagcattctg taacaaagcg ggaccaaagc catgacaaaa acgcgtaaca
4500aaagtgtcta taatcacggc agaaaagtcc acattgatta tttgcacggc
gtcacacttt 4560gctatgccat agcattttta tccataagat tagcggatcc
tacctgacgc tttttatcgc 4620aactctctac tgtttctcca tacccgtttt
tttgggctag cgaattcgag ctcggtaccc 4680cccattctga aatgagctgt
tgacaattaa tcatccggct cgtataatgt gtggaattgt 4740gagcggataa
caatttcaca caggaaacag cgccgctgag aaaaagcgaa gcggcactgc
4800tctttaacaa tttatcagac aatctgtgtg ggcactcgac cggaattatc
gattaacttt 4860attattaaaa attaaagagg tatatattaa tgtatcgatt
aaataaggag gaataaacca 4920tgtgtgcgac ctcttctcaa tttactcaga
ttaccgagca taattcccgt cgttccgcaa 4980actatcagcc aaacctgtgg
aatttcgaat tcctgcaatc cctggagaac gacctgaaag 5040tggaaaagct
ggaggagaaa gcgaccaaac tggaggaaga agttcgctgc atgatcaacc
5100gtgtagacac ccagccgctg tccctgctgg agctgatcga cgatgtgcag
cgcctgggtc 5160tgacctacaa atttgaaaaa gacatcatta aagccctgga
aaacatcgta ctgctggacg 5220aaaacaaaaa gaacaaatct gacctgcacg
caaccgctct gtctttccgt ctgctgcgtc 5280agcacggttt cgaggtttct
caggatgttt ttgagcgttt caaggataaa gaaggtggtt 5340tcagcggtga
actgaaaggt gacgtccaag gcctgctgag cctgtatgaa gcgtcttacc
5400tgggtttcga gggtgagaac ctgctggagg aggcgcgtac cttttccatc
acccacctga 5460agaacaacct gaaagaaggc attaatacca aggttgcaga
acaagtgagc cacgccctgg 5520aactgccata tcaccagcgt ctgcaccgtc
tggaggcacg ttggttcctg gataaatacg 5580aaccgaaaga accgcatcac
cagctgctgc tggagctggc gaagctggat tttaacatgg 5640tacagaccct
gcaccagaaa gagctgcaag atctgtcccg ctggtggacc gagatgggcc
5700tggctagcaa actggatttt gtacgcgacc gcctgatgga agtttatttc
tgggcactgg 5760gtatggcgcc agacccgcag tttggtgaat gtcgcaaagc
tgttactaaa atgtttggtc 5820tggtgacgat catcgatgac gtgtatgacg
tttatggcac tctggacgaa ctgcaactgt 5880tcaccgatgc tgtagagcgc
tgggacgtta acgctattaa caccctgccg gactatatga 5940aactgtgttt
cctggcactg tacaacaccg ttaacgacac gtcctattct attctgaaag
6000agaaaggtca taacaacctg tcctatctga cgaaaagctg gcgtgaactg
tgcaaagcct 6060ttctgcaaga ggcgaaatgg tccaacaaca aaattatccc
ggctttctcc aagtacctgg 6120aaaacgccag cgtttcctcc tccggtgtag
cgctgctggc gccgtcttac ttttccgtat 6180gccagcagca ggaagacatc
tccgaccacg cgctgcgttc cctgaccgac ttccatggtc 6240tggtgcgttc
tagctgcgtt atcttccgcc tgtgcaacga tctggccacc tctgcggcgg
6300agctggaacg tggcgagact accaattcta tcattagcta catgcacgaa
aacgatggta 6360ccagcgagga acaggcccgc gaagaactgc gtaaactgat
cgacgccgaa tggaaaaaga 6420tgaatcgtga acgcgttagc gactccaccc
tgctgcctaa agcgttcatg gaaatcgcag 6480ttaacatggc acgtgtttcc
cactgcacct accagtatgg cgatggtctg ggtcgcccag 6540actacgcgac
tgaaaaccgc atcaaactgc tgctgattga ccctttcccg attaaccagc
6600tgatgtatgt ctaactgcat actagtttca agaggtattt cactcatggc
tatcactggc 6660atctttttcg gcagcgacac cggtaatacc gaaaatatcg
caaaaatgat tcaaaaacag 6720cttggtaaag acgttgccga tgtccatgac
attgcaaaaa gcagcaaaga agatctggaa 6780gcttatgaca ttctgctgct
gggcatccca acctggtatt acggcgaagc gcagtgtgac 6840tgggatgact
tcttcccgac tctcgaagag attgatttca acggcaaact ggttgcgctg
6900tttggttgtg gtgaccagga agattacgcc gaatatttct gcgacgcatt
gggcaccatc 6960cgcgacatca ttgaaccgcg cggtgcaacc atcgttggtc
actggccaac tgcgggctat 7020catttcgaag catcaaaagg tctggcagat
gacgaccact ttgtcggtct ggctatcgac 7080gaagaccgtc agccggaact
gaccgctgaa cgtgtagaaa aatgggttaa acagatttct 7140gaagagttgc
atctcgacga aattctcaat gcctgactgc agggcatgca agcttggctg
7200ttttggcgga tgagagaaga ttttcagcct gatacagatt aaatcagaac
gcagaagcgg 7260tctgataaaa cagaatttgc ctggcggcag tagcgcggtg
gtcccacctg accccatgcc 7320gaactcagaa gtgaaacgcc gtagcgccga
tggtagtgtg gggtctcccc atgcgagagt 7380agggaactgc caggcatcaa
ataaaacgaa aggctcagtc gaaagactgg gcctttcgtt 7440ttatctgttg
tttgtcggtg aacgctctcc tgagtaggac aaatccgccg ggagcggatt
7500tgaacgttgc gaagcaacgg cccggagggt ggcgggcagg acgcccgcca
taaactgcca 7560ggcatcaaat taagcagaag gccatcctga cggatggcct
ttttgcgttt ctacaaactc 7620ttttgtttat ttttctaaat acattcaaat
atgtatccgc tcatgagaca ataaccctga 7680taaatgcttc aataatattg
aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc 7740cttattccct
tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
7800aaagtaaaag atgctgaaga tcagttgggt gca 78335436DNAArtificial
SequenceSynthetic Construct 54ggatgcatac tagtttcaag aggtatttca
ctcatg 365530DNAArtificial SequenceSynthetic Construct 55atcctgcagt
caggcattga gaatttcgtc 30566592DNAArtificial SequenceSynthetic
Construct 56gaattgctcc attttcttct gctatcaaaa taacagactc gtgattttcc
aaacgagctt 60tcaaaaaagc ctctgcccct tgcaaatcgg atgcctgtct ataaaattcc
cgatattggt 120taaacagcgg cgcaatggcg gccgcatctg atgtctttgc
ttggcgaatg ttcatcttat 180ttcttcctcc ctctcaataa ttttttcatt
ctatcccttt tctgtaaagt ttatttttca 240gaatactttt atcatcatgc
tttgaaaaaa tatcacgata atatccattg ttctcacgga 300agcacacgca
ggtcatttga acgaattttt tcgacaggaa tttgccggga ctcaggagca
360tttaacctaa aaaagcatga catttcagca taatgaacat ttactcatgt
ctattttcgt 420tcttttctgt atgaaaatag ttatttcgag tctctacgga
aatagcgaga gatgatatac 480ctaaatagag ataaaatcat ctcaaaaaaa
tgggtctact aaaatattat tccatctatt 540acaataaatt cacagaatag
tcttttaagt aagtctactc tgaatttttt taaaaggaga 600gggtaaagag
tgtgtgcgac ctcttctcaa tttactcaga ttaccgagca taattcccgt
660cgttccgcaa actatcagcc aaacctgtgg aatttcgaat tcctgcaatc
cctggagaac 720gacctgaaag tggaaaagct ggaggagaaa gcgaccaaac
tggaggaaga agttcgctgc 780atgatcaacc gtgtagacac ccagccgctg
tccctgctgg agctgatcga cgatgtgcag 840cgcctgggtc tgacctacaa
atttgaaaaa gacatcatta aagccctgga aaacatcgta 900ctgctggacg
aaaacaaaaa gaacaaatct gacctgcacg caaccgctct gtctttccgt
960ctgctgcgtc agcacggttt cgaggtttct caggatgttt ttgagcgttt
caaggataaa 1020gaaggtggtt tcagcggtga actgaaaggt gacgtccaag
gcctgctgag cctgtatgaa 1080gcgtcttacc tgggtttcga gggtgagaac
ctgctggagg aggcgcgtac cttttccatc 1140acccacctga agaacaacct
gaaagaaggc attaatacca aggttgcaga acaagtgagc 1200cacgccctgg
aactgccata tcaccagcgt ctgcaccgtc tggaggcacg ttggttcctg
1260gataaatacg aaccgaaaga accgcatcac cagctgctgc tggagctggc
gaagctggat 1320tttaacatgg tacagaccct gcaccagaaa gagctgcaag
atctgtcccg ctggtggacc 1380gagatgggcc tggctagcaa actggatttt
gtacgcgacc gcctgatgga agtttatttc 1440tgggcactgg gtatggcgcc
agacccgcag tttggtgaat gtcgcaaagc tgttactaaa 1500atgtttggtc
tggtgacgat catcgatgac gtgtatgacg tttatggcac tctggacgaa
1560ctgcaactgt tcaccgatgc tgtagagcgc tgggacgtta acgctattaa
caccctgccg 1620gactatatga aactgtgttt cctggcactg tacaacaccg
ttaacgacac gtcctattct 1680attctgaaag agaaaggtca taacaacctg
tcctatctga cgaaaagctg gcgtgaactg 1740tgcaaagcct ttctgcaaga
ggcgaaatgg tccaacaaca aaattatccc ggctttctcc 1800aagtacctgg
aaaacgccag cgtttcctcc tccggtgtag cgctgctggc gccgtcttac
1860ttttccgtat gccagcagca ggaagacatc tccgaccacg cgctgcgttc
cctgaccgac 1920ttccatggtc tggtgcgttc tagctgcgtt atcttccgcc
tgtgcaacga tctggccacc 1980tctgcggcgg agctggaacg tggcgagact
accaattcta tcattagcta catgcacgaa 2040aacgatggta ccagcgagga
acaggcccgc gaagaactgc gtaaactgat cgacgccgaa 2100tggaaaaaga
tgaatcgtga acgcgttagc gactccaccc tgctgcctaa agcgttcatg
2160gaaatcgcag ttaacatggc acgtgtttcc cactgcacct accagtatgg
cgatggtctg 2220ggtcgcccag actacgcgac tgaaaaccgc atcaaactgc
tgctgattga ccctttcccg 2280attaaccagc tgatgtatgt ctaaaaaaaa
ccggccttgg ccccgccggt tttttattat 2340ttttcttcct ccgcatgttc
aatccgctcc ataatcgacg gatggctccc tctgaaaatt 2400ttaacgagaa
acggcgggtt gacccggctc agtcccgtaa cggccaagtc ctgaaacgtc
2460tcaatcgccg cttcccggtt tccggtcagc tcaatgccgt aacggtcggc
ggcgttttcc 2520tgataccggg agacggcatt cgtaatcgga tcctctagag
tcgacctgca ggcatgcaag 2580ctttgcctcg cgcgtttcgg tgatgacggt
gaaaacctct gacacatgca gctcccggag 2640acggtcacag cttgtctgta
agcggatgcc gggagcagac aagcccgtca gggcgcgtca 2700gcgggtgttg
gcgggtgtcg gggcgcagcc atgacccagt cacgtagcga tagcggagtg
2760tatactggct taactatgcg gcatcagagc agattgtact gagagtgcac
catatgcggt 2820gtgaaatacc gcacagatgc gtaaggagaa aataccgcat
caggcgctct tccgcttcct 2880cgctcactga ctcgctgcgc tcggtcgttc
ggctgcggcg agcggtatca gctcactcaa 2940aggcggtaat acggttatcc
acagaatcag gggataacgc aggaaagaac atgtgagcaa 3000aaggccagca
aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
3060tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
cgaaacccga 3120caggactata aagataccag gcgtttcccc ctggaagctc
cctcgtgcgc tctcctgttc 3180cgaccctgcc gcttaccgga tacctgtccg
cctttctccc ttcgggaagc gtggcgcttt 3240ctcaatgctc acgctgtagg
tatctcagtt cggtgtaggt cgttcgctcc aagctgggct 3300gtgtgcacga
accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
3360agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
aacaggatta 3420gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
gtggtggcct aactacggct 3480acactagaag gacagtattt ggtatctgcg
ctctgctgaa gccagttacc ttcggaaaaa 3540gagttggtag ctcttgatcc
ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt 3600gcaagcagca
gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
3660cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
atgagattat 3720caaaaaggat cgaagtcggt tcagaaaaag aaggatatgg
atctggagct gtaatataaa 3780aaccttcttc aactaacggg gcaggttagt
gacattagaa aaccgactgt aaaaagtaca 3840gtcggcatta tctcatatta
taaaagccag tcattaggcc tatctgacaa ttcctgaata 3900gagttcataa
acaatcctgc atgataacca tcacaaacag aatgatgtac ctgtaaagat
3960agcggtaaat atattgaatt acctttatta atgaattttc ctgctgtaat
aatgggtaga 4020aggtaattac tattattatt gatatttaag ttaaacccag
taaatgaagt ccatggaata 4080atagaaagag aaaaagcatt ttcaggtata
ggtgttttgg gaaacaattt aaaagaacca 4140ttatatttct ctacatcaga
aaggtataaa tcataaaact ctttgaagtc attctttaca 4200ggagtccaaa
taccagagaa tgttttagat acaccatcaa aaattgtata aagtggctct
4260aacttatccc aataacctaa ctctccgtcg ctattgtaac cagttctaaa
agctgtattt 4320gagtttatca cccttgtcac taagaaaata aatgcagggt
aaaatttata tccttcttgt 4380tttatgtttc ggtataaaac actaatatca
atttctgtgg ttatactaaa agtcgtttgt 4440tggttcaaat aatgattaaa
tatctctttt ctcttccaat tgtctaaatc aattttatta 4500aagttcattt
gatatgcctc ctaaattttt atctaaagtg aatttaggag gcttacttgt
4560ctgctttctt cattagaatc aatccttttt taaagtcaat attactgtaa
cataaatata 4620tattttaaaa atatcccact ttatccaatt ttcgtttgtt
gaactaatgg gtgctttagt 4680tgaagaataa agaccacatt aaaaaatgtg
gtcttttgtg tttttttaaa ggatttgagc 4740gtacgcgaaa aatccttttc
tttctttctt atcttgataa taagggtaac tattgccggt 4800tgtccattca
tggctgaact ctgcttcctc tgttgacatg acacacatca tctcaatatc
4860cgaatagggc ccatcagtct gacgaccaag agagccataa acaccaatag
ccttaacatc 4920atccccatat ttatccaata ttcgttcctt aatttcatga
acaatcttca ttctttcttc 4980tctagtcatt attattggtc cattcactat
tctcattccc ttttcagata attttagatt 5040tgcttttcta aataagaata
tttggagagc accgttctta ttcagctatt aataactcgt 5100cttcctaagc
atccttcaat ccttttaata acaattatag catctaatct tcaacaaact
5160ggcccgtttg ttgaactact ctttaataaa ataatttttc cgttcccaat
tccacattgc 5220aataatagaa aatccatctt catcggcttt ttcgtcatca
tctgtatgaa tcaaatcgcc 5280ttcttctgtg tcatcaaggt ttaatttttt
atgtatttct tttaacaaac caccatagga 5340gattaacctt ttacggtgta
aaccttcctc caaatcagac aaacgtttca aattcttttc 5400ttcatcatcg
gtcataaaat ccgtatcctt tacaggatat tttgcagttt cgtcaattgc
5460cgattgtata tccgatttat atttattttt cggtcgaatc atttgaactt
ttacatttgg 5520atcatagtct aatttcattg cctttttcca aaattgaatc
cattgttttt gattcacgta 5580gttttctgtt attctaaaat aagttggttc
cacacatacc attacatgca tgtgctgatt 5640ataagaatta tctttattat
ttattgtcac atccgttgca cgcataaaac caacaagatt 5700tttattaatt
tttttatatt gcatcattcg gcgaaatcct tgagccatat ctgtcaaact
5760cttatttaat tcttcgccat cataaacatt tttaactgtt aatgtgagaa
acaaccaacg 5820aactgttggc ttttgtttaa taacttcagc aacaaccttt
tgtgactgaa tgccatgttt 5880cattgctctc ctccagttgc acattggaca
aagcctggat ttgcaaaacc acactcgata 5940ccactttctt tcgcctgttt
cacgattttg tttatactct aatatttcag cacaatcttt 6000tactctttca
gcctttttaa attcaagaat atgcagaagt tcaaagtaat caacattagc
6060gattttcttt tctctccatg gtctcacttt tccacttttt gtcttgtcca
ctaaaaccct 6120tgatttttca tctgaataaa tgctactatt aggacacata
atattaaaag aaacccccat 6180ctatttagtt atttgtttag tcacttataa
ctttaacaga tggggttttt ctgtgcaacc 6240aattttaagg gttttcaata
ctttaaaaca catacatacc aacacttcaa cgcacctttc 6300agcaactaaa
ataaaaatga cgttatttct atatgtatca agataagaaa gaacaagttc
6360aaaaccatca aaaaaagaca ccttttcagg tgcttttttt attttataaa
ctcattccct 6420gatctcgact tcgttctttt tttacctctc ggttatgagt
tagttcaaat tcgttctttt 6480taggttctaa atcgtgtttt tcttggaatt
gtgctgtttt atcctttacc ttgtctacaa 6540accccttaaa aacgttttta
aaggctttta agccgtctgt acgttcctta ag 659257176DNAArtificial
SequenceSynthetic Construct 57ctctctttcg gcaacagtcg taactcctgg
gtggagtcga ccagtgccag ggtcgggtat 60ttggcaatat caaaactcat atattccacc
agctatttgt tagtgaataa aagtggttga 120attatttgct caggatgtgg
catngtcaag ggctaatacg actcactata gggctc 17658103DNAArtificial
SequenceSynthetic Construct 58tcgatacctc ggcactggaa gcgctagcgg
actacatcat ccagcgtaat aaataaacaa 60taagtattaa taggcccctg aattaaccct
cactaaaggg cgg 1035922DNAArtificial SequenceSynthetic Construct
59ttgtagacat agtgcagcgc ca 226024DNAArtificial SequenceSynthetic
Construct 60aaagaccgac caagcgacgt ctga 246136DNAArtificial
SequenceSynthetic Construct 61gctgggtacc ctgcccgctt tccagtcggg
aaacct 366236DNAArtificial SequenceSynthetic Construct 62tagaactagt
caaaaaaccc ctcaagaccc gtttag 366317DNAArtificial SequenceSynthetic
Construct 63gtaaaacgac ggccagt 176422DNAArtificial
SequenceSynthetic Construct 64gcactgtctt tccgtctgct gc
226520DNAArtificial SequenceSynthetic Construct 65ctcgtacagg
ctcaggatag 206620DNAArtificial SequenceSynthetic Construct
66ttacgtccca acgctcaact 206736DNAArtificial SequenceSynthetic
Construct 67gctgggtacc ctgcccgctt tccagtcggg aaacct
366836DNAArtificial SequenceSynthetic Construct 68tagaactagt
caaaaaaccc ctcaagaccc gtttag 366917DNAArtificial SequenceSynthetic
Construct 69gtaaaacgac ggccagt 177022DNAArtificial
SequenceSynthetic Construct 70gcactgtctt tccgtctgct gc
227120DNAArtificial SequenceSynthetic Construct 71ctcgtacagg
ctcaggatag 207220DNAArtificial SequenceSynthetic Construct
72ttacgtccca acgctcaact 20738069DNAArtificial SequenceSynthetic
Construct 73accttcggga gcgcctgaag cccgttctgg acgccctggg gccgttgaat
cgggatatgc 60aggccaaggc cgccgcgatc atcaaggccg tgggcgaaaa gctgctgacg
gaacagcggg 120aagtccagcg ccagaaacag gcccagcgcc agcaggaacg
cgggcgcgca catttccccg 180aaaagtgcca cctggcggcg ttgtgacaat
ttaccgaaca actccgcggc cgggaagccg 240atctcggctt gaacgaattg
ttaggtggcg gtacttgggt cgatatcaaa gtgcatcact 300tcttcccgta
tgcccaactt tgtatagaga gccactgcgg gatcgtcacc gtaatctgct
360tgcacgtaga tcacataagc accaagcgcg ttggcctcat gcttgaggag
attgatgagc 420gcggtggcaa tgccctgcct ccggtgctcg ccggagactg
cgagatcata gatatagatc 480tcactacgcg gctgctcaaa cctgggcaga
acgtaagccg cgagagcgcc aacaaccgct 540tcttggtcga aggcagcaag
cgcgatgaat gtcttactac ggagcaagtt cccgaggtaa 600tcggagtccg
gctgatgttg ggagtaggtg gctacgtctc cgaactcacg accgaaaaga
660tcaagagcag cccgcatgga tttgacttgg tcagggccga gcctacatgt
gcgaatgatg 720cccatacttg agccacctaa ctttgtttta gggcgactgc
cctgctgcgt aacatcgttg 780ctgctgcgta acatcgttgc tgctccataa
catcaaacat cgacccacgg cgtaacgcgc 840ttgctgcttg gatgcccgag
gcatagactg tacaaaaaaa cagtcataac aagccatgaa 900aaccgccact
gcgccgttac caccgctgcg ttcggtcaag gttctggacc agttgcgtga
960gcgcatacgc tacttgcatt acagtttacg aaccgaacag gcttatgtca
actgggttcg 1020tgccttcatc cgtttccacg gtgtgcgtcc atgggcaaat
attatacgca aggcgacaag 1080gtgctgatgc cgctggcgat tcaggttcat
catgccgttt gtgatggctt ccatgtcggc 1140agaatgctta atgaattaca
acagttttta tgcatgcgcc caatacgcaa accgcctctc 1200cccgcgcgtt
ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg
1260ggcagtgagc gcaacgcaat taatgtgagt tagctcactc attaggcacc
ccaggcttta 1320cactttatgc ttccggctcg tatgttgtgt ggaattgtga
gcggataaca atttcacaca 1380ggaaacagct atgaccatga ttacgccaag
cgcgcaatta accctcacta aagggaacaa 1440aagctgggta ccctgcccgc
tttccagtcg ggaaacctgt cgtgccagct gcattaatga 1500atcggccaac
gcgcggggag aggcggtttg cgtattgggc gccagggtgg tttttctttt
1560caccagtgag acgggcaaca gctgattgcc cttcaccgcc tggccctgag
agagttgcag 1620caagcggtcc acgctggttt gccccagcag gcgaaaatcc
tgtttgatgg tggttaacgg 1680cgggatataa catgagctgt cttcggtatc
gtcgtatccc actaccgaga tatccgcacc 1740aacgcgcagc ccggactcgg
taatggcgcg cattgcgccc agcgccatct gatcgttggc 1800aaccagcatc
gcagtgggaa cgatgccctc attcagcatt tgcatggttt gttgaaaacc
1860ggacatggca ctccagtcgc cttcccgttc cgctatcggc tgaatttgat
tgcgagtgag 1920atatttatgc cagccagcca gacgcagacg cgccgagaca
gaacttaatg ggcccgctaa 1980cagcgcgatt tgctggtgac ccaatgcgac
cagatgctcc acgcccagtc gcgtaccgtc 2040ttcatgggag aaaataatac
tgttgatggg tgtctggtca gagacatcaa gaaataacgc 2100cggaacatta
gtgcaggcag cttccacagc aatggcatcc tggtcatcca gcggatagtt
2160aatgatcagc ccactgacgc gttgcgcgag aagattgtgc accgccgctt
tacaggcttc 2220gacgccgctt cgttctacca tcgacaccac cacgctggca
cccagttgat cggcgcgaga 2280tttaatcgcc gcgacaattt gcgacggcgc
gtgcagggcc agactggagg tggcaacgcc 2340aatcagcaac gactgtttgc
ccgccagttg ttgtgccacg cggttgggaa tgtaattcag 2400ctccgccatc
gccgcttcca ctttttcccg cgttttcgca gaaacgtggc tggcctggtt
2460caccacgcgg gaaacggtct gataagagac accggcatac tctgcgacat
cgtataacgt 2520tactggtttc acattcacca ccctgaattg actctcttcc
gggcgctatc atgccatacc 2580gcgaaaggtt ttgcgccatt cgatggtgtc
cgggatctcg acgctctccc ttatgcgact 2640cctgcattag gaagcagccc
agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga 2700atggtgcatg
caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac
2760ccacgccgaa acaagcgctc atgagcccga agtggcgagc ccgatcttcc
ccatcggtga 2820tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc
ggtgatgccg gccacgatgc 2880gtccggcgta gaggatcgag atctcgatcc
cgcgaaatta atacgactca ctatagggga 2940attgtgagcg gataacaatt
cccctctaga aataattttg tttaacttta agaaggagat 3000atacatatgg
aagctcgtcg ttctgcgaac tacgaaccta acagctggga ctatgattac
3060ctgctgtcct ccgacacgga cgagtccatc gaagtataca aagacaaagc
gaaaaagctg 3120gaagccgaag ttcgtcgcga gattaataac gaaaaagcag
aatttctgac cctgctggaa 3180ctgattgaca acgtccagcg cctgggcctg
ggttaccgtt tcgagtctga tatccgtggt 3240gcgctggatc gcttcgtttc
ctccggcggc ttcgatgcgg taaccaagac ttccctgcac 3300ggtacggcac
tgtctttccg tctgctgcgt caacacggtt ttgaggtttc tcaggaagcg
3360ttcagcggct tcaaagacca aaacggcaac ttcctggaga acctgaagga
agatatcaaa 3420gctatcctga gcctgtacga ggccagcttc ctggctctgg
aaggcgaaaa catcctggac 3480gaggcgaagg ttttcgcaat ctctcatctg
aaagaactgt ctgaagaaaa gatcggtaaa 3540gagctggcag aacaggtgaa
ccatgcactg gaactgccac tgcatcgccg tactcagcgt 3600ctggaagcag
tatggtctat cgaggcctac cgtaaaaagg aggacgcgaa tcaggttctg
3660ctggagctgg caattctgga ttacaacatg atccagtctg tataccagcg
tgatctgcgt 3720gaaacgtccc gttggtggcg tcgtgtgggt ctggcgacca
aactgcactt tgctcgtgac 3780cgcctgattg agagcttcta ctgggccgtg
ggtgtagcat tcgaaccgca atactccgac 3840tgccgtaact ccgtcgcaaa
aatgttttct ttcgtaacca ttatcgacga tatctacgat 3900gtatacggca
ccctggacga actggagctg tttactgatg cagttgagcg ttgggacgta
3960aacgccatca acgacctgcc ggattacatg aaactgtgct ttctggctct
gtataacact 4020attaacgaaa tcgcctacga caacctgaaa gataaaggtg
agaacatcct gccgtatctg 4080accaaagcct gggctgacct gtgcaacgct
ttcctgcaag aagccaagtg gctgtacaac 4140aaatctactc cgacctttga
cgactacttc ggcaacgcat ggaaatcctc ttctggcccg 4200ctgcaactgg
tgttcgctta cttcgctgtc gtgcagaaca ttaaaaagga agagatcgaa
4260aacctgcaaa aataccatga caccatctct cgtccttccc atatcttccg
tctgtgcaat 4320gacctggcta gcgcgtctgc ggaaattgcg cgtggtgaaa
ccgcaaatag cgtttcttgt 4380tacatgcgca ctaaaggtat ctccgaagaa
ctggctaccg aaagcgtgat gaatctgatc 4440gatgaaacct ggaaaaagat
gaacaaggaa aaactgggtg gtagcctgtt cgcgaaaccg 4500ttcgtggaaa
ccgcgatcaa cctggcacgt caatctcact gcacttatca taacggcgac
4560gcgcatacct ctccggatga gctgacccgc aaacgcgttc tgtctgtaat
cactgaaccg 4620attctgccgt ttgaacgcta aggatccgaa ttcgagctcc
gtcgacaagc ttgcggccgc 4680actcgagcac caccaccacc accactgaga
tccggctgct aacaaagccc gaaaggaagc 4740tgagttggct gctgccaccg
ctgagcaata actagcataa ccccttgggg cctctaaacg 4800ggtcttgagg
ggttttttga ctagttctag agcggccgcc accgcggtgg agctccaatt
4860cgccctatag tgagtcgtat tacgcgcgct cactggccgt cgttttacaa
cgtcgtgact 4920gggaaaaccc tggcgttacc caacttaatc gccttgcagc
acatccccct ttcgccagct 4980ggcgtaatag cgaagaggcc cgcaccgatc
gcccttccca acagttgcgc agcctgaatg 5040gcgaatggaa attgtaagcg
ttaatatttt gttaaaattc gcgttaaatt tttgttaaat 5100cagctcattt
tttaaccaat aggccgactg cgatgagtgg cagggcgggg cgtaattttt
5160ttaaggcagt tattggtgcc cttaaacgcc tggtgctacg cctgaataag
tgataataag 5220cggatgaatg gcagaaattc gaaagcaaat tcgacccggt
cgtcggttca gggcagggtc 5280gttaaatagc cgcttatgtc tattgctggt
ttaccggttt attgactacc ggaagcagtg 5340tgaccgtgtg cttctcaaat
gcctgaggcc agtttgctca ggctctcccc gtggaggtaa 5400taattgacga
tatgatcatt tattctgcct cccagagcct gataaaaacg gtgaatccgt
5460tagcgaggtg ccgccggctt ccattcaggt cgaggtggcc cggctccatg
caccgcgacg 5520caacgcgggg aggcagacaa ggtatagggc ggcgaggcgg
ctacagccga tagtctggaa 5580cagcgcactt acgggttgct gcgcaaccca
agtgctaccg gcgcggcagc gtgacccgtg 5640tcggcggctc caacggctcg
ccatcgtcca gaaaacacgg ctcatcgggc atcggcaggc 5700gctgctgccc
gcgccgttcc cattcctccg tttcggtcaa ggctggcagg tctggttcca
5760tgcccggaat gccgggctgg ctgggcggct cctcgccggg gccggtcggt
agttgctgct 5820cgcccggata cagggtcggg atgcggcgca ggtcgccatg
ccccaacagc gattcgtcct 5880ggtcgtcgtg atcaaccacc acggcggcac
tgaacaccga caggcgcaac tggtcgcggg 5940gctggcccca cgccacgcgg
tcattgacca cgtaggccga cacggtgccg gggccgttga 6000gcttcacgac
ggagatccag cgctcggcca ccaagtcctt gactgcgtat tggaccgtcc
6060gcaaagaacg tccgatgagc ttggaaagtg tcttctggct gaccaccacg
gcgttctggt 6120ggcccatctg cgccacgagg tgatgcagca gcattgccgc
cgtgggtttc ctcgcaataa 6180gcccggccca cgcctcatgc gctttgcgtt
ccgtttgcac ccagtgaccg ggcttgttct 6240tggcttgaat gccgatttct
ctggactgcg tggccatgct tatctccatg cggtagggtg 6300ccgcacggtt
gcggcaccat gcgcaatcag ctgcaacttt tcggcagcgc gacaacaatt
6360atgcgttgcg taaaagtggc agtcaattac agattttctt taacctacgc
aatgagctat 6420tgcggggggt gccgcaatga gctgttgcgt accccccttt
tttaagttgt tgatttttaa 6480gtctttcgca tttcgcccta tatctagttc
tttggtgccc aaagaagggc acccctgcgg 6540ggttccccca cgccttcggc
gcggctcccc ctccggcaaa aagtggcccc tccggggctt 6600gttgatcgac
tgcgcggcct tcggccttgc ccaaggtggc gctgccccct tggaaccccc
6660gcactcgccg ccgtgaggct cggggggcag gcgggcgggc ttcgccttcg
actgccccca 6720ctcgcatagg cttgggtcgt tccaggcgcg tcaaggccaa
gccgctgcgc ggtcgctgcg 6780cgagccttga cccgccttcc acttggtgtc
caaccggcaa gcgaagcgcg caggccgcag 6840gccggaggct tttccccaga
gaaaattaaa aaaattgatg gggcaaggcc gcaggccgcg 6900cagttggagc
cggtgggtat gtggtcgaag gctgggtagc cggtgggcaa tccctgtggt
6960caagctcgtg ggcaggcgca gcctgtccat cagcttgtcc agcagggttg
tccacgggcc 7020gagcgaagcg agccagccgg tggccgctcg cggccatcgt
ccacatatcc acgggctggc 7080aagggagcgc agcgaccgcg cagggcgaag
cccggagagc aagcccgtag ggcgccgcag 7140ccgccgtagg cggtcacgac
tttgcgaagc aaagtctagt gagtatactc aagcattgag 7200tggcccgccg
gaggcaccgc cttgcgctgc ccccgtcgag ccggttggac accaaaaggg
7260aggggcaggc atggcggcat acgcgatcat gcgatgcaag aagctggcga
aaatgggcaa 7320cgtggcggcc agtctcaagc acgcctaccg cgagcgcgag
acgcccaacg ctgacgccag 7380caggacgcca gagaacgagc actgggcggc
cagcagcacc gatgaagcga tgggccgact 7440gcgcgagttg ctgccagaga
agcggcgcaa ggacgctgtg ttggcggtcg agtacgtcat 7500gacggccagc
ccggaatggt ggaagtcggc cagccaagaa cagcaggcgg cgttcttcga
7560gaaggcgcac aagtggctgg cggacaagta cggggcggat cgcatcgtga
cggccagcat 7620ccaccgtgac gaaaccagcc cgcacatgac cgcgttcgtg
gtgccgctga cgcaggacgg 7680caggctgtcg gccaaggagt tcatcggcaa
caaagcgcag atgacccgcg accagaccac 7740gtttgcggcc gctgtggccg
atctagggct gcaacggggc atcgagggca gcaaggcacg 7800tcacacgcgc
attcaggcgt tctacgaggc cctggagcgg ccaccagtgg gccacgtcac
7860catcagcccg caagcggtcg agccacgcgc ctatgcaccg cagggattgg
ccgaaaagct 7920gggaatctca aagcgcgttg agacgccgga agccgtggcc
gaccggctga caaaagcggt 7980tcggcagggg tatgagcctg ccctacaggc
cgccgcagga gcgcgtgaga tgcgcaagaa 8040ggccgatcaa gcccaagaga
cggcccgag 8069745270DNAArtificial SequenceSynthetic Construct
74cgataagcta gcttcacgct gccgcaagca ctcagggcgc aagggctgct aaaggaagcg
60gaacacgtag aaagccagtc cgcagaaacg gtgctgaccc cggatgaatg tcagctactg
120ggctatctgg acaagggaaa acgcaagcgc aaagagaaag caggtagctt
gcagtgggct 180tacatggcga tagctagact gggcggtttt atggacagca
agcgaaccgg aattgccagc 240tggggcgccc tctggtaagg ttgggaagcc
ctgcaaagta aactggatgg ctttcttgcc 300gccaaggatc tgatggcgca
ggggatcaag atctgatcaa gagacaggat gaggatcgtt 360tcgcatgatt
gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct
420attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg
tgttccggct 480gtcagcgcag gggcgcccgg ttctttttgt caagaccgac
ctgtccggtg ccctgaatga 540actccaagac gaggcagcgc ggctatcgtg
gctggccacg acgggcgttc cttgcgcagc 600tgtgctcgac gttgtcactg
aagcgggaag ggactggctg ctattgggcg aagtgccggg 660gcaggatctc
ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc
720aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc
aagcgaaaca 780tcgcatcgag cgagcacgta ctcggatgga agccggtctt
gtcgatcagg atgatctgga 840cgaagagcat caggggctcg cgccagccga
actgttcgcc aggctcaagg cgcggatgcc 900cgacggcgag gatctcgtcg
tgacccatgg cgatgcctgc ttgccgaata tcatggtgga 960aaatggccgc
ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca
1020ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat
gggctgaccg 1080cttcctcgtg ctttacggta tcgccgctcc cgattcgcag
cgcatcgcct tctatcgcct 1140tcttgacgag ttcttctgag cgggactctg
gggttcgcga tgataagctg tcaaacatga 1200gaattacaac ttatatcgta
tggggctgac ttcaggtgct acatttgaag agataaattg 1260cactgaaatc
tagaaatatt ttatctgatt aataagatga tcttcttgag atcgttttgg
1320tctgcgcgta atctcttgct ctgaaaacga aaaaaccgcc ttgcagggcg
gtttttcgaa 1380ggttctctga gctaccaact ctttgaaccg aggtaactgg
cttggaggag cgcagtcacc 1440aaaacttgtc ctttcagttt agccttaacc
ggcgcatgac ttcaagacta actcctctaa 1500atcaattacc agtggctgct
gccagtggtg cttttgcatg tctttccggg ttggactcaa 1560gacgatagtt
accggataag gcgcagcggt cggactgaac ggggggttcg tgcatacagt
1620ccagcttgga gcgaactgcc tacccggaac tgagtgtcag gcgtggaatg
agacaaacgc 1680ggccataaca gcggaatgac accggtaaac cgaaaggcag
gaacaggaga gcgcacgagg 1740gagccgccag gggaaacgcc tggtatcttt
atagtcctgt cgggtttcgc caccactgat 1800ttgagcgtca gatttcgtga
tgcttgtcag gggggcggag cctatggaaa aacggctttg 1860ccttctttcc
tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag
1920ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
gaggaagcgg 1980aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg
gccgattcat taatgcagct 2040ggcacgacag gtttcccgac tggaaagcgg
gcagtgagcg caacgcaatt aatgtgagtt 2100agctcactca ttaggcaccc
caggctttac actttatgct tccggctcgt atgttgtgtg 2160gaattgtgag
cggataacaa tttcacacag gaaacagcta tgaccatgat tacgaattct
2220gttgacaatt aatcatcggc tcgtataatg tgtggaattg tgagcggata
acaatttcac 2280acaggaaaca gattacggat ccatttgagg agtaagccat
gcaaacgttg ccaagcccag 2340ttcaagctac accaacggaa acagctattg
ttagacgcaa aacccgcccg gttccgatag 2400gctccgttgt tattggtggc
ggccatcccg tggctgttca gtcaatgatt aacgaagaca 2460ctctggatat
cgaaggttct gttgctgcaa ttcggcgctt acacgagatc ggttgcgaga
2520tcgtacgtgt gactgtacct tcattagcac acgcgaaagc aatggaagag
attcgggatc 2580ggctttataa aacgtacaaa ccggtcccct tagttgccga
cgtgcatcat aacggaatga 2640aaatcgcgtt agaggttgcc aagtacgtgg
acaatgtgcg cattaatcct ggattatacg 2700tgtttgagaa gccaaaacca
aatcgcacgg agtacactca agctgaattt gacgagattg 2760gcgcgaaaat
ccgtgaaacg ttggaaccac tggtaatttc actgcgggat cagggaaagt
2820cgatgcgcat tggcgttaat catggcagtc tggcggaacg gatgctgttt
acctatggcg 2880ataccccaga gggtatggta gagagtgcac ttgagtttat
acgcatctgt gaaagtctca 2940acttctataa cttagaaatt tcccttaaag
ctagccgcgt cccggttatg atagccgcca 3000atcggcttat ggttaagcgc
atggacgagc tgggtatgga ttatccgttg catctcggag 3060tgactgaggc
aggtgatggt gaatatggcc gtattaaaag cacagcaggc attgcaacac
3120tgctggcgga aggaattgga gacacaatcc gtgtttcatt gactgaagct
ccggaaaagg 3180aaatccccgt gtgctatggc atccttcaag ccctcggtct
ccgccgcacc atggtagaat 3240atgtagcttg cccgtcgtgt ggtcggacat
tgtttaacct ggaagaggtt ctgcacaagg 3300tgagagaagc gactaaacac
ctgacgggac tgaatattgc ggttatggga tgtattgtaa 3360atggacctgg
cgaaatggcc gatgcagact acggctatgt aggtaaacag ccgggatata
3420taagtcttta ccgcggccgg gaagaagtca agaaagtgcc cgaggccgag
ggcgttgcag 3480ctctggtcga actgataaaa gcggatggta gatgggtaga
tccataagtg gagctccccg 3540gtaccgtgga cgaggtttaa tatggcgacg
tataaagtca cactggtccg tccggatggc 3600agcgaaacga ccatcgatgt
tccggaggac gaatacatac tggatgtcgc cgaagaacaa 3660ggtctggatc
tcccgttttc ttgtcgcgcc ggtgcctgct ctacctgtgc tggcaaattg
3720ttggagggag aagtcgatca aagcgaccag agcttcttgg atgacgatca
gatcgaaaaa 3780ggattcgtgc ttacttgtgt ggcctacccc cgttcggact
gcaagatctt gacgaaccaa 3840gaggaggagc tgtactaaga ggtcgacgac
gcatgcatta acagaggtta gtatgtataa 3900tgccactaac tctcgctcac
gtatgttccg gtacgaagtt gtggggctgc gccaaacggc 3960ggagacggag
aaaacaaatt acgcgatcag aaactctggc tcgcagttct ttaatgtgcc
4020ttatgaccgc atgaaccagt ttatgcagca gatcactcgg tggggcggta
aaattgtcag 4080tattcagccc cttaacggaa ccgtggcccc acttgctgca
accacggagc cagctgccaa 4140taacggagct gcacctgtga aagaaaagaa
agtcgatata ccggtcaaca tctaccgtcc 4200caataatccc tgcataggta
aggttattag caacgaggaa ctggtccggg aaggcggtga 4260gggtacggtg
aaacatatta tctttgatat atcggggacc gaattacgtt acttggaagg
4320gcagtcaatc ggtatcattc ccgcgggcac ggacgcgaac ggtaaaccac
ataagctgcg 4380tctgtattcc attgcttcca caagacatgg tgactttcag
gatgacaaga cggtgtcctt 4440atgcgtacgg agattagaat acaaagataa
agagaccggg gagaccattt atggcgtgtg 4500cagttcgtat cttaatcagt
tacagcctgg agatgaagtc aaaatcacag gtcctgttgg 4560gaaagaaatg
cttctctctg acgacccaga agcgactatt attatgctgg ctaccggcac
4620tggaatagcg ccatttcggg catttttatg gcggatgttc aaagagaaca
acccggatta 4680ccagttcaaa ggccttgcgt ggctgttctt tggcgtcgct
tatactgcca atatcctgta 4740taaggacgag cttgaagcta tccaagccca
gtatcccgat cattttcggt taacctacgc 4800gatttcccgt gaacaaaaaa
ccccggacgg agggaaaatg tacatccagg gtcggatcgc 4860agagcacgct
gatgaaatct ggcaactgct gcaaaagaaa aacacccacg tgtacatgtg
4920tggcctgcgt gggatggaac ctggaataga cgaggccatg accgcagcgg
ccgcgaaaaa 4980cggagctgac tggcaggagt ttctgaaagg tacgctgaaa
aaggaaggca gatggcatgt 5040cgaaacttat taactgcagt acaaataaaa
aaggcacgtc agatgacgtg ccttttttct 5100tgaagcttgg cactggccgt
cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc 5160caacttaatc
gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc
5220cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggcg
5270758072DNAArtificial SequenceSynthetic Construct 75accttcggga
gcgcctgaag cccgttctgg acgccctggg gccgttgaat cgggatatgc 60aggccaaggc
cgccgcgatc atcaaggccg tgggcgaaaa gctgctgacg gaacagcggg
120aagtccagcg ccagaaacag gcccagcgcc agcaggaacg cgggcgcgca
catttccccg 180aaaagtgcca cctggcggcg ttgtgacaat ttaccgaaca
actccgcggc cgggaagccg 240atctcggctt gaacgaattg ttaggtggcg
gtacttgggt cgatatcaaa gtgcatcact 300tcttcccgta tgcccaactt
tgtatagaga gccactgcgg gatcgtcacc gtaatctgct 360tgcacgtaga
tcacataagc accaagcgcg ttggcctcat gcttgaggag attgatgagc
420gcggtggcaa tgccctgcct ccggtgctcg ccggagactg cgagatcata
gatatagatc 480tcactacgcg gctgctcaaa cctgggcaga acgtaagccg
cgagagcgcc aacaaccgct 540tcttggtcga aggcagcaag cgcgatgaat
gtcttactac ggagcaagtt cccgaggtaa 600tcggagtccg gctgatgttg
ggagtaggtg gctacgtctc cgaactcacg accgaaaaga 660tcaagagcag
cccgcatgga tttgacttgg tcagggccga gcctacatgt gcgaatgatg
720cccatacttg agccacctaa ctttgtttta gggcgactgc cctgctgcgt
aacatcgttg 780ctgctgcgta acatcgttgc tgctccataa catcaaacat
cgacccacgg cgtaacgcgc 840ttgctgcttg gatgcccgag gcatagactg
tacaaaaaaa cagtcataac aagccatgaa 900aaccgccact gcgccgttac
caccgctgcg ttcggtcaag gttctggacc agttgcgtga 960gcgcatacgc
tacttgcatt acagtttacg aaccgaacag gcttatgtca actgggttcg
1020tgccttcatc cgtttccacg gtgtgcgtcc atgggcaaat attatacgca
aggcgacaag 1080gtgctgatgc cgctggcgat tcaggttcat catgccgttt
gtgatggctt ccatgtcggc 1140agaatgctta atgaattaca acagttttta
tgcatgcgcc caatacgcaa accgcctctc 1200cccgcgcgtt ggccgattca
ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg 1260ggcagtgagc
gcaacgcaat taatgtgagt tagctcactc attaggcacc ccaggcttta
1320cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca
atttcacaca 1380ggaaacagct atgaccatga ttacgccaag cgcgcaatta
accctcacta aagggaacaa 1440aagctgggta ccctgcccgc tttccagtcg
ggaaacctgt cgtgccagct gcattaatga 1500atcggccaac gcgcggggag
aggcggtttg cgtattgggc gccagggtgg tttttctttt 1560caccagtgag
acgggcaaca gctgattgcc cttcaccgcc tggccctgag agagttgcag
1620caagcggtcc acgctggttt gccccagcag gcgaaaatcc tgtttgatgg
tggttaacgg 1680cgggatataa catgagctgt cttcggtatc gtcgtatccc
actaccgaga tatccgcacc 1740aacgcgcagc ccggactcgg taatggcgcg
cattgcgccc agcgccatct gatcgttggc 1800aaccagcatc gcagtgggaa
cgatgccctc attcagcatt tgcatggttt gttgaaaacc 1860ggacatggca
ctccagtcgc cttcccgttc cgctatcggc tgaatttgat tgcgagtgag
1920atatttatgc cagccagcca gacgcagacg cgccgagaca gaacttaatg
ggcccgctaa 1980cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc
acgcccagtc gcgtaccgtc 2040ttcatgggag aaaataatac tgttgatggg
tgtctggtca gagacatcaa gaaataacgc 2100cggaacatta gtgcaggcag
cttccacagc aatggcatcc tggtcatcca gcggatagtt 2160aatgatcagc
ccactgacgc gttgcgcgag aagattgtgc accgccgctt tacaggcttc
2220gacgccgctt cgttctacca tcgacaccac cacgctggca cccagttgat
cggcgcgaga 2280tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc
agactggagg tggcaacgcc 2340aatcagcaac gactgtttgc ccgccagttg
ttgtgccacg cggttgggaa tgtaattcag 2400ctccgccatc gccgcttcca
ctttttcccg cgttttcgca gaaacgtggc tggcctggtt 2460caccacgcgg
gaaacggtct gataagagac accggcatac tctgcgacat cgtataacgt
2520tactggtttc acattcacca ccctgaattg actctcttcc gggcgctatc
atgccatacc 2580gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg
acgctctccc ttatgcgact 2640cctgcattag gaagcagccc agtagtaggt
tgaggccgtt gagcaccgcc gccgcaagga 2700atggtgcatg caaggagatg
gcgcccaaca gtcccccggc cacggggcct gccaccatac 2760ccacgccgaa
acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga
2820tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg
gccacgatgc 2880gtccggcgta gaggatcgag atctcgatcc cgcgaaatta
atacgactca ctatagggga 2940attgtgagcg gataacaatt cccctctaga
aataattttg tttaacttta agaaggagat 3000atacatatga ccgaagctcg
tcgttctgcg aactacgaac ctaacagctg ggactatgat 3060tacctgctgt
cctccgacac ggacgagtcc atcgaagtat acaaagacaa agcgaaaaag
3120ctggaagccg aagttcgtcg cgagattaat aacgaaaaag cagaatttct
gaccctgctg 3180gaactgattg acaacgtcca gcgcctgggc ctgggttacc
gtttcgagtc tgatatccgt 3240ggtgcgctgg atcgcttcgt ttcctccggc
ggcttcgatg cggtaaccaa gacttccctg 3300cacggtacgg cactgtcttt
ccgtctgctg cgtcaacacg gttttgaggt ttctcaggaa 3360gcgttcagcg
gcttcaaaga ccaaaacggc aacttcctgg agaacctgaa ggaagatatc
3420aaagctatcc tgagcctgta cgaggccagc ttcctggctc tggaaggcga
aaacatcctg 3480gacgaggcga aggttttcgc aatctctcat ctgaaagaac
tgtctgaaga aaagatcggt 3540aaagagctgg cagaacaggt gaaccatgca
ctggaactgc cactgcatcg ccgtactcag 3600cgtctggaag cagtatggtc
tatcgaggcc taccgtaaaa aggaggacgc gaatcaggtt 3660ctgctggagc
tggcaattct ggattacaac atgatccagt ctgtatacca gcgtgatctg
3720cgtgaaacgt cccgttggtg gcgtcgtgtg ggtctggcga ccaaactgca
ctttgctcgt 3780gaccgcctga ttgagagctt ctactgggcc gtgggtgtag
cattcgaacc gcaatactcc 3840gactgccgta actccgtcgc aaaaatgttt
tctttcgtaa ccattatcga cgatatctac 3900gatgtatacg gcaccctgga
cgaactggag ctgtttactg atgcagttga gcgttgggac 3960gtaaacgcca
tcaacgacct gccggattac atgaaactgt gctttctggc tctgtataac
4020actattaacg aaatcgccta cgacaacctg aaagataaag gtgagaacat
cctgccgtat 4080ctgaccaaag cctgggctga cctgtgcaac gctttcctgc
aagaagccaa gtggctgtac 4140aacaaatcta ctccgacctt tgacgactac
ttcggcaacg catggaaatc ctcttctggc 4200ccgctgcaac tggtgttcgc
ttacttcgct gtcgtgcaga acattaaaaa
ggaagagatc 4260gaaaacctgc aaaaatacca tgacaccatc tctcgtcctt
cccatatctt ccgtctgtgc 4320aatgacctgg ctagcgcgtc tgcggaaatt
gcgcgtggtg aaaccgcaaa tagcgtttct 4380tgttacatgc gcactaaagg
tatctccgaa gaactggcta ccgaaagcgt gatgaatctg 4440atcgatgaaa
cctggaaaaa gatgaacaag gaaaaactgg gtggtagcct gttcgcgaaa
4500ccgttcgtgg aaaccgcgat caacctggca cgtcaatctc actgcactta
tcataacggc 4560gacgcgcata cctctccgga tgagctgacc cgcaaacgcg
ttctgtctgt aatcactgaa 4620ccgattctgc cgtttgaacg ctaaggatcc
gaattcgagc tccgtcgaca agcttgcggc 4680cgcactcgag caccaccacc
accaccactg agatccggct gctaacaaag cccgaaagga 4740agctgagttg
gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa
4800acgggtcttg aggggttttt tgactagttc tagagcggcc gccaccgcgg
tggagctcca 4860attcgcccta tagtgagtcg tattacgcgc gctcactggc
cgtcgtttta caacgtcgtg 4920actgggaaaa ccctggcgtt acccaactta
atcgccttgc agcacatccc cctttcgcca 4980gctggcgtaa tagcgaagag
gcccgcaccg atcgcccttc ccaacagttg cgcagcctga 5040atggcgaatg
gaaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta
5100aatcagctca ttttttaacc aataggccga ctgcgatgag tggcagggcg
gggcgtaatt 5160tttttaaggc agttattggt gcccttaaac gcctggtgct
acgcctgaat aagtgataat 5220aagcggatga atggcagaaa ttcgaaagca
aattcgaccc ggtcgtcggt tcagggcagg 5280gtcgttaaat agccgcttat
gtctattgct ggtttaccgg tttattgact accggaagca 5340gtgtgaccgt
gtgcttctca aatgcctgag gccagtttgc tcaggctctc cccgtggagg
5400taataattga cgatatgatc atttattctg cctcccagag cctgataaaa
acggtgaatc 5460cgttagcgag gtgccgccgg cttccattca ggtcgaggtg
gcccggctcc atgcaccgcg 5520acgcaacgcg gggaggcaga caaggtatag
ggcggcgagg cggctacagc cgatagtctg 5580gaacagcgca cttacgggtt
gctgcgcaac ccaagtgcta ccggcgcggc agcgtgaccc 5640gtgtcggcgg
ctccaacggc tcgccatcgt ccagaaaaca cggctcatcg ggcatcggca
5700ggcgctgctg cccgcgccgt tcccattcct ccgtttcggt caaggctggc
aggtctggtt 5760ccatgcccgg aatgccgggc tggctgggcg gctcctcgcc
ggggccggtc ggtagttgct 5820gctcgcccgg atacagggtc gggatgcggc
gcaggtcgcc atgccccaac agcgattcgt 5880cctggtcgtc gtgatcaacc
accacggcgg cactgaacac cgacaggcgc aactggtcgc 5940ggggctggcc
ccacgccacg cggtcattga ccacgtaggc cgacacggtg ccggggccgt
6000tgagcttcac gacggagatc cagcgctcgg ccaccaagtc cttgactgcg
tattggaccg 6060tccgcaaaga acgtccgatg agcttggaaa gtgtcttctg
gctgaccacc acggcgttct 6120ggtggcccat ctgcgccacg aggtgatgca
gcagcattgc cgccgtgggt ttcctcgcaa 6180taagcccggc ccacgcctca
tgcgctttgc gttccgtttg cacccagtga ccgggcttgt 6240tcttggcttg
aatgccgatt tctctggact gcgtggccat gcttatctcc atgcggtagg
6300gtgccgcacg gttgcggcac catgcgcaat cagctgcaac ttttcggcag
cgcgacaaca 6360attatgcgtt gcgtaaaagt ggcagtcaat tacagatttt
ctttaaccta cgcaatgagc 6420tattgcgggg ggtgccgcaa tgagctgttg
cgtacccccc ttttttaagt tgttgatttt 6480taagtctttc gcatttcgcc
ctatatctag ttctttggtg cccaaagaag ggcacccctg 6540cggggttccc
ccacgccttc ggcgcggctc cccctccggc aaaaagtggc ccctccgggg
6600cttgttgatc gactgcgcgg ccttcggcct tgcccaaggt ggcgctgccc
ccttggaacc 6660cccgcactcg ccgccgtgag gctcgggggg caggcgggcg
ggcttcgcct tcgactgccc 6720ccactcgcat aggcttgggt cgttccaggc
gcgtcaaggc caagccgctg cgcggtcgct 6780gcgcgagcct tgacccgcct
tccacttggt gtccaaccgg caagcgaagc gcgcaggccg 6840caggccggag
gcttttcccc agagaaaatt aaaaaaattg atggggcaag gccgcaggcc
6900gcgcagttgg agccggtggg tatgtggtcg aaggctgggt agccggtggg
caatccctgt 6960ggtcaagctc gtgggcaggc gcagcctgtc catcagcttg
tccagcaggg ttgtccacgg 7020gccgagcgaa gcgagccagc cggtggccgc
tcgcggccat cgtccacata tccacgggct 7080ggcaagggag cgcagcgacc
gcgcagggcg aagcccggag agcaagcccg tagggcgccg 7140cagccgccgt
aggcggtcac gactttgcga agcaaagtct agtgagtata ctcaagcatt
7200gagtggcccg ccggaggcac cgccttgcgc tgcccccgtc gagccggttg
gacaccaaaa 7260gggaggggca ggcatggcgg catacgcgat catgcgatgc
aagaagctgg cgaaaatggg 7320caacgtggcg gccagtctca agcacgccta
ccgcgagcgc gagacgccca acgctgacgc 7380cagcaggacg ccagagaacg
agcactgggc ggccagcagc accgatgaag cgatgggccg 7440actgcgcgag
ttgctgccag agaagcggcg caaggacgct gtgttggcgg tcgagtacgt
7500catgacggcc agcccggaat ggtggaagtc ggccagccaa gaacagcagg
cggcgttctt 7560cgagaaggcg cacaagtggc tggcggacaa gtacggggcg
gatcgcatcg tgacggccag 7620catccaccgt gacgaaacca gcccgcacat
gaccgcgttc gtggtgccgc tgacgcagga 7680cggcaggctg tcggccaagg
agttcatcgg caacaaagcg cagatgaccc gcgaccagac 7740cacgtttgcg
gccgctgtgg ccgatctagg gctgcaacgg ggcatcgagg gcagcaaggc
7800acgtcacacg cgcattcagg cgttctacga ggccctggag cggccaccag
tgggccacgt 7860caccatcagc ccgcaagcgg tcgagccacg cgcctatgca
ccgcagggat tggccgaaaa 7920gctgggaatc tcaaagcgcg ttgagacgcc
ggaagccgtg gccgaccggc tgacaaaagc 7980ggttcggcag gggtatgagc
ctgccctaca ggccgccgca ggagcgcgtg agatgcgcaa 8040gaaggccgat
caagcccaag agacggcccg ag 8072768102DNAArtificial SequenceSynthetic
Construct 76accttcggga gcgcctgaag cccgttctgg acgccctggg gccgttgaat
cgggatatgc 60aggccaaggc cgccgcgatc atcaaggccg tgggcgaaaa gctgctgacg
gaacagcggg 120aagtccagcg ccagaaacag gcccagcgcc agcaggaacg
cgggcgcgca catttccccg 180aaaagtgcca cctggcggcg ttgtgacaat
ttaccgaaca actccgcggc cgggaagccg 240atctcggctt gaacgaattg
ttaggtggcg gtacttgggt cgatatcaaa gtgcatcact 300tcttcccgta
tgcccaactt tgtatagaga gccactgcgg gatcgtcacc gtaatctgct
360tgcacgtaga tcacataagc accaagcgcg ttggcctcat gcttgaggag
attgatgagc 420gcggtggcaa tgccctgcct ccggtgctcg ccggagactg
cgagatcata gatatagatc 480tcactacgcg gctgctcaaa cctgggcaga
acgtaagccg cgagagcgcc aacaaccgct 540tcttggtcga aggcagcaag
cgcgatgaat gtcttactac ggagcaagtt cccgaggtaa 600tcggagtccg
gctgatgttg ggagtaggtg gctacgtctc cgaactcacg accgaaaaga
660tcaagagcag cccgcatgga tttgacttgg tcagggccga gcctacatgt
gcgaatgatg 720cccatacttg agccacctaa ctttgtttta gggcgactgc
cctgctgcgt aacatcgttg 780ctgctgcgta acatcgttgc tgctccataa
catcaaacat cgacccacgg cgtaacgcgc 840ttgctgcttg gatgcccgag
gcatagactg tacaaaaaaa cagtcataac aagccatgaa 900aaccgccact
gcgccgttac caccgctgcg ttcggtcaag gttctggacc agttgcgtga
960gcgcatacgc tacttgcatt acagtttacg aaccgaacag gcttatgtca
actgggttcg 1020tgccttcatc cgtttccacg gtgtgcgtcc atgggcaaat
attatacgca aggcgacaag 1080gtgctgatgc cgctggcgat tcaggttcat
catgccgttt gtgatggctt ccatgtcggc 1140agaatgctta atgaattaca
acagttttta tgcatgcgcc caatacgcaa accgcctctc 1200cccgcgcgtt
ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg
1260ggcagtgagc gcaacgcaat taatgtgagt tagctcactc attaggcacc
ccaggcttta 1320cactttatgc ttccggctcg tatgttgtgt ggaattgtga
gcggataaca atttcacaca 1380ggaaacagct atgaccatga ttacgccaag
cgcgcaatta accctcacta aagggaacaa 1440aagctgggta ccctgcccgc
tttccagtcg ggaaacctgt cgtgccagct gcattaatga 1500atcggccaac
gcgcggggag aggcggtttg cgtattgggc gccagggtgg tttttctttt
1560caccagtgag acgggcaaca gctgattgcc cttcaccgcc tggccctgag
agagttgcag 1620caagcggtcc acgctggttt gccccagcag gcgaaaatcc
tgtttgatgg tggttaacgg 1680cgggatataa catgagctgt cttcggtatc
gtcgtatccc actaccgaga tatccgcacc 1740aacgcgcagc ccggactcgg
taatggcgcg cattgcgccc agcgccatct gatcgttggc 1800aaccagcatc
gcagtgggaa cgatgccctc attcagcatt tgcatggttt gttgaaaacc
1860ggacatggca ctccagtcgc cttcccgttc cgctatcggc tgaatttgat
tgcgagtgag 1920atatttatgc cagccagcca gacgcagacg cgccgagaca
gaacttaatg ggcccgctaa 1980cagcgcgatt tgctggtgac ccaatgcgac
cagatgctcc acgcccagtc gcgtaccgtc 2040ttcatgggag aaaataatac
tgttgatggg tgtctggtca gagacatcaa gaaataacgc 2100cggaacatta
gtgcaggcag cttccacagc aatggcatcc tggtcatcca gcggatagtt
2160aatgatcagc ccactgacgc gttgcgcgag aagattgtgc accgccgctt
tacaggcttc 2220gacgccgctt cgttctacca tcgacaccac cacgctggca
cccagttgat cggcgcgaga 2280tttaatcgcc gcgacaattt gcgacggcgc
gtgcagggcc agactggagg tggcaacgcc 2340aatcagcaac gactgtttgc
ccgccagttg ttgtgccacg cggttgggaa tgtaattcag 2400ctccgccatc
gccgcttcca ctttttcccg cgttttcgca gaaacgtggc tggcctggtt
2460caccacgcgg gaaacggtct gataagagac accggcatac tctgcgacat
cgtataacgt 2520tactggtttc acattcacca ccctgaattg actctcttcc
gggcgctatc atgccatacc 2580gcgaaaggtt ttgcgccatt cgatggtgtc
cgggatctcg acgctctccc ttatgcgact 2640cctgcattag gaagcagccc
agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga 2700atggtgcatg
caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac
2760ccacgccgaa acaagcgctc atgagcccga agtggcgagc ccgatcttcc
ccatcggtga 2820tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc
ggtgatgccg gccacgatgc 2880gtccggcgta gaggatcgag atctcgatcc
cgcgaaatta atacgactca ctatagggga 2940attgtgagcg gataacaatt
cccctctaga aataattttg tttaacttta agaaggagat 3000atacatatga
ccgaaaatgt gtctttcacc gaaactgaaa ccgaagctcg tcgttctgcg
3060aactacgaac ctaacagctg ggactatgat tacctgctgt cctccgacac
ggacgagtcc 3120atcgaagtat acaaagacaa agcgaaaaag ctggaagccg
aagttcgtcg cgagattaat 3180aacgaaaaag cagaatttct gaccctgctg
gaactgattg acaacgtcca gcgcctgggc 3240ctgggttacc gtttcgagtc
tgatatccgt ggtgcgctgg atcgcttcgt ttcctccggc 3300ggcttcgatg
cggtaaccaa gacttccctg cacggtacgg cactgtcttt ccgtctgctg
3360cgtcaacacg gttttgaggt ttctcaggaa gcgttcagcg gcttcaaaga
ccaaaacggc 3420aacttcctgg agaacctgaa ggaagatatc aaagctatcc
tgagcctgta cgaggccagc 3480ttcctggctc tggaaggcga aaacatcctg
gacgaggcga aggttttcgc aatctctcat 3540ctgaaagaac tgtctgaaga
aaagatcggt aaagagctgg cagaacaggt gaaccatgca 3600ctggaactgc
cactgcatcg ccgtactcag cgtctggaag cagtatggtc tatcgaggcc
3660taccgtaaaa aggaggacgc gaatcaggtt ctgctggagc tggcaattct
ggattacaac 3720atgatccagt ctgtatacca gcgtgatctg cgtgaaacgt
cccgttggtg gcgtcgtgtg 3780ggtctggcga ccaaactgca ctttgctcgt
gaccgcctga ttgagagctt ctactgggcc 3840gtgggtgtag cattcgaacc
gcaatactcc gactgccgta actccgtcgc aaaaatgttt 3900tctttcgtaa
ccattatcga cgatatctac gatgtatacg gcaccctgga cgaactggag
3960ctgtttactg atgcagttga gcgttgggac gtaaacgcca tcaacgacct
gccggattac 4020atgaaactgt gctttctggc tctgtataac actattaacg
aaatcgccta cgacaacctg 4080aaagataaag gtgagaacat cctgccgtat
ctgaccaaag cctgggctga cctgtgcaac 4140gctttcctgc aagaagccaa
gtggctgtac aacaaatcta ctccgacctt tgacgactac 4200ttcggcaacg
catggaaatc ctcttctggc ccgctgcaac tggtgttcgc ttacttcgct
4260gtcgtgcaga acattaaaaa ggaagagatc gaaaacctgc aaaaatacca
tgacaccatc 4320tctcgtcctt cccatatctt ccgtctgtgc aatgacctgg
ctagcgcgtc tgcggaaatt 4380gcgcgtggtg aaaccgcaaa tagcgtttct
tgttacatgc gcactaaagg tatctccgaa 4440gaactggcta ccgaaagcgt
gatgaatctg atcgatgaaa cctggaaaaa gatgaacaag 4500gaaaaactgg
gtggtagcct gttcgcgaaa ccgttcgtgg aaaccgcgat caacctggca
4560cgtcaatctc actgcactta tcataacggc gacgcgcata cctctccgga
tgagctgacc 4620cgcaaacgcg ttctgtctgt aatcactgaa ccgattctgc
cgtttgaacg ctaaggatcc 4680gaattcgagc tccgtcgaca agcttgcggc
cgcactcgag caccaccacc accaccactg 4740agatccggct gctaacaaag
cccgaaagga agctgagttg gctgctgcca ccgctgagca 4800ataactagca
taaccccttg gggcctctaa acgggtcttg aggggttttt tgactagttc
4860tagagcggcc gccaccgcgg tggagctcca attcgcccta tagtgagtcg
tattacgcgc 4920gctcactggc cgtcgtttta caacgtcgtg actgggaaaa
ccctggcgtt acccaactta 4980atcgccttgc agcacatccc cctttcgcca
gctggcgtaa tagcgaagag gcccgcaccg 5040atcgcccttc ccaacagttg
cgcagcctga atggcgaatg gaaattgtaa gcgttaatat 5100tttgttaaaa
ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga
5160ctgcgatgag tggcagggcg gggcgtaatt tttttaaggc agttattggt
gcccttaaac 5220gcctggtgct acgcctgaat aagtgataat aagcggatga
atggcagaaa ttcgaaagca 5280aattcgaccc ggtcgtcggt tcagggcagg
gtcgttaaat agccgcttat gtctattgct 5340ggtttaccgg tttattgact
accggaagca gtgtgaccgt gtgcttctca aatgcctgag 5400gccagtttgc
tcaggctctc cccgtggagg taataattga cgatatgatc atttattctg
5460cctcccagag cctgataaaa acggtgaatc cgttagcgag gtgccgccgg
cttccattca 5520ggtcgaggtg gcccggctcc atgcaccgcg acgcaacgcg
gggaggcaga caaggtatag 5580ggcggcgagg cggctacagc cgatagtctg
gaacagcgca cttacgggtt gctgcgcaac 5640ccaagtgcta ccggcgcggc
agcgtgaccc gtgtcggcgg ctccaacggc tcgccatcgt 5700ccagaaaaca
cggctcatcg ggcatcggca ggcgctgctg cccgcgccgt tcccattcct
5760ccgtttcggt caaggctggc aggtctggtt ccatgcccgg aatgccgggc
tggctgggcg 5820gctcctcgcc ggggccggtc ggtagttgct gctcgcccgg
atacagggtc gggatgcggc 5880gcaggtcgcc atgccccaac agcgattcgt
cctggtcgtc gtgatcaacc accacggcgg 5940cactgaacac cgacaggcgc
aactggtcgc ggggctggcc ccacgccacg cggtcattga 6000ccacgtaggc
cgacacggtg ccggggccgt tgagcttcac gacggagatc cagcgctcgg
6060ccaccaagtc cttgactgcg tattggaccg tccgcaaaga acgtccgatg
agcttggaaa 6120gtgtcttctg gctgaccacc acggcgttct ggtggcccat
ctgcgccacg aggtgatgca 6180gcagcattgc cgccgtgggt ttcctcgcaa
taagcccggc ccacgcctca tgcgctttgc 6240gttccgtttg cacccagtga
ccgggcttgt tcttggcttg aatgccgatt tctctggact 6300gcgtggccat
gcttatctcc atgcggtagg gtgccgcacg gttgcggcac catgcgcaat
6360cagctgcaac ttttcggcag cgcgacaaca attatgcgtt gcgtaaaagt
ggcagtcaat 6420tacagatttt ctttaaccta cgcaatgagc tattgcgggg
ggtgccgcaa tgagctgttg 6480cgtacccccc ttttttaagt tgttgatttt
taagtctttc gcatttcgcc ctatatctag 6540ttctttggtg cccaaagaag
ggcacccctg cggggttccc ccacgccttc ggcgcggctc 6600cccctccggc
aaaaagtggc ccctccgggg cttgttgatc gactgcgcgg ccttcggcct
6660tgcccaaggt ggcgctgccc ccttggaacc cccgcactcg ccgccgtgag
gctcgggggg 6720caggcgggcg ggcttcgcct tcgactgccc ccactcgcat
aggcttgggt cgttccaggc 6780gcgtcaaggc caagccgctg cgcggtcgct
gcgcgagcct tgacccgcct tccacttggt 6840gtccaaccgg caagcgaagc
gcgcaggccg caggccggag gcttttcccc agagaaaatt 6900aaaaaaattg
atggggcaag gccgcaggcc gcgcagttgg agccggtggg tatgtggtcg
6960aaggctgggt agccggtggg caatccctgt ggtcaagctc gtgggcaggc
gcagcctgtc 7020catcagcttg tccagcaggg ttgtccacgg gccgagcgaa
gcgagccagc cggtggccgc 7080tcgcggccat cgtccacata tccacgggct
ggcaagggag cgcagcgacc gcgcagggcg 7140aagcccggag agcaagcccg
tagggcgccg cagccgccgt aggcggtcac gactttgcga 7200agcaaagtct
agtgagtata ctcaagcatt gagtggcccg ccggaggcac cgccttgcgc
7260tgcccccgtc gagccggttg gacaccaaaa gggaggggca ggcatggcgg
catacgcgat 7320catgcgatgc aagaagctgg cgaaaatggg caacgtggcg
gccagtctca agcacgccta 7380ccgcgagcgc gagacgccca acgctgacgc
cagcaggacg ccagagaacg agcactgggc 7440ggccagcagc accgatgaag
cgatgggccg actgcgcgag ttgctgccag agaagcggcg 7500caaggacgct
gtgttggcgg tcgagtacgt catgacggcc agcccggaat ggtggaagtc
7560ggccagccaa gaacagcagg cggcgttctt cgagaaggcg cacaagtggc
tggcggacaa 7620gtacggggcg gatcgcatcg tgacggccag catccaccgt
gacgaaacca gcccgcacat 7680gaccgcgttc gtggtgccgc tgacgcagga
cggcaggctg tcggccaagg agttcatcgg 7740caacaaagcg cagatgaccc
gcgaccagac cacgtttgcg gccgctgtgg ccgatctagg 7800gctgcaacgg
ggcatcgagg gcagcaaggc acgtcacacg cgcattcagg cgttctacga
7860ggccctggag cggccaccag tgggccacgt caccatcagc ccgcaagcgg
tcgagccacg 7920cgcctatgca ccgcagggat tggccgaaaa gctgggaatc
tcaaagcgcg ttgagacgcc 7980ggaagccgtg gccgaccggc tgacaaaagc
ggttcggcag gggtatgagc ctgccctaca 8040ggccgccgca ggagcgcgtg
agatgcgcaa gaaggccgat caagcccaag agacggcccg 8100ag
8102776511DNAArtificial SequenceSynthetic Construct 77cgataagcta
gcttcacgct gccgcaagca ctcagggcgc aagggctgct aaaggaagcg 60gaacacgtag
aaagccagtc cgcagaaacg gtgctgaccc cggatgaatg tcagctactg
120ggctatctgg acaagggaaa acgcaagcgc aaagagaaag caggtagctt
gcagtgggct 180tacatggcga tagctagact gggcggtttt atggacagca
agcgaaccgg aattgccagc 240tggggcgccc tctggtaagg ttgggaagcc
ctgcaaagta aactggatgg ctttcttgcc 300gccaaggatc tgatggcgca
ggggatcaag atctgatcaa gagacaggat gaggatcgtt 360tcgcatgatt
gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct
420attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg
tgttccggct 480gtcagcgcag gggcgcccgg ttctttttgt caagaccgac
ctgtccggtg ccctgaatga 540actccaagac gaggcagcgc ggctatcgtg
gctggccacg acgggcgttc cttgcgcagc 600tgtgctcgac gttgtcactg
aagcgggaag ggactggctg ctattgggcg aagtgccggg 660gcaggatctc
ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc
720aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc
aagcgaaaca 780tcgcatcgag cgagcacgta ctcggatgga agccggtctt
gtcgatcagg atgatctgga 840cgaagagcat caggggctcg cgccagccga
actgttcgcc aggctcaagg cgcggatgcc 900cgacggcgag gatctcgtcg
tgacccatgg cgatgcctgc ttgccgaata tcatggtgga 960aaatggccgc
ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca
1020ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat
gggctgaccg 1080cttcctcgtg ctttacggta tcgccgctcc cgattcgcag
cgcatcgcct tctatcgcct 1140tcttgacgag ttcttctgag cgggactctg
gggttcgcga tgataagctg tcaaacatga 1200gaattacaac ttatatcgta
tggggctgac ttcaggtgct acatttgaag agataaattg 1260cactgaaatc
tagaaatatt ttatctgatt aataagatga tcttcttgag atcgttttgg
1320tctgcgcgta atctcttgct ctgaaaacga aaaaaccgcc ttgcagggcg
gtttttcgaa 1380ggttctctga gctaccaact ctttgaaccg aggtaactgg
cttggaggag cgcagtcacc 1440aaaacttgtc ctttcagttt agccttaacc
ggcgcatgac ttcaagacta actcctctaa 1500atcaattacc agtggctgct
gccagtggtg cttttgcatg tctttccggg ttggactcaa 1560gacgatagtt
accggataag gcgcagcggt cggactgaac ggggggttcg tgcatacagt
1620ccagcttgga gcgaactgcc tacccggaac tgagtgtcag gcgtggaatg
agacaaacgc 1680ggccataaca gcggaatgac accggtaaac cgaaaggcag
gaacaggaga gcgcacgagg 1740gagccgccag gggaaacgcc tggtatcttt
atagtcctgt cgggtttcgc caccactgat 1800ttgagcgtca gatttcgtga
tgcttgtcag gggggcggag cctatggaaa aacggctttg 1860ccttctttcc
tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag
1920ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
gaggaagcgg 1980aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg
gccgattcat taatgcagct 2040ggcacgacag gtttcccgac tggaaagcgg
gcagtgagcg caacgcaatt aatgtgagtt 2100agctcactca ttaggcaccc
caggctttac actttatgct tccggctcgt atgttgtgtg 2160gaattgtgag
cggataacaa tttcacacag gaaacagcta tgaccatgat tacgaattct
2220gttgacaatt aatcatcggc tcgtataatg tgtggaattg tgagcggata
acaatttcac 2280acaggaaaca gattacggat ccatttgagg agtaagccat
gcaaacgttg ccaagcccag 2340ttcaagctac accaacggaa acagctattg
ttagacgcaa aacccgcccg gttccgatag 2400gctccgttgt tattggtggc
ggccatcccg tggctgttca gtcaatgatt aacgaagaca 2460ctctggatat
cgaaggttct gttgctgcaa ttcggcgctt acacgagatc ggttgcgaga
2520tcgtacgtgt gactgtacct tcattagcac acgcgaaagc aatggaagag
attcgggatc 2580ggctttataa aacgtacaaa ccggtcccct tagttgccga
cgtgcatcat aacggaatga 2640aaatcgcgtt agaggttgcc aagtacgtgg
acaatgtgcg cattaatcct ggattatacg 2700tgtttgagaa gccaaaacca
aatcgcacgg agtacactca agctgaattt gacgagattg 2760gcgcgaaaat
ccgtgaaacg ttggaaccac tggtaatttc actgcgggat cagggaaagt
2820cgatgcgcat tggcgttaat catggcagtc tggcggaacg gatgctgttt
acctatggcg 2880ataccccaga gggtatggta gagagtgcac ttgagtttat
acgcatctgt gaaagtctca 2940acttctataa
cttagaaatt tcccttaaag ctagccgcgt cccggttatg atagccgcca
3000atcggcttat ggttaagcgc atggacgagc tgggtatgga ttatccgttg
catctcggag 3060tgactgaggc aggtgatggt gaatatggcc gtattaaaag
cacagcaggc attgcaacac 3120tgctggcgga aggaattgga gacacaatcc
gtgtttcatt gactgaagct ccggaaaagg 3180aaatccccgt gtgctatggc
atccttcaag ccctcggtct ccgccgcacc atggtagaat 3240atgtagcttg
cccgtcgtgt ggtcggacat tgtttaacct ggaagaggtt ctgcacaagg
3300tgagagaagc gactaaacac ctgacgggac tgaatattgc ggttatggga
tgtattgtaa 3360atggacctgg cgaaatggcc gatgcagact acggctatgt
aggtaaacag ccgggatata 3420taagtcttta ccgcggccgg gaagaagtca
agaaagtgcc cgaggccgag ggcgttgcag 3480ctctggtcga actgataaaa
gcggatggta gatgggtaga tccataagtg gagctccagc 3540ccggggcact
ggaggcgtaa atggatacac gtgcgttcaa acgttcactt cattcgtcgg
3600aaaattacca tcgcaaaggc tttggacatg gcgaggaagt taaccagcaa
ttgcagggcg 3660aatatcagtc tagcctcata cagcagatta gagccaatgg
ttatcgctgg cagcagggcg 3720atgttacaat tcgtctggca gaagcgtttg
gcttctgctg gggtgtggaa agagccgtcg 3780ctcttgctta cgaaaccaga
acccatttcc cgaccgagcg catatggata accaacgaaa 3840ttattcacaa
cccctcagtt aatgaacgtc tgcgccaaat ggccgtcgag ttcattcctg
3900tagtgaacgg cgtcaaagat ttttcgggag tacggcccgg cgatgtcgtc
atactgccag 3960catttggggc gtcagttcag gagatgcagt tattaaacga
acgcggttgt actatcgtag 4020atacgacgtg tccgtgggtg agcaaagtat
gcattcggtg gaaaaacata agaaagtttc 4080cttcacgagc attatacacg
gtaaatacaa ccacgaggag actattgcaa catcctcatt 4140tgcgggaact
tacttgatcg tactgaacct cgaagaggcc cgttacgttt gtgactacat
4200tttacatggc ggcgatcgcg ctgcatttat ggcgaaattt gccaaggctt
gctcacctgg 4260ttttgacccg gaccgggatc tggtccgggt agggatagct
aaccaaacaa caatgttaaa 4320aggcgagacc gaacagattg gcaaattgtt
tgagcgcacc atgattcaaa agtatggtcc 4380ggatcgcctt aacgagcact
tcatgtcgtt taatactatt tgcgatgcga cacaggaacg 4440gcaagacgca
atgttgagtt tagtaaaaga gccgttagat ctgatggtag tcattggcgg
4500ttataattct tccaatacta cgcatttgca ggagattgca atcgaacacg
gcattccatc 4560ctatcatatc gactcagcgg atcgtatcgg accaggtaat
cggattgaac ataagccatt 4620gcaccaaaat ccgacagttg ccgaaaattg
gttaccggat cgcccgatca ctatcggcat 4680tacttcaggt gcatcaactc
ccgataaagt tgttgaagag gtgctgaata agatctttgc 4740tttacgcagc
gttgcaacgg ttagttgatc cactagtccc ggtaccgtgg acgaggttta
4800atatggcgac gtataaagtc acactggtcc gtccggatgg cagcgaaacg
accatcgatg 4860ttccggagga cgaatacata ctggatgtcg ccgaagaaca
aggtctggat ctcccgtttt 4920cttgtcgcgc cggtgcctgc tctacctgtg
ctggcaaatt gttggaggga gaagtcgatc 4980aaagcgacca gagcttcttg
gatgacgatc agatcgaaaa aggattcgtg cttacttgtg 5040tggcctaccc
ccgttcggac tgcaagatct tgacgaacca agaggaggag ctgtactaag
5100aggtcgacga cgcatgcatt aacagaggtt agtatgtata atgccactaa
ctctcgctca 5160cgtatgttcc ggtacgaagt tgtggggctg cgccaaacgg
cggagacgga gaaaacaaat 5220tacgcgatca gaaactctgg ctcgcagttc
tttaatgtgc cttatgaccg catgaaccag 5280tttatgcagc agatcactcg
gtggggcggt aaaattgtca gtattcagcc ccttaacgga 5340accgtggccc
cacttgctgc aaccacggag ccagctgcca ataacggagc tgcacctgtg
5400aaagaaaaga aagtcgatat accggtcaac atctaccgtc ccaataatcc
ctgcataggt 5460aaggttatta gcaacgagga actggtccgg gaaggcggtg
agggtacggt gaaacatatt 5520atctttgata tatcggggac cgaattacgt
tacttggaag ggcagtcaat cggtatcatt 5580cccgcgggca cggacgcgaa
cggtaaacca cataagctgc gtctgtattc cattgcttcc 5640acaagacatg
gtgactttca ggatgacaag acggtgtcct tatgcgtacg gagattagaa
5700tacaaagata aagagaccgg ggagaccatt tatggcgtgt gcagttcgta
tcttaatcag 5760ttacagcctg gagatgaagt caaaatcaca ggtcctgttg
ggaaagaaat gcttctctct 5820gacgacccag aagcgactat tattatgctg
gctaccggca ctggaatagc gccatttcgg 5880gcatttttat ggcggatgtt
caaagagaac aacccggatt accagttcaa aggccttgcg 5940tggctgttct
ttggcgtcgc ttatactgcc aatatcctgt ataaggacga gcttgaagct
6000atccaagccc agtatcccga tcattttcgg ttaacctacg cgatttcccg
tgaacaaaaa 6060accccggacg gagggaaaat gtacatccag ggtcggatcg
cagagcacgc tgatgaaatc 6120tggcaactgc tgcaaaagaa aaacacccac
gtgtacatgt gtggcctgcg tgggatggaa 6180cctggaatag acgaggccat
gaccgcagcg gccgcgaaaa acggagctga ctggcaggag 6240tttctgaaag
gtacgctgaa aaaggaaggc agatggcatg tcgaaactta ttaactgcag
6300tacaaataaa aaaggcacgt cagatgacgt gccttttttc ttgaagcttg
gcactggccg 6360tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac
ccaacttaat cgccttgcag 6420cacatccccc tttcgccagc tggcgtaata
gcgaagaggc ccgcaccgat cgcccttccc 6480aacagttgcg cagcctgaat
ggcgaatggc g 6511788069DNAArtificial SequenceSynthetic Construct
78accttcggga gcgcctgaag cccgttctgg acgccctggg gccgttgaat cgggatatgc
60aggccaaggc cgccgcgatc atcaaggccg tgggcgaaaa gctgctgacg gaacagcggg
120aagtccagcg ccagaaacag gcccagcgcc agcaggaacg cgggcgcgca
catttccccg 180aaaagtgcca cctggcggcg ttgtgacaat ttaccgaaca
actccgcggc cgggaagccg 240atctcggctt gaacgaattg ttaggtggcg
gtacttgggt cgatatcaaa gtgcatcact 300tcttcccgta tgcccaactt
tgtatagaga gccactgcgg gatcgtcacc gtaatctgct 360tgcacgtaga
tcacataagc accaagcgcg ttggcctcat gcttgaggag attgatgagc
420gcggtggcaa tgccctgcct ccggtgctcg ccggagactg cgagatcata
gatatagatc 480tcactacgcg gctgctcaaa cctgggcaga acgtaagccg
cgagagcgcc aacaaccgct 540tcttggtcga aggcagcaag cgcgatgaat
gtcttactac ggagcaagtt cccgaggtaa 600tcggagtccg gctgatgttg
ggagtaggtg gctacgtctc cgaactcacg accgaaaaga 660tcaagagcag
cccgcatgga tttgacttgg tcagggccga gcctacatgt gcgaatgatg
720cccatacttg agccacctaa ctttgtttta gggcgactgc cctgctgcgt
aacatcgttg 780ctgctgcgta acatcgttgc tgctccataa catcaaacat
cgacccacgg cgtaacgcgc 840ttgctgcttg gatgcccgag gcatagactg
tacaaaaaaa cagtcataac aagccatgaa 900aaccgccact gcgccgttac
caccgctgcg ttcggtcaag gttctggacc agttgcgtga 960gcgcatacgc
tacttgcatt acagtttacg aaccgaacag gcttatgtca actgggttcg
1020tgccttcatc cgtttccacg gtgtgcgtcc atgggcaaat attatacgca
aggcgacaag 1080gtgctgatgc cgctggcgat tcaggttcat catgccgttt
gtgatggctt ccatgtcggc 1140agaatgctta atgaattaca acagttttta
tgcatgcgcc caatacgcaa accgcctctc 1200cccgcgcgtt ggccgattca
ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg 1260ggcagtgagc
gcaacgcaat taatgtgagt tagctcactc attaggcacc ccaggcttta
1320cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca
atttcacaca 1380ggaaacagct atgaccatga ttacgccaag cgcgcaatta
accctcacta aagggaacaa 1440aagctgggta ccctgcccgc tttccagtcg
ggaaacctgt cgtgccagct gcattaatga 1500atcggccaac gcgcggggag
aggcggtttg cgtattgggc gccagggtgg tttttctttt 1560caccagtgag
acgggcaaca gctgattgcc cttcaccgcc tggccctgag agagttgcag
1620caagcggtcc acgctggttt gccccagcag gcgaaaatcc tgtttgatgg
tggttaacgg 1680cgggatataa catgagctgt cttcggtatc gtcgtatccc
actaccgaga tatccgcacc 1740aacgcgcagc ccggactcgg taatggcgcg
cattgcgccc agcgccatct gatcgttggc 1800aaccagcatc gcagtgggaa
cgatgccctc attcagcatt tgcatggttt gttgaaaacc 1860ggacatggca
ctccagtcgc cttcccgttc cgctatcggc tgaatttgat tgcgagtgag
1920atatttatgc cagccagcca gacgcagacg cgccgagaca gaacttaatg
ggcccgctaa 1980cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc
acgcccagtc gcgtaccgtc 2040ttcatgggag aaaataatac tgttgatggg
tgtctggtca gagacatcaa gaaataacgc 2100cggaacatta gtgcaggcag
cttccacagc aatggcatcc tggtcatcca gcggatagtt 2160aatgatcagc
ccactgacgc gttgcgcgag aagattgtgc accgccgctt tacaggcttc
2220gacgccgctt cgttctacca tcgacaccac cacgctggca cccagttgat
cggcgcgaga 2280tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc
agactggagg tggcaacgcc 2340aatcagcaac gactgtttgc ccgccagttg
ttgtgccacg cggttgggaa tgtaattcag 2400ctccgccatc gccgcttcca
ctttttcccg cgttttcgca gaaacgtggc tggcctggtt 2460caccacgcgg
gaaacggtct gataagagac accggcatac tctgcgacat cgtataacgt
2520tactggtttc acattcacca ccctgaattg actctcttcc gggcgctatc
atgccatacc 2580gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg
acgctctccc ttatgcgact 2640cctgcattag gaagcagccc agtagtaggt
tgaggccgtt gagcaccgcc gccgcaagga 2700atggtgcatg caaggagatg
gcgcccaaca gtcccccggc cacggggcct gccaccatac 2760ccacgccgaa
acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga
2820tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg
gccacgatgc 2880gtccggcgta gaggatcgag atctcgatcc cgcgaaatta
atacgactca ctatagggga 2940attgtgagcg gataacaatt cccctctaga
aataattttg tttaacttta agaaggagat 3000atacatatgg aagctcgtcg
ttctgcgaac tacgaaccta acagctggga ctatgattac 3060ctgctgtcct
ccgacacgga cgagtccatc gaagtataca aagacaaagc gaaaaagctg
3120gaagccgaag ttcgtcgcga gattaataac gaaaaagcag aatttctgac
cctgctggaa 3180ctgattgaca acgtccagcg cctgggcctg ggttaccgtt
tcgagtctga tatccgtggt 3240gcgctggatc gcttcgtttc ctccggcggc
ttcgatgcgg taaccaagac ttccctgcac 3300ggtacggcac tgtctttccg
tctgctgcgt caacacggtt ttgaggtttc tcaggaagcg 3360ttcagcggct
tcaaagacca aaacggcaac ttcctggaga acctgaagga agatatcaaa
3420gctatcctga gcctgtacga ggccagcttc ctggctctgg aaggcgaaaa
catcctggac 3480gaggcgaagg ttttcgcaat ctctcatctg aaagaactgt
ctgaagaaaa gatcggtaaa 3540gagctggcag aacaggtgaa ccatgcactg
gaactgccac tgcatcgccg tactcagcgt 3600ctggaagcag tatggtctat
cgaggcctac cgtaaaaagg aggacgcgaa tcaggttctg 3660ctggagctgg
caattctgga ttacaacatg atccagtctg tataccagcg tgatctgcgt
3720gaaacgtccc gttggtggcg tcgtgtgggt ctggcgacca aactgcactt
tgctcgtgac 3780cgcctgattg agagcttcta ctgggccgtg ggtgtagcat
tcgaaccgca atactccgac 3840tgccgtaact ccgtcgcaaa aatgttttct
ttcgtaacca ttatcgacga tatctacgat 3900gtatacggca ccctggacga
actggagctg tttactgatg cagttgagcg ttgggacgta 3960aacgccatca
acgacctgcc ggattacatg aaactgtgct ttctggctct gtataacact
4020attaacgaaa tcgcctacga caacctgaaa gataaaggtg agaacatcct
gccgtatctg 4080accaaagcct gggctgacct gtgcaacgct ttcctgcaag
aagccaagtg gctgtacaac 4140aaatctactc cgacctttga cgactacttc
ggcaacgcat ggaaatcctc ttctggcccg 4200ctgcaactgg tgttcgctta
cttcgctgtc gtgcagaaca ttaaaaagga agagatcgaa 4260aacctgcaaa
aataccatga caccatctct cgtccttccc atatcttccg tctgtgcaat
4320gacctggcta gcgcgtctgc ggaaattgcg cgtggtgaaa ccgcaaatag
cgtttcttgt 4380tacatgcgca ctaaaggtat ctccgaagaa ctggctaccg
aaagcgtgat gaatctgatc 4440gatgaaacct ggaaaaagat gaacaaggaa
aaactgggtg gtagcctgtt cgcgaaaccg 4500ttcgtggaaa ccgcgatcaa
cctggcacgt caatctcact gcacttatca taacggcgac 4560gcgcatacct
ctccggatga gctgacccgc aaacgcgttc tgtctgtaat cactgaaccg
4620attctgccgt ttgaacgcta aggatccgaa ttcgagctcc gtcgacaagc
ttgcggccgc 4680actcgagcac caccaccacc accactgaga tccggctgct
aacaaagccc gaaaggaagc 4740tgagttggct gctgccaccg ctgagcaata
actagcataa ccccttgggg cctctaaacg 4800ggtcttgagg ggttttttga
ctagttctag agcggccgcc accgcggtgg agctccaatt 4860cgccctatag
tgagtcgtat tacgcgcgct cactggccgt cgttttacaa cgtcgtgact
4920gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct
ttcgccagct 4980ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca
acagttgcgc agcctgaatg 5040gcgaatggaa attgtaagcg ttaatatttt
gttaaaattc gcgttaaatt tttgttaaat 5100cagctcattt tttaaccaat
aggccgactg cgatgagtgg cagggcgggg cgtaattttt 5160ttaaggcagt
tattggtgcc cttaaacgcc tggtgctacg cctgaataag tgataataag
5220cggatgaatg gcagaaattc gaaagcaaat tcgacccggt cgtcggttca
gggcagggtc 5280gttaaatagc cgcttatgtc tattgctggt ttaccggttt
attgactacc ggaagcagtg 5340tgaccgtgtg cttctcaaat gcctgaggcc
agtttgctca ggctctcccc gtggaggtaa 5400taattgacga tatgatcatt
tattctgcct cccagagcct gataaaaacg gtgaatccgt 5460tagcgaggtg
ccgccggctt ccattcaggt cgaggtggcc cggctccatg caccgcgacg
5520caacgcgggg aggcagacaa ggtatagggc ggcgaggcgg ctacagccga
tagtctggaa 5580cagcgcactt acgggttgct gcgcaaccca agtgctaccg
gcgcggcagc gtgacccgtg 5640tcggcggctc caacggctcg ccatcgtcca
gaaaacacgg ctcatcgggc atcggcaggc 5700gctgctgccc gcgccgttcc
cattcctccg tttcggtcaa ggctggcagg tctggttcca 5760tgcccggaat
gccgggctgg ctgggcggct cctcgccggg gccggtcggt agttgctgct
5820cgcccggata cagggtcggg atgcggcgca ggtcgccatg ccccaacagc
gattcgtcct 5880ggtcgtcgtg atcaaccacc acggcggcac tgaacaccga
caggcgcaac tggtcgcggg 5940gctggcccca cgccacgcgg tcattgacca
cgtaggccga cacggtgccg gggccgttga 6000gcttcacgac ggagatccag
cgctcggcca ccaagtcctt gactgcgtat tggaccgtcc 6060gcaaagaacg
tccgatgagc ttggaaagtg tcttctggct gaccaccacg gcgttctggt
6120ggcccatctg cgccacgagg tgatgcagca gcattgccgc cgtgggtttc
ctcgcaataa 6180gcccggccca cgcctcatgc gctttgcgtt ccgtttgcac
ccagtgaccg ggcttgttct 6240tggcttgaat gccgatttct ctggactgcg
tggccatgct tatctccatg cggtagggtg 6300ccgcacggtt gcggcaccat
gcgcaatcag ctgcaacttt tcggcagcgc gacaacaatt 6360atgcgttgcg
taaaagtggc agtcaattac agattttctt taacctacgc aatgagctat
6420tgcggggggt gccgcaatga gctgttgcgt accccccttt tttaagttgt
tgatttttaa 6480gtctttcgca tttcgcccta tatctagttc tttggtgccc
aaagaagggc acccctgcgg 6540ggttccccca cgccttcggc gcggctcccc
ctccggcaaa aagtggcccc tccggggctt 6600gttgatcgac tgcgcggcct
tcggccttgc ccaaggtggc gctgccccct tggaaccccc 6660gcactcgccg
ccgtgaggct cggggggcag gcgggcgggc ttcgccttcg actgccccca
6720ctcgcatagg cttgggtcgt tccaggcgcg tcaaggccaa gccgctgcgc
ggtcgctgcg 6780cgagccttga cccgccttcc acttggtgtc caaccggcaa
gcgaagcgcg caggccgcag 6840gccggaggct tttccccaga gaaaattaaa
aaaattgatg gggcaaggcc gcaggccgcg 6900cagttggagc cggtgggtat
gtggtcgaag gctgggtagc cggtgggcaa tccctgtggt 6960caagctcgtg
ggcaggcgca gcctgtccat cagcttgtcc agcagggttg tccacgggcc
7020gagcgaagcg agccagccgg tggccgctcg cggccatcgt ccacatatcc
acgggctggc 7080aagggagcgc agcgaccgcg cagggcgaag cccggagagc
aagcccgtag ggcgccgcag 7140ccgccgtagg cggtcacgac tttgcgaagc
aaagtctagt gagtatactc aagcattgag 7200tggcccgccg gaggcaccgc
cttgcgctgc ccccgtcgag ccggttggac accaaaaggg 7260aggggcaggc
atggcggcat acgcgatcat gcgatgcaag aagctggcga aaatgggcaa
7320cgtggcggcc agtctcaagc acgcctaccg cgagcgcgag acgcccaacg
ctgacgccag 7380caggacgcca gagaacgagc actgggcggc cagcagcacc
gatgaagcga tgggccgact 7440gcgcgagttg ctgccagaga agcggcgcaa
ggacgctgtg ttggcggtcg agtacgtcat 7500gacggccagc ccggaatggt
ggaagtcggc cagccaagaa cagcaggcgg cgttcttcga 7560gaaggcgcac
aagtggctgg cggacaagta cggggcggat cgcatcgtga cggccagcat
7620ccaccgtgac gaaaccagcc cgcacatgac cgcgttcgtg gtgccgctga
cgcaggacgg 7680caggctgtcg gccaaggagt tcatcggcaa caaagcgcag
atgacccgcg accagaccac 7740gtttgcggcc gctgtggccg atctagggct
gcaacggggc atcgagggca gcaaggcacg 7800tcacacgcgc attcaggcgt
tctacgaggc cctggagcgg ccaccagtgg gccacgtcac 7860catcagcccg
caagcggtcg agccacgcgc ctatgcaccg cagggattgg ccgaaaagct
7920gggaatctca aagcgcgttg agacgccgga agccgtggcc gaccggctga
caaaagcggt 7980tcggcagggg tatgagcctg ccctacaggc cgccgcagga
gcgcgtgaga tgcgcaagaa 8040ggccgatcaa gcccaagaga cggcccgag
80697913661DNAArtificial SequenceSynthetic Construct 79tggcgaatgg
gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 60cagcgtgacc
gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
120ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
tccctttagg 180gttccgattt agtgctttac ggcacctcga ccccaaaaaa
cttgattagg gtgatggttc 240acgtagtggg ccatcgccct gatagacggt
ttttcgccct ttgacgttgg agtccacgtt 300ctttaatagt ggactcttgt
tccaaactgg aacaacactc aaccctatct cggtctattc 360ttttgattta
taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
420acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag
gtggcacttt 480tcggggaaat gtgcgcggaa cccctatttg tttatttttc
taaatacatt caaatatgta 540tccgctcatg aattaattct tagaaaaact
catcgagcat caaatgaaac tgcaatttat 600tcatatcagg attatcaata
ccatattttt gaaaaagccg tttctgtaat gaaggagaaa 660actcaccgag
gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc
720gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta
tcaagtgaga 780aatcaccatg agtgacgact gaatccggtg agaatggcaa
aagtttatgc atttctttcc 840agacttgttc aacaggccag ccattacgct
cgtcatcaaa atcactcgca tcaaccaaac 900cgttattcat tcgtgattgc
gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac 960aattacaaac
aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat
1020tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg
gggatcgcag 1080tggtgagtaa ccatgcatca tcaggagtac ggataaaatg
cttgatggtc ggaagaggca 1140taaattccgt cagccagttt agtctgacca
tctcatctgt aacatcattg gcaacgctac 1200ctttgccatg tttcagaaac
aactctggcg catcgggctt cccatacaat cgatagattg 1260tcgcacctga
ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca
1320tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg
ctcataacac 1380cccttgtatt actgtttatg taagcagaca gttttattgt
tcatgaccaa aatcccttaa 1440cgtgagtttt cgttccactg agcgtcagac
cccgtagaaa agatcaaagg atcttcttga 1500gatccttttt ttctgcgcgt
aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg 1560gtggtttgtt
tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
1620agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca
ccacttcaag 1680aactctgtag caccgcctac atacctcgct ctgctaatcc
tgttaccagt ggctgctgcc 1740agtggcgata agtcgtgtct taccgggttg
gactcaagac gatagttacc ggataaggcg 1800cagcggtcgg gctgaacggg
gggttcgtgc acacagccca gcttggagcg aacgacctac 1860accgaactga
gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
1920aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
gagggagctt 1980ccagggggaa acgcctggta tctttatagt cctgtcgggt
ttcgccacct ctgacttgag 2040cgtcgatttt tgtgatgctc gtcagggggg
cggagcctat ggaaaaacgc cagcaacgcg 2100gcctttttac ggttcctggc
cttttgctgg ccttttgctc acatgttctt tcctgcgtta 2160tcccctgatt
ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
2220agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg
cctgatgcgg 2280tattttctcc ttacgcatct gtgcggtatt tcacaccgca
tatatggtgc actctcagta 2340caatctgctc tgatgccgca tagttaagcc
agtatacact ccgctatcgc tacgtgactg 2400ggtcatggct gcgccccgac
acccgccaac acccgctgac gcgccctgac gggcttgtct 2460gctcccggca
tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag
2520gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat
cagcgtggtc 2580gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc
agctcgttga gtttctccag 2640aagcgttaat gtctggcttc tgataaagcg
ggccatgtta agggcggttt tttcctgttt 2700ggtcactgat gcctccgtgt
aagggggatt tctgttcatg ggggtaatga taccgatgaa 2760acgagagagg
atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg
2820ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa
tcactcaggg 2880tcaatgccag cgcttcgtta atacagatgt aggtgttcca
cagggtagcc agcagcatcc 2940tgcgatgcag atccggaaca taatggtgca
gggcgctgac ttccgcgttt ccagacttta 3000cgaaacacgg aaaccgaaga
ccattcatgt tgttgctcag gtcgcagacg ttttgcagca 3060gcagtcgctt
cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc
3120ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc
gtggggccgc 3180catgccggcg ataatggcct gcttctcgcc gaaacgtttg
gtggcgggac cagtgacgaa 3240ggcttgagcg
agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc
3300gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca
cctgtcctac 3360gagttgcatg ataaagaaga cagtcataag tgcggcgacg
atagtcatgc cccgcgccca 3420ccggaaggag ctgactgggt tgaaggctct
caagggcatc ggtcgagatc ccggtgccta 3480atgagtgagc taacttacat
taattgcgtt gcgctcactg cccgctttcc agtcgggaaa 3540cctgtcgtgc
cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
3600tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga
ttgcccttca 3660ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct
ggtttgcccc agcaggcgaa 3720aatcctgttt gatggtggtt aacggcggga
tataacatga gctgtcttcg gtatcgtcgt 3780atcccactac cgagatatcc
gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg 3840cgcccagcgc
catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca
3900gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc
cgttccgcta 3960tcggctgaat ttgattgcga gtgagatatt tatgccagcc
agccagacgc agacgcgccg 4020agacagaact taatgggccc gctaacagcg
cgatttgctg gtgacccaat gcgaccagat 4080gctccacgcc cagtcgcgta
ccgtcttcat gggagaaaat aatactgttg atgggtgtct 4140ggtcagagac
atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg
4200catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc
gcgagaagat 4260tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc
taccatcgac accaccacgc 4320tggcacccag ttgatcggcg cgagatttaa
tcgccgcgac aatttgcgac ggcgcgtgca 4380gggccagact ggaggtggca
acgccaatca gcaacgactg tttgcccgcc agttgttgtg 4440ccacgcggtt
gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt
4500tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa
gagacaccgg 4560catactctgc gacatcgtat aacgttactg gtttcacatt
caccaccctg aattgactct 4620cttccgggcg ctatcatgcc ataccgcgaa
aggttttgcg ccattcgatg gtgtccggga 4680tctcgacgct ctcccttatg
cgactcctgc attaggaagc agcccagtag taggttgagg 4740ccgttgagca
ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc
4800ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag
cccgaagtgg 4860cgagcccgat cttccccatc ggtgatgtcg gcgatatagg
cgccagcaac cgcacctgtg 4920gcgccggtga tgccggccac gatgcgtccg
gcgtagagga tcgagatctc gatcccgcga 4980aattaatacg actcactata
ggggaattgt gagcggataa caattcccct ctagaaataa 5040ttttgtttaa
ctttaagaag gagatataca tatgagtttt gatattgcca aatacccgac
5100cctggcactg gttgactcca cccaggagtt acgactgttg ccgaaagaga
gtttaccgaa 5160actctgcgac gaactgcgcc gctatttact cgacagcgtg
agccgttcca gcgggcactt 5220cgcctccggg ctgggcacgg tcgaactgac
cgtggcgctg cactatgtct acaacacccc 5280gtttgaccaa ttgatttggg
atgtggggca tcaggcttat ccgcataaaa ttttgaccgg 5340acgccgcgac
aaaatcggca ccatccgtca gaaaggcggt ctgcacccgt tcccgtggcg
5400cggcgaaagc gaatatgacg tattaagcgt cgggcattca tcaacctcca
tcagtgccgg 5460aattggtatt gcggttgctg ccgaaaaaga aggcaaaaat
cgccgcaccg tctgtgtcat 5520tggcgatggc gcgattaccg caggcatggc
gtttgaagcg atgaatcacg cgggcgatat 5580ccgtcctgat atgctggtga
ttctcaacga caatgaaatg tcgatttccg aaaatgtcgg 5640cgcgctcaac
aaccatctgg cacagctgct ttccggtaag ctgtactctt cactgcgcga
5700aggcgggaaa aaagttttct ctggcgtgcc gccaattaaa gagctgctca
aacgcaccga 5760agaacatatt aaaggcatgg tagtgcctgg cacgttgttt
gaagagctgg gctttaacta 5820catcggcccg gtggacggtc acgatgtgct
ggggcttatc accacgctaa agaacatgcg 5880cgacctgaaa ggcccgcagt
tcctgcatat catgaccaaa aaaggtcgtg gttatgaacc 5940ggcagaaaaa
gacccgatca ctttccacgc cgtgcctaaa tttgatccct ccagcggttg
6000tttgccgaaa agtagcggcg gtttgccgag ctattcaaaa atctttggcg
actggttgtg 6060cgaaacggca gcgaaagaca acaagctgat ggcgattact
ccggcgatgc gtgaaggttc 6120cggcatggtc gagttttcac gtaaattccc
ggatcgctac ttcgacgtgg caattgccga 6180gcaacacgcg gtgacctttg
ctgcgggtct ggcgattggt gggtacaaac ccattgtcgc 6240gatttactcc
actttcctgc aacgcgccta tgatcaggtg ctgcatgacg tggcgattca
6300aaaacttccg gtcctgttcg ccatcgaccg cgcgggcatt gttggtgctg
acggtcaaac 6360ccatcagggt gcttttgatc tctcttacct gcgctgcata
ccggaaatgg tcattatgac 6420cccgagcgat gaaaacgaat gtcgccagat
gctctatacc ggctatcact ataacgatgg 6480cccgtcagcg gtgcgctacc
cgcgtggcaa cgcggtcggc gtggaactga cgccgctgga 6540aaaactacca
attggcaaag gcattgtgaa gcgtcgtggc gagaaactgg cgatccttaa
6600ctttggtacg ctgatgccag aagcggcgaa agtcgccgaa tcgctgaacg
ccacgctggt 6660cgatatgcgt tttgtgaaac cgcttgatga agcgttaatt
ctggaaatgg ccgccagcca 6720tgaagcgctg gtcaccgtag aagaaaacgc
cattatgggc ggcgcaggca gcggcgtgaa 6780cgaagtgctg atggcccatc
gtaaaccagt acccgtgctg aacattggcc tgccggactt 6840ctttattccg
caaggaactc aggaagaaat gcgcgccgaa ctcggcctcg atgccgctgg
6900tatggaagcc aaaatcaagg cctggctggc ataagccttc ttaaggtagc
tgctgacaga 6960tatttcgccc ttaaagcttt acaaggagga aaaaaacatg
aagcaactca ccattctggg 7020ctcgaccggc tcgattggtt gcagcacgct
ggacgtggtg cgccataatc ccgaacactt 7080ccgcgtagtt gcgctggtgg
caggcaaaaa tgtcactcgc atggtagaac agtgcctgga 7140attttctccc
cgctatgccg taatggacga tgaagcgagt gcgaaacttc ttaaaacgat
7200gctacagcaa cagggtagcc gcaccgaagt cttaagtggg caacaagccg
cttgcgatat 7260ggcagcgctt gaggatgttg atcaggtgat ggcagccatt
gttggcgctg ctgggctgtt 7320acctacgctt gctgcgatcc gcgcgggtaa
aaccattttg ctggccaata aagaatcact 7380ggttacctgc ggacgtctgt
ttatggacgc cgtaaagcag agcaaagcgc aattgttacc 7440ggtcgatagc
gaacataacg ccatttttca gagtttaccg caacctatcc agcataatct
7500gggatacgct gaccttgagc aaaatggcgt ggtgtccatt ttacttaccg
ggtctggtgg 7560ccctttccgt gagacgccat tgcgcgattt ggcaacaatg
acgccggatc aagcctgccg 7620tcatccgaac tggtcgatgg ggcgtaaaat
ttctgtcgat tcggctacca tgatgaacaa 7680aggtctggaa tacattgaag
cgcgttggct gtttaacgcc agcgccagcc agatggaagt 7740gctgattcac
ccgcagtcag tgattcactc aatggtgcgc tatcaggacg gcagtgttct
7800ggcgcagctg ggggaaccgg atatgcgtac gccaattgcc cacacgatgg
catggccgaa 7860tcgcgtgaac tctggcgtga agccgctcga tttttgcaaa
ctaagtgcgt tgacatttgc 7920cgcaccggat tatgatcgtt atccatgcct
gaaactggcg atggaggcgt tcgaacaagg 7980ccaggcagcg acgacagcat
tgaatgccgc aaacgaaatc accgttgctg cttttcttgc 8040gcaacaaatc
cgctttacgg atatcgctgc gttgaattta tccgtactgg aaaaaatgga
8100tatgcgcgaa ccacaatgtg tggacgatgt gttatctgtt gatgcgaacg
cgcgtgaagt 8160cgccagaaaa gaggtgatgc gtctcgcaag ctgagtccga
ctttgcgata ggcctgcacc 8220cttaacgtcg acacgtaagg aggaaaaaaa
catggcaacc actcatttgg atgtttgcgc 8280cgtggttccg gcggccggat
ttggccgtcg aatgcaaacg gaatgtccta agcaatatct 8340ctcaatcggt
aatcaaacca ttcttgaaca ctcggtgcat gcgctgctgg cgcatccccg
8400tgtgaaacgt gtcgtcattg ccataagtcc tggcgatagc cgttttgcac
aacttcctct 8460ggcgaatcat ccgcaaatca ccgttgtaga tggcggtgat
gagcgtgccg attccgtgct 8520ggcaggtctg aaagccgctg gcgacgcgca
gtgggtattg gtgcatgacg ccgctcgtcc 8580ttgtttgcat caggatgacc
tcgcgcgatt gttggcgttg agcgaaacca gccgcacggg 8640gggcatcctc
gccgcaccag tgcgcgatac tatgaaacgt gccgaaccgg gcaaaaatgc
8700cattgctcat accgttgatc gcaacggctt atggcacgcg ctgacgccgc
aatttttccc 8760tcgtgagctg ttacatgact gtctgacgcg cgctctaaat
gaaggcgcga ctattaccga 8820cgaagcctcg gcgctggaat attgcggatt
ccatcctcag ttggtcgaag gccgtgcgga 8880taacattaaa gtcacgcgcc
cggaagattt ggcactggcc gagttttacc tcacccgaac 8940catccatcag
gagaatacat aatttcggat gcttatacac gccagatatt tcattacgga
9000gctcatacaa ggaggaaaaa aacatgcgga cacagtggcc ctctccggca
aaacttaatc 9060tgtttttata cattaccggt cagcgtgcgg atggttacca
cacgctgcaa acgctgtttc 9120agtttcttga ttacggcgac accatcagca
ttgagcttcg tgacgatggg gatattcgtc 9180tgttaacgcc cgttgaaggc
gtggaacatg aagataacct gatcgttcgc gcagcgcgat 9240tgttgatgaa
aactgcggca gacagcgggc gtcttccgac gggaagcggt gcgaatatca
9300gcattgacaa gcgtttgccg atgggcggcg gtctcggcgg tggttcatcc
aatgccgcga 9360cggtcctggt ggcattaaat catctctggc aatgcgggct
aagcatggat gagctggcgg 9420aaatggggct gacgctgggc gcagatgttc
ctgtctttgt tcgggggcat gccgcgtttg 9480ccgaaggcgt tggtgaaata
ctaacgccgg tggacccgcc agagaagtgg tatctggtgg 9540cgcaccctgg
tgtaagtatt ccgactccgg tgatttttaa agatcctgaa ctcccgcgca
9600atacgccaaa aaggtcaata gaaacgttgc taaaatgtga atttagcaat
gattgcgagg 9660ttatcgcaag aaaacgtttt cgcgaggttg atgcggtgct
ttcctggctg ttagaatacg 9720ccccgtcgcg cctgactggg acaggggcct
gtgtctttgc tgaatttgat acagagtctg 9780aagcccgcca ggtgctagag
caagccccgg aatggctcaa tggctttgtg gcgaaaggcg 9840ctaatctttc
cccattgcac agagccatgc tttaatttgc gattgagatc cggcctgcac
9900ccttaaccgg atccgattca aggaggaaaa aaacatgcga attggacacg
gttttgacgt 9960acatgccttt ggcggtgaag gcccaattat cattggtggc
gtacgcattc cttacgaaaa 10020aggattgctg gcgcattctg atggcgacgt
ggcgctccat gcgttgaccg atgcattgct 10080tggcgcggcg gcgctggggg
atatcggcaa gctgttcccg gataccgatc cggcatttaa 10140aggtgccgat
agccgcgagc tgctacgcga agcctggcgt cgtattcagg cgaagggtta
10200tacccttggc aacgtcgatg tcactatcat cgctcaggca ccgaagatgt
tgccgcacat 10260tccacaaatg cgcgtgttta ttgccgaaga tttgggctgc
cacatggatg atgttaacgt 10320gaaagccact actacggaaa aactgggatt
taccggacgt ggggaaggga ttgcctgtga 10380agcggtggcg ctactcatta
aggcaacaaa atgatttacc gtattattct ttagacaacg 10440gattaagcta
gcacataagg aggaaaaaaa catgcataac caggctccaa ttcaacgtag
10500aaaatcaaca cgtatttacg ttgggaatgt gccgattggc gatggtgctc
ccatcgccgt 10560acagtccatg accaatacgc gtacgacaga cgtcgaagca
acggtcaatc aaatcaaggc 10620gctggaacgc gttggcgctg atatcgtccg
tgtatccgta ccgacgatgg acgcggcaga 10680agcgttcaaa ctcatcaaac
agcaggttaa cgtgccgctg gtggctgaca tccacttcga 10740ctatcgcatt
gcgctgaaag tagcggaata cggcgtcgat tgtctgcgta ttaaccctgg
10800caatatcggt aatgaagagc gtattcgcat ggtggttgac tgtgcgcgcg
ataaaaacat 10860tccgatccgt attggcgtta acgccggatc gctggaaaaa
gatttgcaag aaaagtatgg 10920cgaaccgacg ccgcaggcgt tgctggaatc
tgccatgcgt catgttgatc atctcgatcg 10980cctgaacttc gatcagttca
aagtcagcgt gaaagcgtct gacgtcttcc tcgctgttga 11040gtcttatcgt
ttgctggcaa aacagatcga tcagccgttg catctgggga tcaccgaagc
11100cggtggtgcg cgcagcgggg cagtaaaatc cgccattggt ttaggtctgc
tgctgtctga 11160aggcatcggc gacacgctgc gcgtatcgct ggcggccgat
ccggtcgaag agatcaaagt 11220cggtttcgat attttgaaat cgctgcgtat
ccgttcgcga gggatcaact tcatcgcctg 11280cccgacctgt tcgcgtcagg
aatttgatgt tatcggtacg gttaacgcgc tggagcaacg 11340cctggaagat
atcatcactc cgatggacgt ttcgattatc ggctgcgtgg tgaatggccc
11400aggtgaggcg ctggtttcta cactcggcgt caccggcggc aacaagaaaa
gcggcctcta 11460tgaagatggc gtgcgcaaag accgtctgga caacaacgat
atgatcgacc agctggaagc 11520acgcattcgt gcgaaagcca gtcagctgga
cgaagcgcgt cgaattgacg ttcagcaggt 11580tgaaaaataa ttacaagtaa
atgattcagg ttataactac gttgcggccg caaggaggaa 11640aaaaacatgc
agatcctgtt ggccaacccg cgtggttttt gtgccggggt agaccgcgct
11700atcagcattg ttgaaaacgc gctggccatt tacggcgcac cgatatatgt
ccgtcacgaa 11760gtggtacata accgctatgt ggtcgatagc ttgcgtgagc
gtggggctat ctttattgag 11820cagattagcg aagtaccgga cggcgcgatc
ctgattttct ccgcacacgg tgtttctcag 11880gcggtacgta acgaagcaaa
aagtcgcgat ttgacggtgt ttgatgccac ctgtccgctg 11940gtgaccaaag
tgcacatgga agtcgcccgc gccagtcgcc gtggcgaaga atctattctc
12000atcggtcacg ccgggcaccc ggaagtggaa gggacaatgg gccagtacag
taacccggaa 12060gggggaatgt atctggtcga atcgccggac gatgtgtgga
aactgacggt caaaaacgaa 12120gagaagctct cctttatgac ccagaccacg
ctgtcggtgg atgacacgtc tgatgtgatc 12180gacgcgctgc gtaaacgctt
cccgaaaatt gtcggtccgc gcaaagatga catctgctac 12240gccacgacta
accgtcagga agcggtacgc gccctggcag aacaggcgga agttgtgttg
12300gtggtcggtt cgaaaaactc ctccaactcc aaccgtctgg cggagctggc
ccagcgtatg 12360ggcaaacgcg cgtttttgat tgacgatgcg aaagacatcc
aggaagagtg ggtgaaagag 12420gttaaatgcg tcggcgtgac tgcgggcgca
tcggctccgg atattctggt gcagaatgtg 12480gtggcacgtt tgcagcagct
gggcggtggt gaagccattc cgctggaagg ccgtgaagaa 12540aacattgttt
tcgaagtgcc gaaagagctg cgtgtcgata ttcgtgaagt cgattaattt
12600gcattagcta ttacgtaatt cgtatagtcg gtaccactaa ggaggaaaaa
aacatgactg 12660ccgacaacaa tagtatgccg catggtgcag tatctagtta
cgccaaatta gtgcaaaacc 12720aaacacctga agacattttg gaagagtttc
ctgaaattat tccattacaa caaagaccta 12780atacccgatc tagtgagacg
tcaaatgacg aaagcggaga aacatgtttt tctggtcatg 12840atgaggagca
aattaagtta atgaatgaaa attgtattgt tttggattgg gacgataatg
12900ctattggtgc cggcaccaag aaagtttgtc atttaatgga aaatattgaa
aagggtttac 12960tacatcgtgc attctccgtc tttattttca atgaacaagg
tgaattactt ttacaacaaa 13020gagccactga aaaaataact ttccctgatc
tttggactaa cacatgctgc tctcatccac 13080tatgtattga tgacgaatta
ggtttgaagg gtaagctaga cgataagatt aagggcgcta 13140ttactgcggc
ggtgagaaaa ctagatcatg aattaggtat tccagaagat gaaactaaga
13200caaggggtaa gtttcacttt ttaaacagaa tccattacat ggcaccaagc
aatgaaccgt 13260ggggtgaaca tgaaattgat tacatcctat tttataagat
caacgctaaa gaaaacttga 13320ctgtcaaccc aaacgtcaat gaagttagag
acttcaaatg ggtttcacca aatgatttga 13380aaactatgtt tgctgaccca
agttacaagt ttacgccttg gtttaagatt atttgcgaga 13440attacttatt
caactggtgg gagcaattag atgacctttc tgaagtggaa aatgacaggc
13500aaattcatag aatgctataa gaattcctcg agcaccacca ccaccaccac
tgagatccgg 13560ctgctaacaa agcccgaaag gaagctgagt tggctgctgc
caccgctgag caataactag 13620cataacccct tggggcctct aaacgggtct
tgaggggttt t 136618064DNAArtificial SequenceSynthetic Construct
80gggcgagttt gaggtgaagt aagacatgag actgacatct gaaccctcac taaagggcgg
60ccgc 6481102DNAArtificial SequenceSynthetic Construct
81ttctttttat taagcgcgta acttaacgtc gatcgcgtct tgaagttcct atactttcta
60gagaatagga acttcttacg ccccgccctg ccactcatcg ca
1028221DNAArtificial SequenceSynthetic Construct 82agccaggagt
tgaatatcct g 218321DNAArtificial SequenceSynthetic Construct
83tgatggacac gaggatggtg t 218422DNAArtificial SequenceSynthetic
Construct 84caccatggta tcctgttctg cg 228522DNAArtificial
SequenceSynthetic Construct 85ttaatctact ttcagacctt gc
228637DNAArtificial SequenceSynthetic Construct 86aggaggtggt
ctcaaatgac tgccgacaac aatagta 378750DNAArtificial SequenceSynthetic
Construct 87aggaggtggt ctcagcgctc tgcagttata gcattctatg aatttgcctg
508873DNAArtificial SequenceSynthetic Construct 88gaacaatcac
cggcgcagta acagacgggt aacgcgggag atttttcatg aattaaccct 60cactaaaggg
cgg 738974DNAArtificial SequenceSynthetic Construct 89cgggaagcga
ggcgcttccc atcacgttat tatttttcaa cctgctgaac taatacgact 60cactataggg
ctcg 749073DNAArtificial SequenceSynthetic Construct 90ttttgatatt
gaagtgctgg aaatcgatcc ggcactggag gcgtaacatg aattaaccct 60cactaaaggg
cgg 739174DNAArtificial SequenceSynthetic Construct 91attttcgcat
aacttaggct gctaatgact taatcgactt cacgaatatc taatacgact 60cactataggg
ctcg 749240DNAArtificial SequenceSynthetic Construct 92cggcgcagta
acagacgggt aacgcgggag atttttcatg 409338DNAArtificial
SequenceSynthetic Construct 93cgcttcccat cacgttatta tttttcaacc
tgctgaac 389440DNAArtificial SequenceSynthetic Construct
94gaagtgctgg aaatcgatcc ggcactggag gcgtaacatg 409538DNAArtificial
SequenceSynthetic Construct 95cttaggctgc taatgactta atcgacttca
cgaatatc 3896144DNAArtificial SequenceSynthetic Construct
96gaggaattcg cgagccgtca cgcccttgac natgccacat cctgagcaaa taattcaacc
60actaaacaaa tcaaccgcgt ttcccggagg taaccggatc caaggagata taccatgcat
120aaccaggctc caattcaacg taga 1449738DNAArtificial
SequenceSynthetic Construct 97atatcctgca gttatagcat tctatgaatt
tgcctgtc 389817DNAArtificial SequenceSynthetic Construct
98gtaaaacgac ggccagt 179917DNAArtificial SequenceSynthetic
Construct 99caggaaacag ctatgac 1710021DNAArtificial
SequenceSynthetic Construct 100caggagccag aacgcaactg c
2110121DNAArtificial SequenceSynthetic Construct 101cactttcgcc
tgatgttcac c 2110222DNAArtificial SequenceSynthetic Construct
102cattcagtct cttgcagggg tc 2210321DNAArtificial SequenceSynthetic
Construct 103gcatagtgcc gctcatctgc c 2110435DNAArtificial
SequenceSynthetic Construct 104gaaactgaaa cccatatgga agctcgtcgt
tctgc 3510544DNAArtificial SequenceSynthetic Construct
105cccgcgctta ctcgaggcgt tcaaacggca gaatcggttc agtg
4410630DNAArtificial SequenceSynthetic Construct 106gatcggatcc
attcgccctt aggaggtaaa 3010740DNAArtificial SequenceSynthetic
Construct 107gatcgcggcc gccagctgca ggacgcgttg ttatagcatt
401086909DNAArtificial SequenceSynthetic Construct 108tggcgaatgg
gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 60cagcgtgacc
gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
120ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
tccctttagg 180gttccgattt agtgctttac ggcacctcga ccccaaaaaa
cttgattagg gtgatggttc 240acgtagtggg ccatcgccct gatagacggt
ttttcgccct ttgacgttgg agtccacgtt 300ctttaatagt ggactcttgt
tccaaactgg aacaacactc aaccctatct cggtctattc 360ttttgattta
taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
420acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag
gtggcacttt 480tcggggaaat gtgcgcggaa cccctatttg tttatttttc
taaatacatt caaatatgta 540tccgctcatg aattaattct tagaaaaact
catcgagcat caaatgaaac tgcaatttat 600tcatatcagg attatcaata
ccatattttt gaaaaagccg tttctgtaat gaaggagaaa 660actcaccgag
gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc
720gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta
tcaagtgaga 780aatcaccatg agtgacgact gaatccggtg agaatggcaa
aagtttatgc atttctttcc 840agacttgttc aacaggccag ccattacgct
cgtcatcaaa atcactcgca tcaaccaaac 900cgttattcat tcgtgattgc
gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac 960aattacaaac
aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat
1020tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg
gggatcgcag 1080tggtgagtaa ccatgcatca tcaggagtac ggataaaatg
cttgatggtc ggaagaggca 1140taaattccgt cagccagttt agtctgacca
tctcatctgt aacatcattg gcaacgctac 1200ctttgccatg tttcagaaac
aactctggcg
catcgggctt cccatacaat cgatagattg 1260tcgcacctga ttgcccgaca
ttatcgcgag cccatttata cccatataaa tcagcatcca 1320tgttggaatt
taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac
1380cccttgtatt actgtttatg taagcagaca gttttattgt tcatgaccaa
aatcccttaa 1440cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
agatcaaagg atcttcttga 1500gatccttttt ttctgcgcgt aatctgctgc
ttgcaaacaa aaaaaccacc gctaccagcg 1560gtggtttgtt tgccggatca
agagctacca actctttttc cgaaggtaac tggcttcagc 1620agagcgcaga
taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag
1680aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
ggctgctgcc 1740agtggcgata agtcgtgtct taccgggttg gactcaagac
gatagttacc ggataaggcg 1800cagcggtcgg gctgaacggg gggttcgtgc
acacagccca gcttggagcg aacgacctac 1860accgaactga gatacctaca
gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga 1920aaggcggaca
ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
1980ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
ctgacttgag 2040cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
ggaaaaacgc cagcaacgcg 2100gcctttttac ggttcctggc cttttgctgg
ccttttgctc acatgttctt tcctgcgtta 2160tcccctgatt ctgtggataa
ccgtattacc gcctttgagt gagctgatac cgctcgccgc 2220agccgaacga
ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg
2280tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatatggtgc
actctcagta 2340caatctgctc tgatgccgca tagttaagcc agtatacact
ccgctatcgc tacgtgactg 2400ggtcatggct gcgccccgac acccgccaac
acccgctgac gcgccctgac gggcttgtct 2460gctcccggca tccgcttaca
gacaagctgt gaccgtctcc gggagctgca tgtgtcagag 2520gttttcaccg
tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc
2580gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga
gtttctccag 2640aagcgttaat gtctggcttc tgataaagcg ggccatgtta
agggcggttt tttcctgttt 2700ggtcactgat gcctccgtgt aagggggatt
tctgttcatg ggggtaatga taccgatgaa 2760acgagagagg atgctcacga
tacgggttac tgatgatgaa catgcccggt tactggaacg 2820ttgtgagggt
aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg
2880tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc
agcagcatcc 2940tgcgatgcag atccggaaca taatggtgca gggcgctgac
ttccgcgttt ccagacttta 3000cgaaacacgg aaaccgaaga ccattcatgt
tgttgctcag gtcgcagacg ttttgcagca 3060gcagtcgctt cacgttcgct
cgcgtatcgg tgattcattc tgctaaccag taaggcaacc 3120ccgccagcct
agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc
3180catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac
cagtgacgaa 3240ggcttgagcg agggcgtgca agattccgaa taccgcaagc
gacaggccga tcatcgtcgc 3300gctccagcga aagcggtcct cgccgaaaat
gacccagagc gctgccggca cctgtcctac 3360gagttgcatg ataaagaaga
cagtcataag tgcggcgacg atagtcatgc cccgcgccca 3420ccggaaggag
ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta
3480atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc
agtcgggaaa 3540cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
gggagaggcg gtttgcgtat 3600tgggcgccag ggtggttttt cttttcacca
gtgagacggg caacagctga ttgcccttca 3660ccgcctggcc ctgagagagt
tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa 3720aatcctgttt
gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt
3780atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg
gcgcgcattg 3840cgcccagcgc catctgatcg ttggcaacca gcatcgcagt
gggaacgatg ccctcattca 3900gcatttgcat ggtttgttga aaaccggaca
tggcactcca gtcgccttcc cgttccgcta 3960tcggctgaat ttgattgcga
gtgagatatt tatgccagcc agccagacgc agacgcgccg 4020agacagaact
taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat
4080gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg
atgggtgtct 4140ggtcagagac atcaagaaat aacgccggaa cattagtgca
ggcagcttcc acagcaatgg 4200catcctggtc atccagcgga tagttaatga
tcagcccact gacgcgttgc gcgagaagat 4260tgtgcaccgc cgctttacag
gcttcgacgc cgcttcgttc taccatcgac accaccacgc 4320tggcacccag
ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca
4380gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc
agttgttgtg 4440ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc
ttccactttt tcccgcgttt 4500tcgcagaaac gtggctggcc tggttcacca
cgcgggaaac ggtctgataa gagacaccgg 4560catactctgc gacatcgtat
aacgttactg gtttcacatt caccaccctg aattgactct 4620cttccgggcg
ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga
4680tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag
taggttgagg 4740ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg
agatggcgcc caacagtccc 4800ccggccacgg ggcctgccac catacccacg
ccgaaacaag cgctcatgag cccgaagtgg 4860cgagcccgat cttccccatc
ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg 4920gcgccggtga
tgccggccac gatgcgtccg gcgtagagga tcgagatctc gatcccgcga
4980aattaatacg actcactata ggggaattgt gagcggataa caattcccct
ctagaaataa 5040ttttgtttaa ctttaagaag gagatataca tatggaagct
cgtcgttctg cgaactacga 5100acctaacagc tgggactatg attacctgct
gtcctccgac acggacgagt ccatcgaagt 5160atacaaagac aaagcgaaaa
agctggaagc cgaagttcgt cgcgagatta ataacgaaaa 5220agcagaattt
ctgaccctgc tggaactgat tgacaacgtc cagcgcctgg gcctgggtta
5280ccgtttcgag tctgatatcc gtggtgcgct ggatcgcttc gtttcctccg
gcggcttcga 5340tgcggtaacc aagacttccc tgcacggtac ggcactgtct
ttccgtctgc tgcgtcaaca 5400cggttttgag gtttctcagg aagcgttcag
cggcttcaaa gaccaaaacg gcaacttcct 5460ggagaacctg aaggaagata
tcaaagctat cctgagcctg tacgaggcca gcttcctggc 5520tctggaaggc
gaaaacatcc tggacgaggc gaaggttttc gcaatctctc atctgaaaga
5580actgtctgaa gaaaagatcg gtaaagagct ggcagaacag gtgaaccatg
cactggaact 5640gccactgcat cgccgtactc agcgtctgga agcagtatgg
tctatcgagg cctaccgtaa 5700aaaggaggac gcgaatcagg ttctgctgga
gctggcaatt ctggattaca acatgatcca 5760gtctgtatac cagcgtgatc
tgcgtgaaac gtcccgttgg tggcgtcgtg tgggtctggc 5820gaccaaactg
cactttgctc gtgaccgcct gattgagagc ttctactggg ccgtgggtgt
5880agcattcgaa ccgcaatact ccgactgccg taactccgtc gcaaaaatgt
tttctttcgt 5940aaccattatc gacgatatct acgatgtata cggcaccctg
gacgaactgg agctgtttac 6000tgatgcagtt gagcgttggg acgtaaacgc
catcaacgac ctgccggatt acatgaaact 6060gtgctttctg gctctgtata
acactattaa cgaaatcgcc tacgacaacc tgaaagataa 6120aggtgagaac
atcctgccgt atctgaccaa agcctgggct gacctgtgca acgctttcct
6180gcaagaagcc aagtggctgt acaacaaatc tactccgacc tttgacgact
acttcggcaa 6240cgcatggaaa tcctcttctg gcccgctgca actggtgttc
gcttacttcg ctgtcgtgca 6300gaacattaaa aaggaagaga tcgaaaacct
gcaaaaatac catgacacca tctctcgtcc 6360ttcccatatc ttccgtctgt
gcaatgacct ggctagcgcg tctgcggaaa ttgcgcgtgg 6420tgaaaccgca
aatagcgttt cttgttacat gcgcactaaa ggtatctccg aagaactggc
6480taccgaaagc gtgatgaatc tgatcgatga aacctggaaa aagatgaaca
aggaaaaact 6540gggtggtagc ctgttcgcga aaccgttcgt ggaaaccgcg
atcaacctgg cacgtcaatc 6600tcactgcact tatcataacg gcgacgcgca
tacctctccg gatgagctga cccgcaaacg 6660cgttctgtct gtaatcactg
aaccgattct gccgtttgaa cgctaaggat ccgaattcga 6720gctccgtcga
caagcttgcg gccgcactcg agcaccacca ccaccaccac tgagatccgg
6780ctgctaacaa agcccgaaag gaagctgagt tggctgctgc caccgctgag
caataactag 6840cataacccct tggggcctct aaacgggtct tgaggggttt
tttgctgaaa ggaggaacta 6900tatccggat 69091099685DNAArtificial
SequenceSynthetic Construct 109tggcgaatgg gacgcgccct gtagcggcgc
attaagcgcg gcgggtgtgg tggttacgcg 60cagcgtgacc gctacacttg ccagcgccct
agcgcccgct cctttcgctt tcttcccttc 120ctttctcgcc acgttcgccg
gctttccccg tcaagctcta aatcgggggc tccctttagg 180gttccgattt
agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
240acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
agtccacgtt 300ctttaatagt ggactcttgt tccaaactgg aacaacactc
aaccctatct cggtctattc 360ttttgattta taagggattt tgccgatttc
ggcctattgg ttaaaaaatg agctgattta 420acaaaaattt aacgcgaatt
ttaacaaaat attaacgttt acaatttcag gtggcacttt 480tcggggaaat
gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
540tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac
tgcaatttat 600tcatatcagg attatcaata ccatattttt gaaaaagccg
tttctgtaat gaaggagaaa 660actcaccgag gcagttccat aggatggcaa
gatcctggta tcggtctgcg attccgactc 720gtccaacatc aatacaacct
attaatttcc cctcgtcaaa aataaggtta tcaagtgaga 780aatcaccatg
agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc
840agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca
tcaaccaaac 900cgttattcat tcgtgattgc gcctgagcga gacgaaatac
gcgatcgctg ttaaaaggac 960aattacaaac aggaatcgaa tgcaaccggc
gcaggaacac tgccagcgca tcaacaatat 1020tttcacctga atcaggatat
tcttctaata cctggaatgc tgttttcccg gggatcgcag 1080tggtgagtaa
ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca
1140taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg
gcaacgctac 1200ctttgccatg tttcagaaac aactctggcg catcgggctt
cccatacaat cgatagattg 1260tcgcacctga ttgcccgaca ttatcgcgag
cccatttata cccatataaa tcagcatcca 1320tgttggaatt taatcgcggc
ctagagcaag acgtttcccg ttgaatatgg ctcataacac 1380cccttgtatt
actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa
1440cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
atcttcttga 1500gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
aaaaaccacc gctaccagcg 1560gtggtttgtt tgccggatca agagctacca
actctttttc cgaaggtaac tggcttcagc 1620agagcgcaga taccaaatac
tgtccttcta gtgtagccgt agttaggcca ccacttcaag 1680aactctgtag
caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
1740agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
ggataaggcg 1800cagcggtcgg gctgaacggg gggttcgtgc acacagccca
gcttggagcg aacgacctac 1860accgaactga gatacctaca gcgtgagcta
tgagaaagcg ccacgcttcc cgaagggaga 1920aaggcggaca ggtatccggt
aagcggcagg gtcggaacag gagagcgcac gagggagctt 1980ccagggggaa
acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
2040cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
cagcaacgcg 2100gcctttttac ggttcctggc cttttgctgg ccttttgctc
acatgttctt tcctgcgtta 2160tcccctgatt ctgtggataa ccgtattacc
gcctttgagt gagctgatac cgctcgccgc 2220agccgaacga ccgagcgcag
cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg 2280tattttctcc
ttacgcatct gtgcggtatt tcacaccgca tatatggtgc actctcagta
2340caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc
tacgtgactg 2400ggtcatggct gcgccccgac acccgccaac acccgctgac
gcgccctgac gggcttgtct 2460gctcccggca tccgcttaca gacaagctgt
gaccgtctcc gggagctgca tgtgtcagag 2520gttttcaccg tcatcaccga
aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc 2580gtgaagcgat
tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag
2640aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt
tttcctgttt 2700ggtcactgat gcctccgtgt aagggggatt tctgttcatg
ggggtaatga taccgatgaa 2760acgagagagg atgctcacga tacgggttac
tgatgatgaa catgcccggt tactggaacg 2820ttgtgagggt aaacaactgg
cggtatggat gcggcgggac cagagaaaaa tcactcaggg 2880tcaatgccag
cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc
2940tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt
ccagacttta 3000cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag
gtcgcagacg ttttgcagca 3060gcagtcgctt cacgttcgct cgcgtatcgg
tgattcattc tgctaaccag taaggcaacc 3120ccgccagcct agccgggtcc
tcaacgacag gagcacgatc atgcgcaccc gtggggccgc 3180catgccggcg
ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa
3240ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga
tcatcgtcgc 3300gctccagcga aagcggtcct cgccgaaaat gacccagagc
gctgccggca cctgtcctac 3360gagttgcatg ataaagaaga cagtcataag
tgcggcgacg atagtcatgc cccgcgccca 3420ccggaaggag ctgactgggt
tgaaggctct caagggcatc ggtcgagatc ccggtgccta 3480atgagtgagc
taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
3540cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
gtttgcgtat 3600tgggcgccag ggtggttttt cttttcacca gtgagacggg
caacagctga ttgcccttca 3660ccgcctggcc ctgagagagt tgcagcaagc
ggtccacgct ggtttgcccc agcaggcgaa 3720aatcctgttt gatggtggtt
aacggcggga tataacatga gctgtcttcg gtatcgtcgt 3780atcccactac
cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg
3840cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg
ccctcattca 3900gcatttgcat ggtttgttga aaaccggaca tggcactcca
gtcgccttcc cgttccgcta 3960tcggctgaat ttgattgcga gtgagatatt
tatgccagcc agccagacgc agacgcgccg 4020agacagaact taatgggccc
gctaacagcg cgatttgctg gtgacccaat gcgaccagat 4080gctccacgcc
cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct
4140ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc
acagcaatgg 4200catcctggtc atccagcgga tagttaatga tcagcccact
gacgcgttgc gcgagaagat 4260tgtgcaccgc cgctttacag gcttcgacgc
cgcttcgttc taccatcgac accaccacgc 4320tggcacccag ttgatcggcg
cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca 4380gggccagact
ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg
4440ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt
tcccgcgttt 4500tcgcagaaac gtggctggcc tggttcacca cgcgggaaac
ggtctgataa gagacaccgg 4560catactctgc gacatcgtat aacgttactg
gtttcacatt caccaccctg aattgactct 4620cttccgggcg ctatcatgcc
ataccgcgaa aggttttgcg ccattcgatg gtgtccggga 4680tctcgacgct
ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg
4740ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc
caacagtccc 4800ccggccacgg ggcctgccac catacccacg ccgaaacaag
cgctcatgag cccgaagtgg 4860cgagcccgat cttccccatc ggtgatgtcg
gcgatatagg cgccagcaac cgcacctgtg 4920gcgccggtga tgccggccac
gatgcgtccg gcgtagagga tcgagatctc gatcccgcga 4980aattaatacg
actcactata ggggaattgt gagcggataa caattcccct ctagaaataa
5040ttttgtttaa ctttaagaag gagatataca tatggaagct cgtcgttctg
cgaactacga 5100acctaacagc tgggactatg attacctgct gtcctccgac
acggacgagt ccatcgaagt 5160atacaaagac aaagcgaaaa agctggaagc
cgaagttcgt cgcgagatta ataacgaaaa 5220agcagaattt ctgaccctgc
tggaactgat tgacaacgtc cagcgcctgg gcctgggtta 5280ccgtttcgag
tctgatatcc gtggtgcgct ggatcgcttc gtttcctccg gcggcttcga
5340tgcggtaacc aagacttccc tgcacggtac ggcactgtct ttccgtctgc
tgcgtcaaca 5400cggttttgag gtttctcagg aagcgttcag cggcttcaaa
gaccaaaacg gcaacttcct 5460ggagaacctg aaggaagata tcaaagctat
cctgagcctg tacgaggcca gcttcctggc 5520tctggaaggc gaaaacatcc
tggacgaggc gaaggttttc gcaatctctc atctgaaaga 5580actgtctgaa
gaaaagatcg gtaaagagct ggcagaacag gtgaaccatg cactggaact
5640gccactgcat cgccgtactc agcgtctgga agcagtatgg tctatcgagg
cctaccgtaa 5700aaaggaggac gcgaatcagg ttctgctgga gctggcaatt
ctggattaca acatgatcca 5760gtctgtatac cagcgtgatc tgcgtgaaac
gtcccgttgg tggcgtcgtg tgggtctggc 5820gaccaaactg cactttgctc
gtgaccgcct gattgagagc ttctactggg ccgtgggtgt 5880agcattcgaa
ccgcaatact ccgactgccg taactccgtc gcaaaaatgt tttctttcgt
5940aaccattatc gacgatatct acgatgtata cggcaccctg gacgaactgg
agctgtttac 6000tgatgcagtt gagcgttggg acgtaaacgc catcaacgac
ctgccggatt acatgaaact 6060gtgctttctg gctctgtata acactattaa
cgaaatcgcc tacgacaacc tgaaagataa 6120aggtgagaac atcctgccgt
atctgaccaa agcctgggct gacctgtgca acgctttcct 6180gcaagaagcc
aagtggctgt acaacaaatc tactccgacc tttgacgact acttcggcaa
6240cgcatggaaa tcctcttctg gcccgctgca actggtgttc gcttacttcg
ctgtcgtgca 6300gaacattaaa aaggaagaga tcgaaaacct gcaaaaatac
catgacacca tctctcgtcc 6360ttcccatatc ttccgtctgt gcaatgacct
ggctagcgcg tctgcggaaa ttgcgcgtgg 6420tgaaaccgca aatagcgttt
cttgttacat gcgcactaaa ggtatctccg aagaactggc 6480taccgaaagc
gtgatgaatc tgatcgatga aacctggaaa aagatgaaca aggaaaaact
6540gggtggtagc ctgttcgcga aaccgttcgt ggaaaccgcg atcaacctgg
cacgtcaatc 6600tcactgcact tatcataacg gcgacgcgca tacctctccg
gatgagctga cccgcaaacg 6660cgttctgtct gtaatcactg aaccgattct
gccgtttgaa cgctaaggat ccattcgccc 6720ttaggaggta aaaaaacatg
agttttgata ttgccaaata cccgaccctg gcactggtcg 6780actccaccca
ggagttacga ctgttgccga aagagagttt accgaaactc tgcgacgaac
6840tgcgccgcta tttactcgac agcgtgagcc gttccagcgg gcacttcgcc
tccgggctgg 6900gcacggtcga actgaccgtg gcgctgcact atgtctacaa
caccccgttt gaccaattga 6960tttgggatgt ggggcatcag gcttatccgc
ataaaatttt gaccggacgc cgcgacaaaa 7020tcggcaccat ccgtcagaaa
ggcggtctgc acccgttccc gtggcgcggc gaaagcgaat 7080atgacgtatt
aagcgtcggg cattcatcaa cctccatcag tgccggaatt ggtattgcgg
7140ttgctgccga aaaagaaggc aaaaatcgcc gcaccgtctg tgtcattggc
gatggcgcga 7200ttaccgcagg catggcgttt gaagcgatga atcacgcggg
cgatatccgt cctgatatgc 7260tggtgattct caacgacaat gaaatgtcga
tttccgaaaa tgtcggcgcg ctcaacaacc 7320atctggcaca gctgctttcc
ggtaagcttt actcttcact gcgcgaaggc gggaaaaaag 7380ttttctctgg
cgtgccgcca attaaagagc tgctcaaacg caccgaagaa catattaaag
7440gcatggtagt gcctggcacg ttgtttgaag agctgggctt taactacatc
ggcccggtgg 7500acggtcacga tgtgctgggg cttatcacca cgctaaagaa
catgcgcgac ctgaaaggcc 7560cgcagttcct gcatatcatg accaaaaaag
gtcgtggtta tgaaccggca gaaaaagacc 7620cgatcacttt ccacgccgtg
cctaaatttg atccctccag cggttgtttg ccgaaaagta 7680gcggcggttt
gccgagctat tcaaaaatct ttggcgactg gttgtgcgaa acggcagcga
7740aagacaacaa gctgatggcg attactccgg cgatgcgtga aggttccggc
atggtcgagt 7800tttcacgtaa attcccggat cgctacttcg acgtggcaat
tgccgagcaa cacgcggtga 7860cctttgctgc gggtctggcg attggtgggt
acaaacccat tgtcgcgatt tactccactt 7920tcctgcaacg cgcctatgat
caggtgctgc atgacgtggc gattcaaaag cttccggtcc 7980tgttcgccat
cgaccgcgcg ggcattgttg gtgctgacgg tcaaacccat cagggtgctt
8040ttgatctctc ttacctgcgc tgcataccgg aaatggtcat tatgaccccg
agcgatgaaa 8100acgaatgtcg ccagatgctc tataccggct atcactataa
cgatggcccg tcagcggtgc 8160gctacccgcg tggcaacgcg gtcggcgtgg
aactgacgcc gctggaaaaa ctaccaattg 8220gcaaaggcat tgtgaagcgt
cgtggcgaga aactggcgat ccttaacttt ggtacgctga 8280tgccagaagc
ggcgaaagtc gccgaatcgc tgaacgccac gctggtcgat atgcgttttg
8340tgaaaccgct tgatgaagcg ttaattctgg aaatggccgc cagccatgaa
gcgctggtca 8400ccgtagaaga aaacgccatt atgggcggcg caggcagcgg
cgtgaacgaa gtgctgatgg 8460cccatcgtaa accagtaccc gtgctgaaca
ttggcctgcc ggacttcttt attccgcaag 8520gaactcagga agaaatgcgc
gccgaactcg gcctcgatgc cgctggtatg gaagccaaaa 8580tcaaggcctg
gctggcataa ctgcatcgcc cttaggaggt aaaaaaaaat gactgccgac
8640aacaatagta tgccccatgg tgcagtatct agttacgcca aattagtgca
aaaccaaaca 8700cctgaagaca ttttggaaga gtttcctgaa attattccat
tacaacaaag acctaatacc 8760cgatctagtg agacgtcaaa tgacgaaagc
ggagaaacat gtttttctgg tcatgatgag 8820gagcaaatta agttaatgaa
tgaaaattgt attgttttgg attgggacga taatgctatt 8880ggtgccggta
ccaagaaagt ttgtcattta atggaaaata ttgaaaaggg tttactacat
8940cgtgcattct ccgtctttat tttcaatgaa caaggtgaat tacttttaca
acaaagagcc 9000actgaaaaaa taactttccc tgatctttgg actaacacat
gctgctctca tccactatgt 9060attgatgacg aattaggttt gaagggtaag
ctagacgata agattaaggg cgctattact 9120gcggcggtga gaaaactaga
tcatgaatta ggtattccag aagatgaaac taagacaagg 9180ggtaagtttc
actttttaaa cagaatccat tacatggcac caagcaatga accatggggt
9240gaacatgaaa ttgattacat cctattttat
aagatcaacg ctaaagaaaa cttgactgtc 9300aacccaaacg tcaatgaagt
tagagacttc aaatgggttt caccaaatga tttgaaaact 9360atgtttgctg
acccaagtta caagtttacg ccttggttta agattatttg cgagaattac
9420ttattcaact ggtgggagca attagatgac ctttctgaag tggaaaatga
caggcaaatt 9480catagaatgc tataacaacg cgtcctgcag ctggcggccg
cactcgagca ccaccaccac 9540caccactgag atccggctgc taacaaagcc
cgaaaggaag ctgagttggc tgctgccacc 9600gctgagcaat aactagcata
accccttggg gcctctaaac gggtcttgag gggttttttg 9660ctgaaaggag
gaactatatc cggat 96851109685DNAArtificial SequenceSynthetic
Construct 110tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
tggttacgcg 60cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
tcttcccttc 120ctttctcgcc acgttcgccg gctttccccg tcaagctcta
aatcgggggc tccctttagg 180gttccgattt agtgctttac ggcacctcga
ccccaaaaaa cttgattagg gtgatggttc 240acgtagtggg ccatcgccct
gatagacggt ttttcgccct ttgacgttgg agtccacgtt 300ctttaatagt
ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
360ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg
agctgattta 420acaaaaattt aacgcgaatt ttaacaaaat attaacgttt
acaatttcag gtggcacttt 480tcggggaaat gtgcgcggaa cccctatttg
tttatttttc taaatacatt caaatatgta 540tccgctcatg aattaattct
tagaaaaact catcgagcat caaatgaaac tgcaatttat 600tcatatcagg
attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa
660actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg
attccgactc 720gtccaacatc aatacaacct attaatttcc cctcgtcaaa
aataaggtta tcaagtgaga 780aatcaccatg agtgacgact gaatccggtg
agaatggcaa aagtttatgc atttctttcc 840agacttgttc aacaggccag
ccattacgct cgtcatcaaa atcactcgca tcaaccaaac 900cgttattcat
tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac
960aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca
tcaacaatat 1020tttcacctga atcaggatat tcttctaata cctggaatgc
tgttttcccg gggatcgcag 1080tggtgagtaa ccatgcatca tcaggagtac
ggataaaatg cttgatggtc ggaagaggca 1140taaattccgt cagccagttt
agtctgacca tctcatctgt aacatcattg gcaacgctac 1200ctttgccatg
tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg
1260tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa
tcagcatcca 1320tgttggaatt taatcgcggc ctagagcaag acgtttcccg
ttgaatatgg ctcataacac 1380cccttgtatt actgtttatg taagcagaca
gttttattgt tcatgaccaa aatcccttaa 1440cgtgagtttt cgttccactg
agcgtcagac cccgtagaaa agatcaaagg atcttcttga 1500gatccttttt
ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
1560gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
tggcttcagc 1620agagcgcaga taccaaatac tgtccttcta gtgtagccgt
agttaggcca ccacttcaag 1680aactctgtag caccgcctac atacctcgct
ctgctaatcc tgttaccagt ggctgctgcc 1740agtggcgata agtcgtgtct
taccgggttg gactcaagac gatagttacc ggataaggcg 1800cagcggtcgg
gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
1860accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
cgaagggaga 1920aaggcggaca ggtatccggt aagcggcagg gtcggaacag
gagagcgcac gagggagctt 1980ccagggggaa acgcctggta tctttatagt
cctgtcgggt ttcgccacct ctgacttgag 2040cgtcgatttt tgtgatgctc
gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg 2100gcctttttac
ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
2160tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac
cgctcgccgc 2220agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
cggaagagcg cctgatgcgg 2280tattttctcc ttacgcatct gtgcggtatt
tcacaccgca tatatggtgc actctcagta 2340caatctgctc tgatgccgca
tagttaagcc agtatacact ccgctatcgc tacgtgactg 2400ggtcatggct
gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct
2460gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca
tgtgtcagag 2520gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg
taaagctcat cagcgtggtc 2580gtgaagcgat tcacagatgt ctgcctgttc
atccgcgtcc agctcgttga gtttctccag 2640aagcgttaat gtctggcttc
tgataaagcg ggccatgtta agggcggttt tttcctgttt 2700ggtcactgat
gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa
2760acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt
tactggaacg 2820ttgtgagggt aaacaactgg cggtatggat gcggcgggac
cagagaaaaa tcactcaggg 2880tcaatgccag cgcttcgtta atacagatgt
aggtgttcca cagggtagcc agcagcatcc 2940tgcgatgcag atccggaaca
taatggtgca gggcgctgac ttccgcgttt ccagacttta 3000cgaaacacgg
aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca
3060gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag
taaggcaacc 3120ccgccagcct agccgggtcc tcaacgacag gagcacgatc
atgcgcaccc gtggggccgc 3180catgccggcg ataatggcct gcttctcgcc
gaaacgtttg gtggcgggac cagtgacgaa 3240ggcttgagcg agggcgtgca
agattccgaa taccgcaagc gacaggccga tcatcgtcgc 3300gctccagcga
aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac
3360gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc
cccgcgccca 3420ccggaaggag ctgactgggt tgaaggctct caagggcatc
ggtcgagatc ccggtgccta 3480atgagtgagc taacttacat taattgcgtt
gcgctcactg cccgctttcc agtcgggaaa 3540cctgtcgtgc cagctgcatt
aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat 3600tgggcgccag
ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca
3660ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc
agcaggcgaa 3720aatcctgttt gatggtggtt aacggcggga tataacatga
gctgtcttcg gtatcgtcgt 3780atcccactac cgagatatcc gcaccaacgc
gcagcccgga ctcggtaatg gcgcgcattg 3840cgcccagcgc catctgatcg
ttggcaacca gcatcgcagt gggaacgatg ccctcattca 3900gcatttgcat
ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta
3960tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc
agacgcgccg 4020agacagaact taatgggccc gctaacagcg cgatttgctg
gtgacccaat gcgaccagat 4080gctccacgcc cagtcgcgta ccgtcttcat
gggagaaaat aatactgttg atgggtgtct 4140ggtcagagac atcaagaaat
aacgccggaa cattagtgca ggcagcttcc acagcaatgg 4200catcctggtc
atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat
4260tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac
accaccacgc 4320tggcacccag ttgatcggcg cgagatttaa tcgccgcgac
aatttgcgac ggcgcgtgca 4380gggccagact ggaggtggca acgccaatca
gcaacgactg tttgcccgcc agttgttgtg 4440ccacgcggtt gggaatgtaa
ttcagctccg ccatcgccgc ttccactttt tcccgcgttt 4500tcgcagaaac
gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg
4560catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg
aattgactct 4620cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg
ccattcgatg gtgtccggga 4680tctcgacgct ctcccttatg cgactcctgc
attaggaagc agcccagtag taggttgagg 4740ccgttgagca ccgccgccgc
aaggaatggt gcatgcaagg agatggcgcc caacagtccc 4800ccggccacgg
ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg
4860cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac
cgcacctgtg 4920gcgccggtga tgccggccac gatgcgtccg gcgtagagga
tcgagatctc gatcccgcga 4980aattaatacg actcactata ggggaattgt
gagcggataa caattcccct ctagaaataa 5040ttttgtttaa ctttaagaag
gagatataca tatggaagct cgtcgttctg cgaactacga 5100acctaacagc
tgggactatg attacctgct gtcctccgac acggacgagt ccatcgaagt
5160atacaaagac aaagcgaaaa agctggaagc cgaagttcgt cgcgagatta
ataacgaaaa 5220agcagaattt ctgaccctgc tggaactgat tgacaacgtc
cagcgcctgg gcctgggtta 5280ccgtttcgag tctgatatcc gtggtgcgct
ggatcgcttc gtttcctccg gcggcttcga 5340tgcggtaacc aagacttccc
tgcacggtac ggcactgtct ttccgtctgc tgcgtcaaca 5400cggttttgag
gtttctcagg aagcgttcag cggcttcaaa gaccaaaacg gcaacttcct
5460ggagaacctg aaggaagata tcaaagctat cctgagcctg tacgaggcca
gcttcctggc 5520tctggaaggc gaaaacatcc tggacgaggc gaaggttttc
gcaatctctc atctgaaaga 5580actgtctgaa gaaaagatcg gtaaagagct
ggcagaacag gtgaaccatg cactggaact 5640gccactgcat cgccgtactc
agcgtctgga agcagtatgg tctatcgagg cctaccgtaa 5700aaaggaggac
gcgaatcagg ttctgctgga gctggcaatt ctggattaca acatgatcca
5760gtctgtatac cagcgtgatc tgcgtgaaac gtcccgttgg tggcgtcgtg
tgggtctggc 5820gaccaaactg cactttgctc gtgaccgcct gattgagagc
ttctactggg ccgtgggtgt 5880agcattcgaa ccgcaatact ccgactgccg
taactccgtc gcaaaaatgt tttctttcgt 5940aaccattatc gacgatatct
acgatgtata cggcaccctg gacgaactgg agctgtttac 6000tgatgcagtt
gagcgttggg acgtaaacgc catcaacgac ctgccggatt acatgaaact
6060gtgctttctg gctctgtata acactattaa cgaaatcgcc tacgacaacc
tgaaagataa 6120aggtgagaac atcctgccgt atctgaccaa agcctgggct
gacctgtgca acgctttcct 6180gcaagaagcc aagtggctgt acaacaaatc
tactccgacc tttgacgact acttcggcaa 6240cgcatggaaa tcctcttctg
gcccgctgca actggtgttc gcttacttcg ctgtcgtgca 6300gaacattaaa
aaggaagaga tcgaaaacct gcaaaaatac catgacacca tctctcgtcc
6360ttcccatatc ttccgtctgt gcaatgacct ggctagcgcg tctgcggaaa
ttgcgcgtgg 6420tgaaaccgca aatagcgttt cttgttacat gcgcactaaa
ggtatctccg aagaactggc 6480taccgaaagc gtgatgaatc tgatcgatga
aacctggaaa aagatgaaca aggaaaaact 6540gggtggtagc ctgttcgcga
aaccgttcgt ggaaaccgcg atcaacctgg cacgtcaatc 6600tcactgcact
tatcataacg gcgacgcgca tacctctccg gatgagctga cccgcaaacg
6660cgttctgtct gtaatcactg aaccgattct gccgtttgaa cgctaaggat
ccattcgccc 6720ttaggaggta aaaaaacatg agttttgata ttgccaaata
cccgaccctg gcactggtcg 6780actccaccca ggagttacga ctgttgccga
aagagagttt accgaaactc tgcgacgaac 6840tgcgccgcta tttactcgac
agcgtgagcc gttccagcgg gcacttcgcc tccgggctgg 6900gcacggtcga
actgaccgtg gcgctgcact atgtctacaa caccccgttt gaccaattga
6960tttgggatgt ggggcatcag gcttatccgc ataaaatttt gaccggacgc
cgcgacaaaa 7020tcggcaccat ccgtcagaaa ggcggtctgc acccgttccc
gtggcgcggc gaaagcgaat 7080atgacgtatt aagcgtcggg cattcatcaa
cctccatcag tgccggaatt ggtattgcgg 7140ttgctgccga aaaagaaggc
aaaaatcgcc gcaccgtctg tgtcattggc gatggcgcga 7200ttaccgcagg
catggcgttt gaagcgatga atcacgcggg cgatatccgt cctgatatgc
7260tggtgattct caacgacaat gaaatgtcga tttccgaaaa tgtcggcgcg
ctcaacaacc 7320atctggcaca gctgctttcc ggtaagcttt actcttcact
gcgcgaaggc gggaaaaaag 7380ttttctctgg cgtgccgcca attaaagagc
tgctcaaacg caccgaagaa catattaaag 7440gcatggtagt gcctggcacg
ttgtttgaag agctgggctt taactacatc ggcccggtgg 7500acggtcacga
tgtgctgggg cttatcacca cgctaaagaa catgcgcgac ctgaaaggcc
7560cgcagttcct gcatatcatg accaaaaaag gtcgtggtta tgaaccggca
gaaaaagacc 7620cgatcacttt ccacgccgtg cctaaatttg atccctccag
cggttgtttg ccgaaaagta 7680gcggcggttt gccgagctat tcaaaaatct
ttggcgactg gttgtgcgaa acggcagcga 7740aagacaacaa gctgatggcg
attactccgg cgatgcgtga aggttccggc atggtcgagt 7800tttcacgtaa
attcccggat cgctacttcg acgtggcaat tgccgagcaa cacgcggtga
7860cctttgctgc gggtctggcg attggtgggt acaaacccat tgtcgcgatt
tactccactt 7920tcctgcaacg cgcctatgat caggtgctgc atgacgtggc
gattcaaaag cttccggtcc 7980tgttcgccat cgaccgcgcg ggcattgttg
gtgctgacgg tcaaacccat cagggtgctt 8040ttgatctctc ttacctgcgc
tgcataccgg aaatggtcat tatgaccccg agcgatgaaa 8100acgaatgtcg
ccagatgctc tataccggct atcactataa cgatggcccg tcagcggtgc
8160gctacccgcg tggcaacgcg gtcggcgtgg aactgacgcc gctggaaaaa
ctaccaattg 8220gcaaaggcat tgtgaagcgt cgtggcgaga aactggcgat
ccttaacttt ggtacgctga 8280tgccagaagc ggcgaaagtc gccgaatcgc
tgaacgccac gctggtcgat atgcgttttg 8340tgaaaccgct tgatgaagcg
ttaattctgg aaatggccgc cagccatgaa gcgctggtca 8400ccgtagaaga
aaacgccatt atgggcggcg caggcagcgg cgtgaacgaa gtgctgatgg
8460cccatcgtaa accagtaccc gtgctgaaca ttggcctgcc ggacttcttt
attccgcaag 8520gaactcagga agaaatgcgc gccgaactcg gcctcgatgc
cgctggtatg gaagccaaaa 8580tcaaggcctg gctggcataa ctgcatcgcc
cttaggaggt aaaaaaaaat gactgccgac 8640aacaatagta tgccccatgg
tgcagtatct agttacgcca aattagtgca aaaccaaaca 8700cctgaagaca
ttttggaaga gtttcctgaa attattccat tacaacaaag acctaatacc
8760cgatctagtg agacgtcaaa tgacgaaagc ggagaaacat gtttttctgg
tcatgatgag 8820gagcaaatta agttaatgaa tgaaaattgt attgttttgg
attgggacga taatgctatt 8880ggtgccggta ccaagaaagt ttgtcattta
atggaaaata ttgaaaaggg tttactacat 8940cgtgcattct ccgtctttat
tttcaatgaa caaggtgaat tacttttaca acaaagagcc 9000actgaaaaaa
taactttccc tgatctttgg actaacacat gctgctctca tccactatgt
9060attgatgacg aattaggttt gaagggtaag ctagacgata agattaaggg
cgctattact 9120gcggcggtga gaaaactaga tcatgaatta ggtattccag
aagatgaaac taagacaagg 9180ggtaagtttc actttttaaa cagaatccat
tacatggcac caagcaatga accatggggt 9240gaacatgaaa ttgattacat
cctattttat aagatcaacg ctaaagaaaa cttgactgtc 9300aacccaaacg
tcaatgaagt tagagacttc aaatgggttt caccaaatga tttgaaaact
9360atgtttgctg acccaagtta caagtttacg ccttggttta agattatttg
cgagaattac 9420ttattcaact ggtgggagca attagatgac ctttctgaag
tggaaaatga caggcaaatt 9480catagaatgc tataacaacg cgtcctgcag
ctggcggccg cactcgagca ccaccaccac 9540caccactgag atccggctgc
taacaaagcc cgaaaggaag ctgagttggc tgctgccacc 9600gctgagcaat
aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg
9660ctgaaaggag gaactatatc cggat 968511132DNAArtificial
SequenceSynthetic Construct 111cagcagcagg gatccgacgc gttgttatag ca
3211232DNAArtificial SequenceSynthetic Construct 112cagcagcagc
atatgactgc cgacaacaat ag 3211325DNAArtificial SequenceSynthetic
Construct 113gaaacctaca tccaatcttt tgccc 2511425DNAArtificial
SequenceSynthetic Construct 114gctatgcttc attagatcct tatcg
2511524DNAArtificial SequenceSynthetic Construct 115ttgccaatca
tatgattgaa aatc 2411623DNAArtificial SequenceSynthetic Construct
116gaaatagccc cattagaagt atc 2311726DNAArtificial SequenceSynthetic
Construct 117atgacaattg ggattgataa aattag 2611820DNAArtificial
SequenceSynthetic Construct 118cttaaatcat ttaaaatagc
2011926DNAArtificial SequenceSynthetic Construct 119atgaaaacag
tagttattat tgatgc 2612033DNAArtificial SequenceSynthetic Construct
120cgatctagaa aggcccagtc tttcgactga gcc 3312134DNAArtificial
SequenceSynthetic Construct 121atgctcgagc tgttgacaat taatcatccg
gctc 34122452DNAArtificial SequenceSynthetic Construct
122aaagaccgac caagcgacgt ctgagagctc cctggcgaat tcggtaccaa
taaaagagct 60ttattttcat gatctgtgtg ttggtttttg tgtgcggcgc ggaagttcct
attctctaga 120aagtatagga acttcctcga gccctatagt gagtcgtatt
agcccttgac aatgccacat 180cctgagcaaa taattcaacc acttttattc
actaacaaat agctggtgga atatatgaag 240caactcacca ttctgggctc
gaccggctcg attggttgca gcacgctgga cgtggtgcgc 300cataatcccg
aacacttccg cgtagttgcg ctggtggcag gcaaaaatgt cactcgcatg
360gtagaacagt gcctggaatt ctctccccgc tatgccgtaa tggacgatga
agcgagtgcg 420aaacttctta aaacgatgct acagcaacag gg
452123337DNAArtificial SequenceSynthetic Construct 123aaagaccgac
caagcgacgt ctgagagctc cctggcgaat tcggtaccaa taaaagagct 60ttattttcat
gatctgtgtg ttggtttttg tgtgcggcgc ggaagttcct attctctaga
120aagtatagga acttcctcga gccctatagt gagtcgtatt aagataacca
tctgcggtga 180taaattatct ctggcggtgt tgacataaat accactggcg
gtgatactga gcacatcagc 240aggacgcact gcaaaggagg taaaaaaaca
tgagttttga tattgccaaa tacccgaccc 300tggcactggt cgactccacc
caggagttac gactgtt 33712422DNAArtificial SequenceSynthetic
Construct 124ccctgttgct gtagcatcgt tt 22125200DNAArtificial
SequenceSynthetic Construct 125aacagtcgta actcctgggt ggagtcgacc
agtgccaggg tcgggtattt ggcaatatca 60aaactcatgt ttttttacct cctttgcagt
gcgtcctgct gatgtgctca gtatcaccgc 120cagtggtatt tangtcaaca
ccgccagaga taatttatca ccgcagatgg ttatcttaat 180acgactcact
atagggctcg 200126103DNAArtificial SequenceSynthetic Construct
126acaaaaacgc cgctcagtag atccttgcgg atcggctggc ggcgttttgc
tttttattct 60gtctcaactc tggatgtttc aattaaccct cactaaaggg cgg
103127177DNAArtificial SequenceSynthetic Construct 127tgttcgggat
tatggcgcac cacgtccagc gtgctgcaac caatcgagcc ggtcgagccc 60agaatggtga
gttgcttcat atattccacc agctatttgt tagtgaataa aagtggttga
120attatttgct caggatgtgg catngtcaag ggctaatacg actcactata gggctcg
177128103DNAArtificial SequenceSynthetic Construct 128tcgatacctc
ggcactggaa gcgctagcgg actacatcat ccagcgtaat aaataaacaa 60taagtattaa
taggcccctg aattaaccct cactaaaggg cgg 1031291702DNAArtificial
SequenceSynthetic Construct 129actaaagggc ggccgcgaag ttcctattct
ctagaaagta taggaacttc attctaccgg 60gtaggggagg cgcttttccc aaggcagtct
ggagcatgcg ctttagcagc cccgctgggc 120acttggcgct acacaagtgg
cctctggcct cgcacacatt ccacatccac cggtaggcgc 180caaccggctc
cgttctttgg tggccccttc gcgccacctt ccactcctcc cctagtcagg
240aagttccccc ccgccccgca gctcgcgtcg tgcaggacgt gacaaatgga
agtagcacgt 300ctcactagtc tcgtgcagat ggacagcacc gctgagcaat
ggaagcgggt aggcctttgg 360ggcagcggcc aatagcagct ttgctccttc
gctttctggg ctcagaggct gggaaggggt 420gggtccgggg gcgggctcag
gggcgggctc aggggcgggg cgggcgcccg aaggtcctcc 480ggaggcccgg
cattctgcac gcttcaaaag cgcacgtctg ccgcgctgtt ctcctcttcc
540tcatctccgg gcctttcgac ctgcagcagc acgtgttgac aattaatcat
cggcatagta 600tatcggcata gtataatacg acaaggtgag gaactaaacc
atgggatcgg ccattgaaca 660agatggattg cacgcaggtt ctccggccgc
ttgggtggag aggctattcg gctatgactg 720ggcacaacag acgatcggct
gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg 780cccggttctt
tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aggacgaggc
840agcgcggcta tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc
tcgacgttgt 900cactgaagcg ggaagggact ggctgctatt gggcgaagtg
ccggggcagg atctcctgtc 960atctcacctt gctcctgccg agaaagtatc
catcatggct gatgcaatgc ggcggctgca 1020tacgcttgat ccggctacct
gcccattcga ccaccaagcg aaacatcgca tcgagcgagc 1080acgtactcgg
atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg
1140gctcgcgcca gccgaactgt tcgccaggct caaggcgcgc atgcccgacg
gcgaggatct 1200cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg
gtggaaaatg gccgcttttc 1260tggattcatc gactgtggcc ggctgggtgt
ggcggaccgc tatcaggaca tagcgttggc 1320tacccgtgat attgctgaag
agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta 1380cggtatcgcc
gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt
1440ctgagcggga ctctggggtt cgaataaaga ccgaccaagc gacgtctgag
agctccctgg 1500cgaattcggt accaataaaa gagctttatt ttcatgatct
gtgtgttggt ttttgtgtgc 1560ggcgcggaag ttcctattct ctagaaagta
taggaacttc ctcgagccct atagtgagtc 1620gtattagccc ttgacnatgc
cacatcctga gcaaataatt caaccacttt tattcactaa 1680caaatagctg
gtggaatata tg 17021301724DNAArtificial SequenceSynthetic Construct
130cgcgaagttc ctattctcta gaaagtatag gaacttcatt ctaccgggta
ggggaggcgc 60ttttcccaag gcagtctgga gcatgcgctt tagcagcccc gctgggcact
tggcgctaca 120caagtggcct ctggcctcgc acacattcca catccaccgg
taggcgccaa ccggctccgt 180tctttggtgg ccccttcgcg ccaccttcca
ctcctcccct agtcaggaag ttcccccccg 240ccccgcagct cgcgtcgtgc
aggacgtgac aaatggaagt agcacgtctc actagtctcg 300tgcagatgga
cagcaccgct gagcaatgga agcgggtagg cctttggggc agcggccaat
360agcagctttg ctccttcgct ttctgggctc agaggctggg aaggggtggg
tccgggggcg 420ggctcagggg cgggctcagg ggcggggcgg gcgcccgaag
gtcctccgga ggcccggcat 480tctgcacgct tcaaaagcgc acgtctgccg
cgctgttctc ctcttcctca tctccgggcc 540tttcgacctg cagcagcacg
tgttgacaat taatcatcgg catagtatat cggcatagta 600taatacgaca
aggtgaggaa ctaaaccatg ggatcggcca ttgaacaaga tggattgcac
660gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc
acaacagacg 720atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc
aggggcgccc ggttcttttt 780gtcaagaccg acctgtccgg tgccctgaat
gaactgcagg acgaggcagc gcggctatcg 840tggctggcca cgacgggcgt
tccttgcgca gctgtgctcg acgttgtcac tgaagcggga 900agggactggc
tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct
960cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac
gcttgatccg 1020gctacctgcc cattcgacca ccaagcgaaa catcgcatcg
agcgagcacg tactcggatg 1080gaagccggtc ttgtcgatca ggatgatctg
gacgaagagc atcaggggct cgcgccagcc 1140gaactgttcg ccaggctcaa
ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat 1200ggcgatgcct
gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac
1260tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac
ccgtgatatt 1320gctgaagagc ttggcggcga atgggctgac cgcttcctcg
tgctttacgg tatcgccgct 1380cccgattcgc agcgcatcgc cttctatcgc
cttcttgacg agttcttctg agcgggactc 1440tggggttcga ataaagaccg
accaagcgac gtctgagagc tccctggcga attcggtacc 1500aataaaagag
ctttattttc atgatctgtg tgttggtttt tgtgtgcggc gcggaagttc
1560ctattctcta gaaagtatag gaacttcctc gagccctata gtgagtcgta
ttaagataac 1620catctgcggt gataaattat ctctggcggt gttgacntaa
ataccactgg cggtgatact 1680gagcacatca gcaggacgca ctgcaaagga
ggtaaaaaaa catg 172413118DNAArtificial SequenceSynthetic Construct
131ggcgatagaa ggcgatgc 1813220DNAArtificial SequenceSynthetic
Construct 132gagcgcccaa tacgcaaacc 2013321DNAArtificial
SequenceSynthetic Construct 133cacgacaggt ttcccgactg g
2113421DNAArtificial SequenceSynthetic Construct 134ggactcaaga
cgatagttac c 2113522DNAArtificial SequenceSynthetic Construct
135gtgatattgc tgaagagctt gg 2213620DNAArtificial SequenceSynthetic
Construct 136gaactccaag acgaggcagc 2013720DNAArtificial
SequenceSynthetic Construct 137cgtcgtttta caacgtcgtg
20138112DNAArtificial SequenceSynthetic Construct 138ggatccgtaa
tctgtttcct gtgtgaaatt gttatccgct cacaattcca cacattatac 60gagccgatga
ttaattgtca acagaattcc tttccagtcg ggaaacctgt cg
11213979DNAArtificial SequenceSynthetic Construct 139taactttaag
gaggtataca tatggagctc acgcgtgcgg ccgcctcgag ctgcagtaca 60aataaaaaag
gcacgtcag 7914094DNAArtificial SequenceSynthetic Construct
140ggttaatcat ttcactcttc aattatctat aatgatgagt gatcagaatt
acatgtgaga 60aattaattaa ccctcactaa agggcggccg cgaa
94141100DNAArtificial SequenceSynthetic Construct 141atattccacc
agctatttgt tagtgaataa aagtggttga attatttgct caggatgtgg 60catngtcaag
ggctaatacg actcactata gggctcgagg 100142100DNAArtificial
SequenceSynthetic Construct 142gcccttgacn atgccacatc ctgagcaaat
aattcaacca cttttattca ctaacaaata 60gctggtggaa tatatgactg ccgacaacaa
tagtatgccc 10014382DNAArtificial SequenceSynthetic Construct
143gatgcgtcca gtaaaataag cattacgtta tgctcataac cccggcaaat
gtcggggttt 60tttatagcat tctatgaatt tg 8214421DNAArtificial
SequenceSynthetic Construct 144actgaaacgt tttcatcgct c
2114535DNAArtificial SequenceSynthetic Construct 145gatgcgtcca
gtaaaataag cattacgtta tgctc 3514621DNAArtificial SequenceSynthetic
Construct 146gtcaggctgg aatactcttc g 2114720DNAArtificial
SequenceSynthetic Construct 147gacgctttcg ccaagtcagg
2014830DNAArtificial SequenceSynthetic Construct 148gaggaataaa
ccatggaagc tcgtcgttct 3014930DNAArtificial SequenceSynthetic
Construct 149agaacgacga gcttccatgg tttattcctc 3015023DNAArtificial
SequenceSynthetic Construct 150gacagcttat catcgactgc acg
2315122DNAArtificial SequenceSynthetic Construct 151gcactgtctt
tccgtctgct gc 2215220DNAArtificial SequenceSynthetic Construct
152ctcgtacagg ctcaggatag 2015320DNAArtificial SequenceSynthetic
Construct 153ttacgtccca acgctcaact 2015420DNAArtificial
SequenceSynthetic Construct 154cttcggcaac gcatggaaat
2015523DNAArtificial SequenceSynthetic Construct 155ccaggcaaat
tctgttttat cag 23156544PRTArtificial SequenceSynthetic Construct
156Met Glu Ala Arg Arg Ser Ala Asn Tyr Glu Pro Asn Ser Trp Asp Tyr1
5 10 15 Asp Tyr Leu Leu Ser Ser Asp Thr Asp Glu Ser Ile Glu Val Tyr
Lys 20 25 30 Asp Lys Ala Lys Lys Leu Glu Ala Glu Val Arg Arg Glu
Ile Asn Asn 35 40 45 Glu Lys Ala Glu Phe Leu Thr Leu Leu Glu Leu
Ile Asp Asn Val Gln 50 55 60 Arg Leu Gly Leu Gly Tyr Arg Phe Glu
Ser Asp Ile Arg Gly Ala Leu65 70 75 80 Asp Arg Phe Val Ser Ser Gly
Gly Phe Asp Ala Val Thr Lys Thr Ser 85 90 95 Leu His Gly Thr Ala
Leu Ser Phe Arg Leu Leu Arg Gln His Gly Phe 100 105 110 Glu Val Ser
Gln Glu Ala Phe Ser Gly Phe Lys Asp Gln Asn Gly Asn 115 120 125 Phe
Leu Glu Asn Leu Lys Glu Asp Ile Lys Ala Ile Leu Ser Leu Tyr 130 135
140 Glu Ala Ser Phe Leu Ala Leu Glu Gly Glu Asn Ile Leu Asp Glu
Ala145 150 155 160 Lys Val Phe Ala Ile Ser His Leu Lys Glu Leu Ser
Glu Glu Lys Ile 165 170 175 Gly Lys Glu Leu Ala Glu Gln Val Asn His
Ala Leu Glu Leu Pro Leu 180 185 190 His Arg Arg Thr Gln Arg Leu Glu
Ala Val Trp Ser Ile Glu Ala Tyr 195 200 205 Arg Lys Lys Glu Asp Ala
Asn Gln Val Leu Leu Glu Leu Ala Ile Leu 210 215 220 Asp Tyr Asn Met
Ile Gln Ser Val Tyr Gln Arg Asp Leu Arg Glu Thr225 230 235 240 Ser
Arg Trp Trp Arg Arg Val Gly Leu Ala Thr Lys Leu His Phe Ala 245 250
255 Arg Asp Arg Leu Ile Glu Ser Phe Tyr Trp Ala Val Gly Val Ala Phe
260 265 270 Glu Pro Gln Tyr Ser Asp Cys Arg Asn Ser Val Ala Lys Met
Phe Ser 275 280 285 Phe Val Thr Ile Ile Asp Asp Ile Tyr Asp Val Tyr
Gly Thr Leu Asp 290 295 300 Glu Leu Glu Leu Phe Thr Asp Ala Val Glu
Arg Trp Asp Val Asn Ala305 310 315 320 Ile Asn Asp Leu Pro Asp Tyr
Met Lys Leu Cys Phe Leu Ala Leu Tyr 325 330 335 Asn Thr Ile Asn Glu
Ile Ala Tyr Asp Asn Leu Lys Asp Lys Gly Glu 340 345 350 Asn Ile Leu
Pro Tyr Leu Thr Lys Ala Trp Ala Asp Leu Cys Asn Ala 355 360 365 Phe
Leu Gln Glu Ala Lys Trp Leu Tyr Asn Lys Ser Thr Pro Thr Phe 370 375
380 Asp Asp Tyr Phe Gly Asn Ala Trp Lys Ser Ser Ser Gly Pro Leu
Gln385 390 395 400 Leu Val Phe Ala Tyr Phe Ala Val Val Gln Asn Ile
Lys Lys Glu Glu 405 410 415 Ile Glu Asn Leu Gln Lys Tyr His Asp Thr
Ile Ser Arg Pro Ser His 420 425 430 Ile Phe Arg Leu Cys Asn Asp Leu
Ala Ser Ala Ser Ala Glu Ile Ala 435 440 445 Arg Gly Glu Thr Ala Asn
Ser Val Ser Cys Tyr Met Arg Thr Lys Gly 450 455 460 Ile Ser Glu Glu
Leu Ala Thr Glu Ser Val Met Asn Leu Ile Asp Glu465 470 475 480 Thr
Trp Lys Lys Met Asn Lys Glu Lys Leu Gly Gly Ser Leu Phe Ala 485 490
495 Lys Pro Phe Val Glu Thr Ala Ile Asn Leu Ala Arg Gln Ser His Cys
500 505 510 Thr Tyr His Asn Gly Asp Ala His Thr Ser Pro Asp Glu Leu
Thr Arg 515 520 525 Lys Arg Val Leu Ser Val Ile Thr Glu Pro Ile Leu
Pro Phe Glu Arg 530 535 540 1576020DNAArtificial SequenceSynthetic
Construct 157gtttgacagc ttatcatcga ctgcacggtg caccaatgct tctggcgtca
ggcagccatc 60ggaagctgtg gtatggctgt gcaggtcgta aatcactgca taattcgtgt
cgctcaaggc 120gcactcccgt tctggataat gttttttgcg ccgacatcat
aacggttctg gcaaatattc 180tgaaatgagc tgttgacaat taatcatccg
gctcgtataa tgtgtggaat tgtgagcgga 240taacaatttc acacaggaaa
cagcgccgct gagaaaaagc gaagcggcac tgctctttaa 300caatttatca
gacaatctgt gtgggcactc gaccggaatt atcgattaac tttattatta
360aaaattaaag aggtatatat taatgtatcg attaaataag gaggaataaa
ccatggaagc 420tcgtcgttct gcgaactacg aacctaacag ctgggactat
gattacctgc tgtcctccga 480cacggacgag tccatcgaag tatacaaaga
caaagcgaaa aagctggaag ccgaagttcg 540tcgcgagatt aataacgaaa
aagcagaatt tctgaccctg ctggaactga ttgacaacgt 600ccagcgcctg
ggcctgggtt accgtttcga gtctgatatc cgtggtgcgc tggatcgctt
660cgtttcctcc ggcggcttcg atgcggtaac caagacttcc ctgcacggta
cggcactgtc 720tttccgtctg ctgcgtcaac acggttttga ggtttctcag
gaagcgttca gcggcttcaa 780agaccaaaac ggcaacttcc tggagaacct
gaaggaagat atcaaagcta tcctgagcct 840gtacgaggcc agcttcctgg
ctctggaagg cgaaaacatc ctggacgagg cgaaggtttt 900cgcaatctct
catctgaaag aactgtctga agaaaagatc ggtaaagagc tggcagaaca
960ggtgaaccat gcactggaac tgccactgca tcgccgtact cagcgtctgg
aagcagtatg 1020gtctatcgag gcctaccgta aaaaggagga cgcgaatcag
gttctgctgg agctggcaat 1080tctggattac aacatgatcc agtctgtata
ccagcgtgat ctgcgtgaaa cgtcccgttg 1140gtggcgtcgt gtgggtctgg
cgaccaaact gcactttgct cgtgaccgcc tgattgagag 1200cttctactgg
gccgtgggtg tagcattcga accgcaatac tccgactgcc gtaactccgt
1260cgcaaaaatg ttttctttcg taaccattat cgacgatatc tacgatgtat
acggcaccct 1320ggacgaactg gagctgttta ctgatgcagt tgagcgttgg
gacgtaaacg ccatcaacga 1380cctgccggat tacatgaaac tgtgctttct
ggctctgtat aacactatta acgaaatcgc 1440ctacgacaac ctgaaagata
aaggtgagaa catcctgccg tatctgacca aagcctgggc 1500tgacctgtgc
aacgctttcc tgcaagaagc caagtggctg tacaacaaat ctactccgac
1560ctttgacgac tacttcggca acgcatggaa atcctcttct ggcccgctgc
aactggtgtt 1620cgcttacttc gctgtcgtgc agaacattaa aaaggaagag
atcgaaaacc tgcaaaaata 1680ccatgacacc atctctcgtc cttcccatat
cttccgtctg tgcaatgacc tggctagcgc 1740gtctgcggaa attgcgcgtg
gtgaaaccgc aaatagcgtt tcttgttaca tgcgcactaa 1800aggtatctcc
gaagaactgg ctaccgaaag cgtgatgaat ctgatcgatg aaacctggaa
1860aaagatgaac aaggaaaaac tgggtggtag cctgttcgcg aaaccgttcg
tggaaaccgc 1920gatcaacctg gcacgtcaat ctcactgcac ttatcataac
ggcgacgcgc atacctctcc 1980ggatgagctg acccgcaaac gcgttctgtc
tgtaatcact gaaccgattc tgccgtttga 2040acgctaactg cagctggtac
catatgggaa ttcgaagctt tctagaacaa aaactcatct 2100cagaagagga
tctgaatagc gccgtcgacc atcatcatca tcatcattga gtttaaacgg
2160tctccagctt ggctgttttg gcggatgaga gaagattttc agcctgatac
agattaaatc 2220agaacgcaga agcggtctga taaaacagaa tttgcctggc
ggcagtagcg cggtggtccc 2280acctgacccc atgccgaact cagaagtgaa
acgccgtagc gccgatggta gtgtggggtc 2340tccccatgcg agagtaggga
actgccaggc atcaaataaa acgaaaggct cagtcgaaag 2400actgggcctt
tcgttttatc tgttgtttgt cggtgaacgc tctcctgagt aggacaaatc
2460cgccgggagc ggatttgaac gttgcgaagc aacggcccgg agggtggcgg
gcaggacgcc 2520cgccataaac tgccaggcat caaattaagc agaaggccat
cctgacggat ggcctttttg 2580cgtttctaca aactcttttt gtttattttt
ctaaatacat tcaaatatgt atccgctcat 2640gagacaataa ccctgataaa
tgcttcaata atattgaaaa aggaagagta tgagtattca 2700acatttccgt
gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca
2760cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
gagtgggtta 2820catcgaactg gatctcaaca gcggtaagat ccttgagagt
tttcgccccg aagaacgttt 2880tccaatgatg agcactttta aagttctgct
atgtggcgcg gtattatccc gtgttgacgc 2940cgggcaagag caactcggtc
gccgcataca ctattctcag aatgacttgg ttgagtactc 3000accagtcaca
gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc
3060cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
gaggaccgaa 3120ggagctaacc gcttttttgc acaacatggg ggatcatgta
actcgccttg atcgttggga 3180accggagctg aatgaagcca taccaaacga
cgagcgtgac accacgatgc ctgtagcaat 3240ggcaacaacg ttgcgcaaac
tattaactgg cgaactactt actctagctt cccggcaaca 3300attaatagac
tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc
3360ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc
gcggtatcat 3420tgcagcactg gggccagatg gtaagccctc ccgtatcgta
gttatctaca cgacggggag 3480tcaggcaact atggatgaac gaaatagaca
gatcgctgag ataggtgcct cactgattaa 3540gcattggtaa ctgtcagacc
aagtttactc atatatactt tagattgatt taaaacttca 3600tttttaattt
aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
3660ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
aaggatcttc 3720ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
acaaaaaaac caccgctacc 3780agcggtggtt tgtttgccgg atcaagagct
accaactctt tttccgaagg taactggctt 3840cagcagagcg cagataccaa
atactgtcct tctagtgtag ccgtagttag gccaccactt 3900caagaactct
gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
3960tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
taccggataa 4020ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
cccagcttgg agcgaacgac 4080ctacaccgaa ctgagatacc tacagcgtga
gctatgagaa agcgccacgc ttcccgaagg 4140gagaaaggcg gacaggtatc
cggtaagcgg cagggtcgga acaggagagc gcacgaggga 4200gcttccaggg
ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
4260tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
acgccagcaa 4320cgcggccttt ttacggttcc tggccttttg ctggcctttt
gctcacatgt tctttcctgc 4380gttatcccct gattctgtgg ataaccgtat
taccgccttt gagtgagctg ataccgctcg 4440ccgcagccga acgaccgagc
gcagcgagtc agtgagcgag gaagcggaag agcgcctgat 4500gcggtatttt
ctccttacgc atctgtgcgg tatttcacac cgcatatggt gcactctcag
4560tacaatctgc tctgatgccg catagttaag ccagtataca ctccgctatc
gctacgtgac 4620tgggtcatgg ctgcgccccg acacccgcca acacccgctg
acgcgccctg acgggcttgt 4680ctgctcccgg catccgctta cagacaagct
gtgaccgtct ccgggagctg catgtgtcag 4740aggttttcac cgtcatcacc
gaaacgcgcg aggcagcaga tcaattcgcg cgcgaaggcg 4800aagcggcatg
catttacgtt gacaccatcg aatggtgcaa aacctttcgc ggtatggcat
4860gatagcgccc ggaagagagt caattcaggg tggtgaatgt gaaaccagta
acgttatacg 4920atgtcgcaga gtatgccggt gtctcttatc agaccgtttc
ccgcgtggtg aaccaggcca 4980gccacgtttc tgcgaaaacg cgggaaaaag
tggaagcggc gatggcggag ctgaattaca 5040ttcccaaccg cgtggcacaa
caactggcgg gcaaacagtc gttgctgatt ggcgttgcca 5100cctccagtct
ggccctgcac gcgccgtcgc aaattgtcgc ggcgattaaa tctcgcgccg
5160atcaactggg tgccagcgtg gtggtgtcga tggtagaacg aagcggcgtc
gaagcctgta 5220aagcggcggt gcacaatctt ctcgcgcaac gcgtcagtgg
gctgatcatt aactatccgc 5280tggatgacca ggatgccatt gctgtggaag
ctgcctgcac taatgttccg gcgttatttc 5340ttgatgtctc tgaccagaca
cccatcaaca gtattatttt ctcccatgaa gacggtacgc 5400gactgggcgt
ggagcatctg gtcgcattgg gtcaccagca aatcgcgctg ttagcgggcc
5460cattaagttc tgtctcggcg cgtctgcgtc tggctggctg gcataaatat
ctcactcgca 5520atcaaattca gccgatagcg gaacgggaag gcgactggag
tgccatgtcc ggttttcaac 5580aaaccatgca aatgctgaat gagggcatcg
ttcccactgc gatgctggtt gccaacgatc 5640agatggcgct gggcgcaatg
cgcgccatta ccgagtccgg gctgcgcgtt ggtgcggata 5700tctcggtagt
gggatacgac gataccgaag acagctcatg ttatatcccg ccgtcaacca
5760ccatcaaaca ggattttcgc ctgctggggc aaaccagcgt ggaccgcttg
ctgcaactct 5820ctcagggcca ggcggtgaag ggcaatcagc tgttgcccgt
ctcactggtg aaaagaaaaa 5880ccaccctggc gcccaatacg caaaccgcct
ctccccgcgc gttggccgat tcattaatgc 5940agctggcacg acaggtttcc
cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg 6000agttagcgcg
aattgatctg 602015821DNAArtificial SequenceSynthetic Construct
158tgattccgca agactgcctg t 2115921DNAArtificial SequenceSynthetic
Construct 159ttcggtatta ccggtgtcgc t 2116067DNAArtificial
SequenceSynthetic Construct 160ctatgattgc ctttatccgt gggcaatttt
ccacccccat aattaaccct cactaaaggg 60cggccgc 67161152DNAArtificial
SequenceSynthetic Construct 161aagatgccag tgatagccat gagtgaaata
acctcttgaa ggttacctcc gggaaacgcg 60gttgatttgt ttagtggttg aattatttgc
tcaggatgtg gcatngtcaa gggcgtgacg 120gctcgctaat acgactcact
atagggctcg ag 15216238DNAArtificial SequenceSynthetic Construct
162agataaccat ctgcggtgat aaattatctc tggcggtg 3816321DNAArtificial
SequenceSynthetic Construct 163ggtttagttc ctcaccttgt c
2116431DNAArtificial SequenceSynthetic Construct 164cgaggtcgac
gcgagccgtc acgcccttga c 3116544DNAArtificial SequenceSynthetic
Construct 165gctctcgcga gagcccgcgg tcaggcattg agaatttcgt cgag
4416652DNAArtificial SequenceSynthetic Construct 166tccaccgcgg
gctcgaagga gatataccat gcataaccag gctccaattc aa 5216734DNAArtificial
SequenceSynthetic Construct 167gctctcgcga ttatttttca acctgctgaa
cgtc 3416820DNAArtificial SequenceSynthetic Construct 168gttgtaaaac
gacggccagt 2016950DNAArtificial SequenceSynthetic Construct
169tacgggatcc atttgaggag taagccatgc ataaccaggc tccaattcaa
5017037DNAArtificial SequenceSynthetic Construct 170gctggagctc
cacttatttt tcaacctgct gaacgtc 3717122DNAArtificial
SequenceSynthetic Construct 171gatgatcaac atgacgcatg gc
2217220DNAArtificial SequenceSynthetic Construct 172cattccgatc
cgtattggcg 2017321DNAArtificial SequenceSynthetic Construct
173tcacacagga aacagctatg a 2117433DNAArtificial SequenceSynthetic
Construct 174cagtctatta atatgaagca aacagtttat atc
3317535DNAArtificial SequenceSynthetic Construct 175tagcagccgg
atccttagtg tgcgttaacc accac 351768911DNAArtificial
SequenceSynthetic Construct 176accttcggga gcgcctgaag cccgttctgg
acgccctggg gccgttgaat cgggatatgc 60aggccaaggc cgccgcgatc atcaaggccg
tgggcgaaaa gctgctgacg gaacagcggg 120aagtccagcg ccagaaacag
gcccagcgcc agcaggaacg cgggcgcgca catttccccg 180aaaagtgcca
cctggcggcg ttgtgacaat ttaccgaaca actccgcggc cgggaagccg
240atctcggctt gaacgaattg ttaggtggcg gtacttgggt cgatatcaaa
gtgcatcact 300tcttcccgta tgcccaactt tgtatagaga gccactgcgg
gatcgtcacc gtaatctgct 360tgcacgtaga tcacataagc accaagcgcg
ttggcctcat gcttgaggag attgatgagc 420gcggtggcaa tgccctgcct
ccggtgctcg ccggagactg cgagatcata gatatagatc 480tcactacgcg
gctgctcaaa cctgggcaga acgtaagccg cgagagcgcc aacaaccgct
540tcttggtcga aggcagcaag cgcgatgaat gtcttactac ggagcaagtt
cccgaggtaa 600tcggagtccg gctgatgttg ggagtaggtg gctacgtctc
cgaactcacg accgaaaaga 660tcaagagcag cccgcatgga tttgacttgg
tcagggccga gcctacatgt gcgaatgatg 720cccatacttg agccacctaa
ctttgtttta gggcgactgc cctgctgcgt aacatcgttg 780ctgctgcgta
acatcgttgc tgctccataa catcaaacat cgacccacgg cgtaacgcgc
840ttgctgcttg gatgcccgag gcatagactg tacaaaaaaa cagtcataac
aagccatgaa 900aaccgccact gcgccgttac caccgctgcg ttcggtcaag
gttctggacc agttgcgtga 960gcgcatacgc tacttgcatt acagtttacg
aaccgaacag gcttatgtca actgggttcg 1020tgccttcatc cgtttccacg
gtgtgcgtcc atgggcaaat attatacgca aggcgacaag 1080gtgctgatgc
cgctggcgat tcaggttcat catgccgttt gtgatggctt ccatgtcggc
1140agaatgctta atgaattaca acagttttta tgcatgcgcc caatacgcaa
accgcctctc 1200cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca
ggtttcccga ctggaaagcg 1260ggcagtgagc gcaacgcaat taatgtgagt
tagctcactc attaggcacc ccaggcttta 1320cactttatgc ttccggctcg
tatgttgtgt ggaattgtga gcggataaca atttcacaca 1380ggaaacagct
atgaccatga ttacgccaag cgcgcaatta accctcacta aagggaacaa
1440aagctgggta ccgggccccc cctcgagctg ttgacaatta atcatccggc
tcgtataatg 1500tgtggaattg tgagcggata acaatttcac acaggaaaca
gcgccgctga gaaaaagcga 1560agcggcactg ctctttaaca atttatcaga
caatctgtgt gggcactcga ccggaattat 1620cgattaactt tattattaaa
aattaaagag gtatatatta atgtatcgat taaataagga 1680ggaataaacc
atggatccga gctcaggagg taaaaaaaca tgaaaacagt agttattatt
1740gatgcattac gaacaccaat tggaaaatat aaaggcagct taagtcaagt
aagtgccgta 1800gacttaggaa cacatgttac aacacaactt ttaaaaagac
attccactat ttctgaagaa 1860attgatcaag taatctttgg aaatgtttta
caagctggaa atggccaaaa tcccgcacga 1920caaatagcaa taaacagcgg
tttgtctcat gaaattcccg caatgacggt taatgaggtc 1980tgcggatcag
gaatgaaggc cgttattttg gcgaaacaat tgattcaatt aggagaagcg
2040gaagttttaa ttgctggcgg gattgagaat atgtcccaag cacctaaatt
acaacgtttt 2100aattacgaaa cagaaagcta cgatgcgcct ttttctagta
tgatgtatga tggattaacg 2160gatgccttta gtggtcaggc aatgggctta
actgctgaaa atgtggccga aaagtatcat 2220gtaactagag aagagcaaga
tcaattttct gtacattcac aattaaaagc agctcaagca 2280caagcagaag
ggatattcgc tgacgaaata gccccattag aagtatcagg aacgcttgtg
2340gagaaagatg aagggattcg ccctaattcg agcgttgaga agctaggaac
gcttaaaaca 2400gtttttaaag aagacggtac tgtaacagca gggaatgcat
caaccattaa tgatggggct 2460tctgctttga ttattgcttc acaagaatat
gccgaagcac acggtcttcc ttatttagct 2520attattcgag acagtgtgga
agtcggtatt gatccagcct atatgggaat ttcgccgatt 2580aaagccattc
aaaaactgtt agcgcgcaat caacttacta cggaagaaat tgatctgtat
2640gaaatcaacg aagcatttgc agcaacttca atcgtggtcc aaagagaact
ggctttacca 2700gaggaaaagg tcaacattta tggtggcggt atttcattag
gtcatgcgat tggtgccaca 2760ggtgctcgtt tattaacgag tttaagttat
caattaaatc aaaaagaaaa gaaatatgga 2820gtggcttctt tatgtatcgg
cggtggctta ggactcgcta tgctactaga gagacctcag 2880caaaaaaaaa
acagccgatt ttatcaaatg agtcctgagg aacgcctggc ttctcttctt
2940aatgaaggcc agatttctgc tgatacaaaa aaagaatttg aaaatacggc
tttatcttcg 3000cagattgcca atcatatgat tgaaaatcaa atcagtgaaa
cagaagtgcc gatgggcgtt 3060ggcttacatt taacagtgga cgaaactgat
tatttggtac caatggcgac agaagagccc 3120tcagttattg cggctttgag
taatggtgca aaaatagcac aaggatttaa aacagtgaat 3180caacaacgct
taatgcgtgg acaaatcgtt ttttacgatg ttgcagatcc cgagtcattg
3240attgataaac tacaagtaag agaagcggaa gtttttcaac aagcagagtt
aagttatcca 3300tctatcgtta aacggggcgg cggcttaaga gatttgcaat
atcgtacttt tgatgaatca 3360tttgtatctg tcgacttttt agtagatgtt
aaggatgcaa tgggggcaaa tatcgttaac 3420gctatgttgg aaggtgtggc
cgagttgttc cgtgaatggt ttgcggagca aaagatttta 3480ttcagtattt
taagtaatta tgccacggag tcggttgtta cgatgaaaac ggctattcca
3540gtttcacgtt taagtaaggg gagcaatggc cgggaaattg ctgaaaaaat
tgttttagct 3600tcacgctatg cttcattaga tccttatcgg gcagtcacgc
ataacaaagg aatcatgaat 3660ggcattgaag ctgtagtttt agctacagga
aatgatacac gcgctgttag cgcttcttgt 3720catgcttttg cggtgaagga
aggtcgctac caaggcttga ctagttggac gctggatggc 3780gaacaactaa
ttggtgaaat ttcagttccg cttgctttag ccacggttgg cggtgccaca
3840aaagtcttac ctaaatctca agcagctgct gatttgttag cagtgacgga
tgcaaaagaa 3900ctaagtcgag tagtagcggc tgttggtttg gcacaaaatt
tagcggcgtt acgggcctta 3960gtctctgaag gaattcaaaa aggacacatg
gctctacaag cacgttcttt agcgatgacg 4020gtcggagcta ctggtaaaga
agttgaggca gtcgctcaac aattaaaacg tcaaaaaacg 4080atgaaccaag
accgagccat ggctatttta aatgatttaa gaaaacaata aaggaggtaa
4140aaaaacatga caattgggat tgataaaatt agtttttttg tgccccctta
ttatattgat 4200atgacggcac tggctgaagc cagaaatgta gaccctggaa
aatttcatat tggtattggg 4260caagaccaaa tggcggtgaa cccaatcagc
caagatattg tgacatttgc agccaatgcc 4320gcagaagcga tcttgaccaa
agaagataaa gaggccattg atatggtgat tgtcgggact 4380gagtccagta
tcgatgagtc aaaagcggcc gcagttgtct tacatcgttt aatggggatt
4440caacctttcg ctcgctcttt cgaaatcaag gaagcttgtt acggagcaac
agcaggctta 4500cagttagcta agaatcacgt agccttacat ccagataaaa
aagtcttggt cgtagcggca 4560gatattgcaa aatatggctt aaattctggc
ggtgagccta cacaaggagc tggggcggtt 4620gcaatgttag ttgctagtga
accgcgcatt ttggctttaa aagaggataa tgtgatgctg 4680acgcaagata
tctatgactt ttggcgtcca acaggccacc cgtatcctat ggtcgatggt
4740cctttgtcaa acgaaaccta catccaatct tttgcccaag tctgggatga
acataaaaaa 4800cgaaccggtc ttgattttgc agattatgat gctttagcgt
tccatattcc ttacacaaaa 4860atgggcaaaa aagccttatt agcaaaaatc
tccgaccaaa ctgaagcaga acaggaacga 4920attttagccc gttatgaaga
aagtatcgtc tatagtcgtc gcgtaggaaa cttgtatacg 4980ggttcacttt
atctgggact catttccctt ttagaaaatg caacgacttt aaccgcaggc
5040aatcaaattg gtttattcag ttatggttct ggtgctgtcg ctgaattttt
cactggtgaa 5100ttagtagctg gttatcaaaa tcatttacaa aaagaaactc
atttagcact gctggataat 5160cggacagaac tttctatcgc tgaatatgaa
gccatgtttg cagaaacttt agacacagac 5220attgatcaaa cgttagaaga
tgaattaaaa tatagtattt ctgctattaa taataccgtt 5280cgttcttatc
gaaactaaag atctgcagct ggtaccatat gggaattcga agcttgggcc
5340cgaacaaaaa ctcatctcag aagaggatct gaatagcgcc gtcgaccatc
atcatcatca 5400tcattgagtt taaacggtct ccagcttggc tgttttggcg
gatgagagaa gattttcagc 5460ctgatacaga ttaaatcaga acgcagaagc
ggtctgataa aacagaattt gcctggcggc 5520agtagcgcgg tggtcccacc
tgaccccatg ccgaactcag aagtgaaacg ccgtagcgcc 5580gatggtagtg
tggggtctcc ccatgcgaga gtagggaact gccaggcatc aaataaaacg
5640aaaggctcag tcgaaagact gggcctttct agagcggccg ccaccgcggt
ggagctccaa 5700ttcgccctat agtgagtcgt attacgcgcg ctcactggcc
gtcgttttac aacgtcgtga 5760ctgggaaaac cctggcgtta cccaacttaa
tcgccttgca gcacatcccc ctttcgccag 5820ctggcgtaat agcgaagagg
cccgcaccga tcgcccttcc caacagttgc gcagcctgaa 5880tggcgaatgg
aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa
5940atcagctcat tttttaacca ataggccgac tgcgatgagt ggcagggcgg
ggcgtaattt 6000ttttaaggca gttattggtg cccttaaacg cctggtgcta
cgcctgaata agtgataata 6060agcggatgaa tggcagaaat tcgaaagcaa
attcgacccg gtcgtcggtt cagggcaggg 6120tcgttaaata gccgcttatg
tctattgctg gtttaccggt ttattgacta ccggaagcag 6180tgtgaccgtg
tgcttctcaa atgcctgagg ccagtttgct caggctctcc ccgtggaggt
6240aataattgac gatatgatca tttattctgc ctcccagagc ctgataaaaa
cggtgaatcc 6300gttagcgagg tgccgccggc ttccattcag gtcgaggtgg
cccggctcca tgcaccgcga 6360cgcaacgcgg ggaggcagac aaggtatagg
gcggcgaggc ggctacagcc gatagtctgg 6420aacagcgcac ttacgggttg
ctgcgcaacc caagtgctac cggcgcggca gcgtgacccg 6480tgtcggcggc
tccaacggct cgccatcgtc cagaaaacac ggctcatcgg gcatcggcag
6540gcgctgctgc ccgcgccgtt cccattcctc cgtttcggtc aaggctggca
ggtctggttc 6600catgcccgga atgccgggct ggctgggcgg ctcctcgccg
gggccggtcg gtagttgctg 6660ctcgcccgga tacagggtcg ggatgcggcg
caggtcgcca tgccccaaca gcgattcgtc 6720ctggtcgtcg tgatcaacca
ccacggcggc actgaacacc gacaggcgca actggtcgcg 6780gggctggccc
cacgccacgc ggtcattgac cacgtaggcc gacacggtgc cggggccgtt
6840gagcttcacg acggagatcc agcgctcggc caccaagtcc ttgactgcgt
attggaccgt 6900ccgcaaagaa cgtccgatga gcttggaaag tgtcttctgg
ctgaccacca cggcgttctg 6960gtggcccatc tgcgccacga ggtgatgcag
cagcattgcc gccgtgggtt tcctcgcaat 7020aagcccggcc cacgcctcat
gcgctttgcg ttccgtttgc acccagtgac cgggcttgtt 7080cttggcttga
atgccgattt ctctggactg cgtggccatg cttatctcca tgcggtaggg
7140tgccgcacgg ttgcggcacc atgcgcaatc agctgcaact tttcggcagc
gcgacaacaa 7200ttatgcgttg cgtaaaagtg gcagtcaatt acagattttc
tttaacctac gcaatgagct 7260attgcggggg gtgccgcaat gagctgttgc
gtacccccct tttttaagtt gttgattttt 7320aagtctttcg catttcgccc
tatatctagt tctttggtgc ccaaagaagg gcacccctgc 7380ggggttcccc
cacgccttcg gcgcggctcc ccctccggca aaaagtggcc cctccggggc
7440ttgttgatcg actgcgcggc cttcggcctt gcccaaggtg gcgctgcccc
cttggaaccc 7500ccgcactcgc cgccgtgagg ctcggggggc aggcgggcgg
gcttcgcctt cgactgcccc 7560cactcgcata ggcttgggtc gttccaggcg
cgtcaaggcc aagccgctgc gcggtcgctg 7620cgcgagcctt gacccgcctt
ccacttggtg tccaaccggc aagcgaagcg cgcaggccgc 7680aggccggagg
cttttcccca gagaaaatta aaaaaattga tggggcaagg ccgcaggccg
7740cgcagttgga gccggtgggt atgtggtcga aggctgggta gccggtgggc
aatccctgtg 7800gtcaagctcg tgggcaggcg cagcctgtcc atcagcttgt
ccagcagggt tgtccacggg 7860ccgagcgaag cgagccagcc ggtggccgct
cgcggccatc gtccacatat ccacgggctg 7920gcaagggagc gcagcgaccg
cgcagggcga agcccggaga gcaagcccgt agggcgccgc 7980agccgccgta
ggcggtcacg actttgcgaa gcaaagtcta gtgagtatac tcaagcattg
8040agtggcccgc cggaggcacc gccttgcgct gcccccgtcg agccggttgg
acaccaaaag 8100ggaggggcag gcatggcggc atacgcgatc atgcgatgca
agaagctggc gaaaatgggc 8160aacgtggcgg ccagtctcaa gcacgcctac
cgcgagcgcg agacgcccaa cgctgacgcc 8220agcaggacgc cagagaacga
gcactgggcg gccagcagca ccgatgaagc gatgggccga 8280ctgcgcgagt
tgctgccaga gaagcggcgc aaggacgctg tgttggcggt cgagtacgtc
8340atgacggcca gcccggaatg gtggaagtcg gccagccaag aacagcaggc
ggcgttcttc 8400gagaaggcgc acaagtggct ggcggacaag tacggggcgg
atcgcatcgt gacggccagc 8460atccaccgtg acgaaaccag cccgcacatg
accgcgttcg tggtgccgct gacgcaggac 8520ggcaggctgt cggccaagga
gttcatcggc aacaaagcgc agatgacccg cgaccagacc 8580acgtttgcgg
ccgctgtggc cgatctaggg ctgcaacggg gcatcgaggg cagcaaggca
8640cgtcacacgc gcattcaggc gttctacgag gccctggagc ggccaccagt
gggccacgtc 8700accatcagcc cgcaagcggt cgagccacgc gcctatgcac
cgcagggatt ggccgaaaag 8760ctgggaatct caaagcgcgt tgagacgccg
gaagccgtgg ccgaccggct gacaaaagcg 8820gttcggcagg ggtatgagcc
tgccctacag gccgccgcag gagcgcgtga gatgcgcaag 8880aaggccgatc
aagcccaaga gacggcccga g 8911177113DNAArtificial SequenceSynthetic
Construct 177tggcaaatat tctgaaatga gctgttgaca attaatcatc gaactagtta
actagtacgc 60aagttcacgt aaaaagggta tcgacatggt atcctgttct gcgccgggta
aga 11317873DNAArtificial SequenceSynthetic Construct 178caagaaaaaa
ggcacgtcat ctgacgtgcc ttttttattt gtattaatct actttcagac 60cttgctcggt
cgg 7317921DNAArtificial SequenceSynthetic Construct 179gatacgtatg
tttctacctt c 2118021DNAArtificial SequenceSynthetic Construct
180gaaggtagaa acatacgtat c 2118124DNAArtificial SequenceSynthetic
Construct 181gatagtaacg gctgcgctgc tacc 2418248DNAArtificial
SequenceSynthetic Construct 182gggcccgttt aaactttaac tagactttaa
tctactttca gaccttgc 481839945DNAArtificial SequenceSynthetic
Construct 183accttcggga gcgcctgaag cccgttctgg acgccctggg gccgttgaat
cgggatatgc 60aggccaaggc cgccgcgatc atcaaggccg tgggcgaaaa gctgctgacg
gaacagcggg 120aagtccagcg ccagaaacag gcccagcgcc agcaggaacg
cgggcgcgca catttccccg 180aaaagtgcca cctggcggcg ttgtgacaat
ttaccgaaca actccgcggc cgggaagccg 240atctcggctt gaacgaattg
ttaggtggcg gtacttgggt cgatatcaaa gtgcatcact 300tcttcccgta
tgcccaactt tgtatagaga gccactgcgg gatcgtcacc gtaatctgct
360tgcacgtaga tcacataagc accaagcgcg ttggcctcat gcttgaggag
attgatgagc 420gcggtggcaa tgccctgcct ccggtgctcg ccggagactg
cgagatcata gatatagatc 480tcactacgcg gctgctcaaa cctgggcaga
acgtaagccg cgagagcgcc aacaaccgct 540tcttggtcga aggcagcaag
cgcgatgaat gtcttactac ggagcaagtt cccgaggtaa 600tcggagtccg
gctgatgttg ggagtaggtg gctacgtctc cgaactcacg accgaaaaga
660tcaagagcag cccgcatgga tttgacttgg tcagggccga gcctacatgt
gcgaatgatg 720cccatacttg agccacctaa ctttgtttta gggcgactgc
cctgctgcgt aacatcgttg 780ctgctgcgta acatcgttgc tgctccataa
catcaaacat cgacccacgg cgtaacgcgc 840ttgctgcttg gatgcccgag
gcatagactg tacaaaaaaa cagtcataac aagccatgaa 900aaccgccact
gcgccgttac caccgctgcg ttcggtcaag gttctggacc agttgcgtga
960gcgcatacgc tacttgcatt acagtttacg aaccgaacag gcttatgtca
actgggttcg 1020tgccttcatc cgtttccacg gtgtgcgtcc atgggcaaat
attatacgca aggcgacaag 1080gtgctgatgc cgctggcgat tcaggttcat
catgccgttt gtgatggctt ccatgtcggc 1140agaatgctta atgaattaca
acagttttta tgcatgcgcc caatacgcaa accgcctctc 1200cccgcgcgtt
ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg
1260ggcagtgagc gcaacgcaat taatgtgagt tagctcactc attaggcacc
ccaggcttta 1320cactttatgc ttccggctcg tatgttgtgt ggaattgtga
gcggataaca atttcacaca 1380ggaaacagct atgaccatga ttacgccaag
cgcgcaatta accctcacta aagggaacaa 1440aagctgggta ccgggccccc
cctcgagctg ttgacaatta atcatccggc tcgtataatg 1500tgtggaattg
tgagcggata acaatttcac acaggaaaca gcgccgctga gaaaaagcga
1560agcggcactg ctctttaaca atttatcaga caatctgtgt gggcactcga
ccggaattat 1620cgattaactt tattattaaa aattaaagag gtatatatta
atgtatcgat taaataagga 1680ggaataaacc atggatccga gctcaggagg
taaaaaaaca tgaaaacagt agttattatt 1740gatgcattac gaacaccaat
tggaaaatat aaaggcagct taagtcaagt aagtgccgta 1800gacttaggaa
cacatgttac aacacaactt ttaaaaagac attccactat ttctgaagaa
1860attgatcaag taatctttgg aaatgtttta caagctggaa atggccaaaa
tcccgcacga 1920caaatagcaa taaacagcgg tttgtctcat gaaattcccg
caatgacggt taatgaggtc 1980tgcggatcag gaatgaaggc cgttattttg
gcgaaacaat tgattcaatt aggagaagcg 2040gaagttttaa ttgctggcgg
gattgagaat atgtcccaag cacctaaatt acaacgtttt 2100aattacgaaa
cagaaagcta cgatgcgcct ttttctagta tgatgtatga tggattaacg
2160gatgccttta gtggtcaggc aatgggctta actgctgaaa atgtggccga
aaagtatcat 2220gtaactagag aagagcaaga tcaattttct gtacattcac
aattaaaagc agctcaagca 2280caagcagaag ggatattcgc tgacgaaata
gccccattag aagtatcagg aacgcttgtg 2340gagaaagatg aagggattcg
ccctaattcg agcgttgaga agctaggaac gcttaaaaca 2400gtttttaaag
aagacggtac tgtaacagca gggaatgcat caaccattaa tgatggggct
2460tctgctttga ttattgcttc acaagaatat gccgaagcac acggtcttcc
ttatttagct 2520attattcgag acagtgtgga agtcggtatt gatccagcct
atatgggaat ttcgccgatt 2580aaagccattc aaaaactgtt agcgcgcaat
caacttacta cggaagaaat tgatctgtat 2640gaaatcaacg aagcatttgc
agcaacttca atcgtggtcc aaagagaact ggctttacca 2700gaggaaaagg
tcaacattta tggtggcggt atttcattag gtcatgcgat tggtgccaca
2760ggtgctcgtt tattaacgag tttaagttat caattaaatc aaaaagaaaa
gaaatatgga 2820gtggcttctt tatgtatcgg cggtggctta ggactcgcta
tgctactaga gagacctcag 2880caaaaaaaaa acagccgatt ttatcaaatg
agtcctgagg aacgcctggc ttctcttctt 2940aatgaaggcc agatttctgc
tgatacaaaa aaagaatttg aaaatacggc tttatcttcg 3000cagattgcca
atcatatgat tgaaaatcaa atcagtgaaa cagaagtgcc gatgggcgtt
3060ggcttacatt taacagtgga cgaaactgat tatttggtac caatggcgac
agaagagccc 3120tcagttattg cggctttgag taatggtgca aaaatagcac
aaggatttaa aacagtgaat 3180caacaacgct taatgcgtgg acaaatcgtt
ttttacgatg ttgcagatcc cgagtcattg 3240attgataaac tacaagtaag
agaagcggaa gtttttcaac aagcagagtt aagttatcca 3300tctatcgtta
aacggggcgg cggcttaaga gatttgcaat atcgtacttt tgatgaatca
3360tttgtatctg tcgacttttt agtagatgtt aaggatgcaa tgggggcaaa
tatcgttaac 3420gctatgttgg aaggtgtggc cgagttgttc cgtgaatggt
ttgcggagca aaagatttta 3480ttcagtattt taagtaatta tgccacggag
tcggttgtta cgatgaaaac ggctattcca 3540gtttcacgtt taagtaaggg
gagcaatggc cgggaaattg ctgaaaaaat tgttttagct 3600tcacgctatg
cttcattaga tccttatcgg gcagtcacgc ataacaaagg aatcatgaat
3660ggcattgaag ctgtagtttt agctacagga aatgatacac gcgctgttag
cgcttcttgt 3720catgcttttg cggtgaagga aggtcgctac caaggcttga
ctagttggac gctggatggc 3780gaacaactaa ttggtgaaat ttcagttccg
cttgctttag ccacggttgg cggtgccaca 3840aaagtcttac ctaaatctca
agcagctgct gatttgttag cagtgacgga tgcaaaagaa 3900ctaagtcgag
tagtagcggc tgttggtttg gcacaaaatt tagcggcgtt acgggcctta
3960gtctctgaag gaattcaaaa aggacacatg gctctacaag cacgttcttt
agcgatgacg 4020gtcggagcta ctggtaaaga agttgaggca gtcgctcaac
aattaaaacg tcaaaaaacg 4080atgaaccaag accgagccat ggctatttta
aatgatttaa gaaaacaata aaggaggtaa 4140aaaaacatga caattgggat
tgataaaatt agtttttttg tgccccctta ttatattgat 4200atgacggcac
tggctgaagc cagaaatgta gaccctggaa aatttcatat tggtattggg
4260caagaccaaa tggcggtgaa cccaatcagc caagatattg tgacatttgc
agccaatgcc 4320gcagaagcga tcttgaccaa agaagataaa gaggccattg
atatggtgat tgtcgggact 4380gagtccagta tcgatgagtc aaaagcggcc
gcagttgtct tacatcgttt aatggggatt 4440caacctttcg ctcgctcttt
cgaaatcaag gaagcttgtt acggagcaac agcaggctta 4500cagttagcta
agaatcacgt agccttacat ccagataaaa aagtcttggt cgtagcggca
4560gatattgcaa aatatggctt aaattctggc ggtgagccta cacaaggagc
tggggcggtt 4620gcaatgttag ttgctagtga accgcgcatt ttggctttaa
aagaggataa tgtgatgctg 4680acgcaagata tctatgactt ttggcgtcca
acaggccacc cgtatcctat ggtcgatggt 4740cctttgtcaa acgaaaccta
catccaatct tttgcccaag tctgggatga acataaaaaa 4800cgaaccggtc
ttgattttgc agattatgat gctttagcgt tccatattcc ttacacaaaa
4860atgggcaaaa aagccttatt agcaaaaatc tccgaccaaa ctgaagcaga
acaggaacga 4920attttagccc gttatgaaga aagtatcgtc tatagtcgtc
gcgtaggaaa cttgtatacg 4980ggttcacttt atctgggact catttccctt
ttagaaaatg caacgacttt aaccgcaggc 5040aatcaaattg gtttattcag
ttatggttct ggtgctgtcg ctgaattttt cactggtgaa 5100ttagtagctg
gttatcaaaa tcatttacaa aaagaaactc atttagcact gctggataat
5160cggacagaac tttctatcgc tgaatatgaa gccatgtttg cagaaacttt
agacacagac 5220attgatcaaa cgttagaaga tgaattaaaa tatagtattt
ctgctattaa taataccgtt 5280cgttcttatc gaaactaaag atctgcagct
ggtaccatat gggaattcga agcttgggcc 5340cgaacaaaaa ctcatctcag
aagaggatct gaatagcgcc gtcgaccatc atcatcatca 5400tcattgagtt
taaacggtct ccagcttggc tgttttggcg gatgagagaa gattttcagc
5460ctgatacaga ttaaatcaga acgcagaagc ggtctgataa aacagaattt
gcctggcggc 5520agtagcgcgg tggtcccacc tgaccccatg ccgaactcag
aagtgaaacg ccgtagcgcc 5580gatggtagtg tggggtctcc ccatgcgaga
gtagggaact gccaggcatc aaataaaacg 5640aaaggctcag tcgaaagact
gggcctttct agagcggccg ccaccgcggt ggagctccaa 5700ttcgccctat
agtgagtcgt attacgcgcg ctcactggcc gtcgttttac aacgtcgtga
5760ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc
ctttcgccag 5820ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc
caacagttgc gcagcctgaa 5880tggcgaatgg aaattgtaag cgttaatatt
ttgttaaaat tcgcgttaaa tttttgttaa 5940atcagctcat tttttaacca
ataggccgac tgcgatgagt ggcagggcgg ggcgtaattt 6000ttttaaggca
gttattggtg cccttaaacg cctggtgcta cgcctgaata agtgataata
6060agcggatgaa tggcagaaat tcgaaagcaa attcgacccg gtcgtcggtt
cagggcaggg 6120tcgttaaata gccgcttatg tctattgctg gtttaccggt
ttattgacta ccggaagcag 6180tgtgaccgtg tgcttctcaa atgcctgagg
ccagtttgct caggctctcc ccgtggaggt 6240aataattgac gatatgatca
tttattctgc ctcccagagc ctgataaaaa cggtgaatcc 6300gttagcgagg
tgccgccggc ttccattcag gtcgaggtgg cccggctcca tgcaccgcga
6360cgcaacgcgg ggaggcagac aaggtatagg gcggcgaggc ggctacagcc
gatagtctgg 6420aacagcgcac ttacgggttg ctgcgcaacc caagtgctac
cggcgcggca gcgtgacccg 6480tgtcggcggc tccaacggct cgccatcgtc
cagaaaacac ggctcatcgg gcatcggcag 6540gcgctgctgc ccgcgccgtt
cccattcctc cgtttcggtc aaggctggca ggtctggttc 6600catgcccgga
atgccgggct ggctgggcgg ctcctcgccg gggccggtcg gtagttgctg
6660ctcgcccgga tacagggtcg ggatgcggcg caggtcgcca tgccccaaca
gcgattcgtc 6720ctggtcgtcg tgatcaacca ccacggcggc actgaacacc
gacaggcgca actggtcgcg 6780gggctggccc cacgccacgc ggtcattgac
cacgtaggcc gacacggtgc cggggccgtt 6840gagcttcacg acggagatcc
agcgctcggc caccaagtcc ttgactgcgt attggaccgt 6900ccgcaaagaa
cgtccgatga gcttggaaag tgtcttctgg ctgaccacca cggcgttctg
6960gtggcccatc tgcgccacga ggtgatgcag cagcattgcc gccgtgggtt
tcctcgcaat 7020aagcccggcc cacgcctcat gcgctttgcg ttccgtttgc
acccagtgac cgggcttgtt 7080cttggcttga atgccgattt ctctggactg
cgtggccatg cttatctcca tgcggtaggg 7140tgccgcacgg ttgcggcacc
atgcgcaatc agctgcaact tttcggcagc gcgacaacaa 7200ttatgcgttg
cgtaaaagtg gcagtcaatt acagattttc tttaacctac gcaatgagct
7260attgcggggg gtgccgcaat gagctgttgc gtacccccct tttttaagtt
gttgattttt 7320aagtctttcg catttcgccc tatatctagt tctttggtgc
ccaaagaagg gcacccctgc 7380ggggttcccc cacgccttcg gcgcggctcc
ccctccggca aaaagtggcc cctccggggc 7440ttgttgatcg actgcgcggc
cttcggcctt gcccaaggtg gcgctgcccc cttggaaccc 7500ccgcactcgc
cgccgtgagg ctcggggggc aggcgggcgg gcttcgcctt cgactgcccc
7560cactcgcata ggcttgggtc gttccaggcg cgtcaaggcc aagccgctgc
gcggtcgctg 7620cgcgagcctt gacccgcctt ccacttggtg tccaaccggc
aagcgaagcg cgcaggccgc 7680aggccggagg cttttcccca gagaaaatta
aaaaaattga tggggcaagg ccgcaggccg 7740cgcagttgga gccggtgggt
atgtggtcga aggctgggta gccggtgggc aatccctgtg 7800gtcaagctcg
tgggcaggcg cagcctgtcc atcagcttgt ccagcagggt tgtccacggg
7860ccgagcgaag cgagccagcc ggtggccgct cgcggccatc gtccacatat
ccacgggctg 7920gcaagggagc gcagcgaccg cgcagggcga agcccggaga
gcaagcccgt agggctggca 7980aatattctga aatgagctgt tgacaattaa
tcatcgaact agttaactag tacgcaagtt 8040cacgtaaaaa gggtatcgac
atggtatcct gttctgcgcc gggtaagatt tacctgttcg 8100gtgaacacgc
cgtagtttat ggcgaaactg caattgcgtg tgcggtggaa ctgcgtaccc
8160gtgttcgcgc ggaactcaat gactctatca ctattcagag ccagatcggc
cgcaccggtc 8220tggatttcga aaagcaccct tatgtgtctg cggtaattga
gaaaatgcgc aaatctattc 8280ctattaacgg tgttttcttg accgtcgatt
ccgacatccc ggtgggctcc ggtctgggta 8340gcagcgcagc cgttactatc
gcgtctattg gtgcgctgaa cgagctgttc ggctttggcc 8400tcagcctgca
agaaatcgct aaactgggcc acgaaatcga aattaaagta cagggtgccg
8460cgtccccaac cgatacgtat gtttctacct tcggcggcgt ggttaccatc
ccggaacgtc 8520gcaaactgaa aactccggac tgcggcattg tgattggcga
taccggcgtt ttctcctcca 8580ccaaagagtt agtagctaac gtacgtcagc
tgcgcgaaag ctacccggat ttgatcgaac 8640cgctgatgac ctctattggc
aaaatctctc gtatcggcga acaactggtt ctgtctggcg 8700actacgcatc
catcggccgc ctgatgaacg tcaaccaggg tctcctggac gccctgggcg
8760ttaacatctt agaactgagc cagctgatct attccgctcg tgcggcaggt
gcgtttggcg 8820ctaaaatcac gggcgctggc ggcggtggct gtatggttgc
gctgaccgct ccggaaaaat 8880gcaaccaagt ggcagaagcg gtagcaggcg
ctggcggtaa agtgactatc actaaaccga 8940ccgagcaagg tctgaaagta
gattaataca aataaaaaag gcacgtcaga tgacgtgcct 9000tttttcttgg
ccgcagccgc cgtaggcggt cacgactttg cgaagcaaag tctagtgagt
9060atactcaagc attgagtggc ccgccggagg caccgccttg cgctgccccc
gtcgagccgg 9120ttggacacca aaagggaggg gcaggcatgg cggcatacgc
gatcatgcga tgcaagaagc 9180tggcgaaaat gggcaacgtg gcggccagtc
tcaagcacgc ctaccgcgag cgcgagacgc 9240ccaacgctga cgccagcagg
acgccagaga acgagcactg ggcggccagc agcaccgatg 9300aagcgatggg
ccgactgcgc gagttgctgc cagagaagcg gcgcaaggac gctgtgttgg
9360cggtcgagta cgtcatgacg gccagcccgg aatggtggaa gtcggccagc
caagaacagc 9420aggcggcgtt cttcgagaag gcgcacaagt ggctggcgga
caagtacggg gcggatcgca 9480tcgtgacggc cagcatccac cgtgacgaaa
ccagcccgca catgaccgcg ttcgtggtgc 9540cgctgacgca ggacggcagg
ctgtcggcca aggagttcat cggcaacaaa gcgcagatga 9600cccgcgacca
gaccacgttt gcggccgctg tggccgatct agggctgcaa cggggcatcg
9660agggcagcaa ggcacgtcac acgcgcattc aggcgttcta cgaggccctg
gagcggccac 9720cagtgggcca cgtcaccatc agcccgcaag cggtcgagcc
acgcgcctat gcaccgcagg 9780gattggccga aaagctggga atctcaaagc
gcgttgagac gccggaagcc gtggccgacc 9840ggctgacaaa agcggttcgg
caggggtatg agcctgccct acaggccgcc gcaggagcgc 9900gtgagatgcg
caagaaggcc gatcaagccc aagagacggc ccgag 994518435DNAArtificial
SequenceSynthetic Construct 184ttttgcggcc gcaattaacc ctcactaaag
ggcgg 35185103DNAArtificial SequenceSynthetic Construct
185gatcgatatc cctgcaggaa attgttatcc gctcacaatt ccacacatta
tacgagccgg 60atgattaatt gtcaacagct aatacgactc actatagggc tcg
1031868983DNAArtificial SequenceSynthetic Construct 186caagaaaaat
gccccgctta cgcagggcat ccatttatta ctcaaccgta accgattttg 60ccaggttacg
cggctggtca acgtcggtgc ctttgatcag cgcgacatgg taagccagca
120gctgcagcgg aacggtgtag aagatcggtg caatcacctc ttccacatgc
ggcatctcga 180tgatgtgcat gttatcgcta cttacaaaac ccgcatcctg
atcggcgaag acatacaact 240gaccgccacg cgcgcgaact tcttcaatgt
tggatttcag tttttccagc aattcgttgt 300tcggtgcaac aacaataacc
ggcatatcgg catcaattag cgccagcgga ccgtgtttca 360gttcgccagc
agcgtaggct tcagcgtgaa tgtaagagat ctctttcaac ttcaatgcgc
420cttccagcgc gattgggtac tgatcgccac ggcccaggaa cagcgcgtga
tgtttgtcag 480agaaatcttc tgccagcgct tcaatgcgtt tgtcctgaga
cagcatctgc tcaatacggc 540tcggcagcgc ctgcagacca tgcacgatgt
catgttcaat ggaggcatcc agacctttca 600ggcgagacag cttcgccacc
agcatcaaca gcacagttaa ctgagtggtg aatgctttag 660tggatgccac
gccgatttct gtacccgcgt tggtcattag cgccagatcg gattcgcgca
720ccagagaaga acccggaacg ttacagattg ccagtgaacc aaggtaaccc
agctctttcg 780acagacgcag gccagccagg gtatccgcgg tttcgccaga
ctgtgacacg atcgcccttc 840ccaacagttg cgcagcctat acgtacggca
gtttaaggtt tacacctata aaagagagag 900ccgttatcgt ctgtttgtgg
atgtacagag tgatattatt gacacgccgg ggcgacggat 960ggtgatcccc
ctggccagtg cacgtctgct gtcagataaa gtctcccgtg aactttaccc
1020ggtggtgcat atcggggatg aaagctggcg catgatgacc accgatatgg
ccagtgtgcc 1080ggtctccgtt atcggggaag aagtggctga tctcagccac
cgcgaaaatg acatcaaaaa 1140cgccattaac ctgatgttct ggggaatata
aatgtcaggc atgagattat caaaaaggat 1200cttcacctag atccttttca
cgtagaaagc cagtccgcag aaacggtgct gaccccggat 1260gaatgtcagc
tactgggcta tctggacaag ggaaaacgca agcgcaaaga gaaagcaggt
1320agcttgcagt gggcttacat ggcgatagct agactgggcg gttttatgga
cagcaagcga 1380accggaattg ccagctgggg cgccctctgg taaggttggg
aagccctgca aagtaaactg 1440gatggctttc tcgccgccaa ggatctgatg
gcgcagggga tcaagctctg atcaagagac 1500aggatgagga tcgtttcgca
tgattgaaca agatggattg cacgcaggtt ctccggccgc 1560ttgggtggag
aggctattcg gctatgactg ggcacaacag acaatcggct gctctgatgc
1620cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga
ccgacctgtc 1680cggtgccctg aatgaactgc aagacgaggc agcgcggcta
tcgtggctgg ccacgacggg 1740cgttccttgc gcagctgtgc tcgacgttgt
cactgaagcg ggaagggact ggctgctatt 1800gggcgaagtg ccggggcagg
atctcctgtc atctcacctt gctcctgccg agaaagtatc 1860catcatggct
gatgcaatgc ggcggctgca tacgcttgat ccggctacct gcccattcga
1920ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg
gtcttgtcga 1980tcaggatgat ctggacgaag agcatcaggg gctcgcgcca
gccgaactgt tcgccaggct 2040caaggcgagc atgcccgacg gcgaggatct
cgtcgtgacc catggcgatg cctgcttgcc 2100gaatatcatg gtggaaaatg
gccgcttttc tggattcatc gactgtggcc ggctgggtgt 2160ggcggaccgc
tatcaggaca tagcgttggc tacccgtgat attgctgaag agcttggcgg
2220cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt
cgcagcgcat 2280cgccttctat cgccttcttg acgagttctt ctgaattatt
aacgcttaca atttcctgat 2340gcggtatttt ctccttacgc atctgtgcgg
tatttcacac cgcatacagg tggcactttt 2400cggggaaatg tgcgcggaac
ccctatttgt ttatttttct aaatacattc aaatatgtat 2460ccgctcatga
gacaataacc ctgataaatg cttcaataat agcacgtgag gagggccacc
2520atggccaagt tgaccagtgc cgttccggtg ctcaccgcgc gcgacgtcgc
cggagcggtc 2580gagttctgga ccgaccggct cgggttctcc cctagtaacg
gccgccagtg tgctggaatt 2640caggcagttc aacctgttga tagtacgtac
taagctctca tgtttcacgt actaagctct 2700catgtttaac gtactaagct
ctcatgttta acgaactaaa ccctcatggc taacgtacta 2760agctctcatg
gctaacgtac taagctctca tgtttcacgt actaagctct catgtttgaa
2820caataaaatt aatataaatc agcaacttaa atagcctcta aggttttaag
ttttataaga 2880aaaaaaagaa tatataaggc ttttaaagct tttaaggttt
aacggttgtg gacaacaagc 2940cagggatgta acgcactgag aagcccttag
agcctctcaa agcaattttc agtgacacag 3000gaacacttaa cggctgacag
cctgaattct gcagatatct gtttttccac tcttcgttca 3060ctttcgccag
gtagctggtg aagacgaagg aagtcccgga gccatctgcg cggcgtacta
3120cagcaatgtt ttgtgaaggc agtttcagac ccggattcag tttggcgatg
gcttcatcat 3180cccacttctt gattttgccc aggtagatgt cgccgagggt
tttaccatcc agcaccagtt 3240cgccagactt cagccctgga atgttaaccg
ccagcaccac gccgccaatc acggtcggga 3300actggaacag accttcctga
gccagttttt cgtcagacag cggcgcgtca gaggcaccaa 3360aatcaacggt
attagcgata atctgtttta cgccaccgga agaaccgata ccctggtagt
3420taactttatt accggtttct ttctggtaag tgtcagccca tttggcatac
accggcgcag 3480ggaaggttgc acctgcacct gtcaggcttg cttctgcaaa
cacagagaaa gcactcatcg 3540ataaggtcgc ggcgacaaca gttgcgacgg
tggtacgcat aactttcata atgtctcctg 3600ggaggattca taaagcattg
tttgttggct acgagaagca aaataggaca aacaggtgac 3660agttatatgt
aaggaatatg acagttttat gacagagaga taaagtcttc agtctgattt
3720aaataagcgt tgatattcag tcaattacaa acattaataa cgaagagatg
acagaaaaat 3780tttcattctg tgacagagaa aaagtagccg aagatgacgg
tttgtcacat ggagttggca 3840ggatgtttga ttaaaagcgg ccgcgaagtt
cctattctct agaaagtata ggaacttcat 3900tctaccgggt aggggaggcg
cttttcccaa ggcagtctgg agcatgcgct ttagcagccc 3960cgctgggcac
ttggcgctac acaagtggcc tctggcctcg cacacattcc acatccaccg
4020gtaggcgcca accggctccg ttctttggtg gccccttcgc gccaccttcc
actcctcccc 4080tagtcaggaa gttccccccc gccccgcagc tcgcgtcgtg
caggacgtga caaatggaag 4140tagcacgtct cactagtctc gtgcagatgg
acagcaccgc tgagcaatgg aagcgggtag 4200gcctttgggg cagcggccaa
tagcagcttt gctccttcgc tttctgggct cagaggctgg 4260gaaggggtgg
gtccgggggc gggctcaggg gcgggctcag gggcggggcg ggcgcccgaa
4320ggtcctccgg aggcccggca ttctgcacgc ttcaaaagcg cacgtctgcc
gcgctgttct 4380cctcttcctc atctccgggc ctttcgacct gcagcagcac
gtgttgacaa ttaatcatcg 4440gcatagtata tcggcatagt ataatacgac
aaggtgagga actaaaccat ggagaaaaaa 4500atcactggat ataccaccgt
tgatatatcc caatggcatc gtaaagaaca ttttgaggca 4560tttcagtcag
ttgctcaatg tacctataac cagaccgttc agctggatat tacggccttt
4620ttaaagaccg taaagaaaaa taagcacaag ttttatccgg cctttattca
cattcttgcc 4680cgcctgatga atgctcatcc ggaattccgt atggcaatga
aagacggtga gctggtgata 4740tgggatagtg ttcacccttg ttacaccgtt
ttccatgagc aaactgaaac gttttcatcg 4800ctctggagtg aataccacga
cgatttccgg cagtttctac acatatattc gcaagatgtg 4860gcgtgttacg
gtgaaaacct ggcctatttc cctaaagggt ttattgagaa tatgtttttc
4920gtctcagcca atccctgggt gagtttcacc agttttgatt taaacgtggc
caatatggac 4980aacttcttcg cccccgtttt caccatgggc aaatattata
cgcaaggcga caaggtgctg 5040atgccgctgg cgattcaggt tcatcatgcc
gtttgtgatg gcttccatgt cggcagaatg 5100cttaatgaat tacaacagta
ctgcgatgag tggcagggcg gggcgtaagc gggactctgg 5160ggttcgaata
aagaccgacc aagcgacgtc tgagagctcc ctggcgaatt cggtaccaat
5220aaaagagctt tattttcatg atctgtgtgt tggtttttgt gtgcggcgcg
gaagttccta 5280ttctctagaa agtataggaa cttcctcgag ccctatagtg
agtcgtatta gctgttgaca 5340attaatcatc cggctcgtat aatgtgtgga
attgtgagcg gataacaatt tcctgcaggg 5400atcctgcacc cttaaggagg
aaaaaaacat gtcagagttg agagccttca gtgccccagg 5460gaaagcgtta
ctagctggtg gatatttagt tttagataca aaatatgaag catttgtagt
5520cggattatcg gcaagaatgc atgctgtagc ccatccttac ggttcattgc
aagggtctga 5580taagtttgaa gtgcgtgtga aaagtaaaca atttaaagat
ggggagtggc tgtaccatat 5640aagtcctaaa agtggcttca ttcctgtttc
gataggcgga tctaagaacc ctttcattga 5700aaaagttatc gctaacgtat
ttagctactt taaacctaac atggacgact actgcaatag 5760aaacttgttc
gttattgata ttttctctga tgatgcctac cattctcagg aggatagcgt
5820taccgaacat cgtggcaaca gaagattgag ttttcattcg cacagaattg
aagaagttcc 5880caaaacaggg ctgggctcct cggcaggttt agtcacagtt
ttaactacag ctttggcctc 5940cttttttgta tcggacctgg aaaataatgt
agacaaatat agagaagtta ttcataattt 6000agcacaagtt gctcattgtc
aagctcaggg taaaattgga agcgggtttg atgtagcggc 6060ggcagcatat
ggatctatca gatatagaag attcccaccc gcattaatct ctaatttgcc
6120agatattgga agtgctactt acggcagtaa actggcgcat ttggttgatg
aagaagactg 6180gaatattacg attaaaagta accatttacc ttcgggatta
actttatgga tgggcgatat 6240taagaatggt tcagaaacag taaaactggt
ccagaaggta aaaaattggt atgattcgca 6300tatgccagaa agcttgaaaa
tatatacaga actcgatcat gcaaattcta gatttatgga 6360tggactatct
aaactagatc gcttacacga gactcatgac gattacagcg atcagatatt
6420tgagtctctt gagaggaatg actgtacctg tcaaaagtat cctgaaatca
cagaagttag 6480agatgcagtt gccacaatta gacgttcctt tagaaaaata
actaaagaat ctggtgccga 6540tatcgaacct cccgtacaaa ctagcttatt
ggatgattgc cagaccttaa aaggagttct 6600tacttgctta atacctggtg
ctggtggtta tgacgccatt gcagtgatta ctaagcaaga 6660tgttgatctt
agggctcaaa ccgctaatga caaaagattt tctaaggttc aatggctgga
6720tgtaactcag gctgactggg gtgttaggaa agaaaaagat ccggaaactt
atcttgataa 6780ataacttaag gtagctgcat gcagaattcg cccttaagga
ggaaaaaaaa atgaccgttt 6840acacagcatc cgttaccgca cccgtcaaca
tcgcaaccct taagtattgg gggaaaaggg 6900acacgaagtt gaatctgccc
accaattcgt ccatatcagt gactttatcg caagatgacc 6960tcagaacgtt
gacctctgcg gctactgcac ctgagtttga acgcgacact ttgtggttaa
7020atggagaacc acacagcatc gacaatgaaa gaactcaaaa ttgtctgcgc
gacctacgcc 7080aattaagaaa ggaaatggaa tcgaaggacg cctcattgcc
cacattatct caatggaaac 7140tccacattgt ctccgaaaat aactttccta
cagcagctgg tttagcttcc tccgctgctg 7200gctttgctgc attggtctct
gcaattgcta agttatacca attaccacag tcaacttcag 7260aaatatctag
aatagcaaga aaggggtctg gttcagcttg tagatcgttg tttggcggat
7320acgtggcctg ggaaatggga aaagctgaag atggtcatga ttccatggca
gtacaaatcg 7380cagacagctc tgactggcct cagatgaaag cttgtgtcct
agttgtcagc gatattaaaa 7440aggatgtgag ttccactcag ggtatgcaat
tgaccgtggc aacctccgaa ctatttaaag 7500aaagaattga acatgtcgta
ccaaagagat ttgaagtcat gcgtaaagcc attgttgaaa 7560aagatttcgc
cacctttgca aaggaaacaa tgatggattc caactctttc catgccacat
7620gtttggactc tttccctcca atattctaca tgaatgacac ttccaagcgt
atcatcagtt 7680ggtgccacac cattaatcag ttttacggag aaacaatcgt
tgcatacacg tttgatgcag 7740gtccaaatgc tgtgttgtac tacttagctg
aaaatgagtc gaaactcttt gcatttatct 7800ataaattgtt tggctctgtt
cctggatggg acaagaaatt tactactgag cagcttgagg 7860ctttcaacca
tcaatttgaa tcatctaact ttactgcacg tgaattggat cttgagttgc
7920aaaaggatgt tgccagagtg attttaactc aagtcggttc aggcccacaa
gaaacaaacg 7980aatctttgat tgacgcaaag actggtctac caaaggaata
agatcaattc gctgcatcgc 8040ccttaggagg taaaaaaaaa tgactgccga
caacaatagt atgccccatg gtgcagtatc 8100tagttacgcc aaattagtgc
aaaaccaaac acctgaagac attttggaag agtttcctga 8160aattattcca
ttacaacaaa gacctaatac ccgatctagt gagacgtcaa atgacgaaag
8220cggagaaaca tgtttttctg gtcatgatga ggagcaaatt aagttaatga
atgaaaattg 8280tattgttttg gattgggacg ataatgctat tggtgccggt
accaagaaag tttgtcattt 8340aatggaaaat
attgaaaagg gtttactaca tcgtgcattc tccgtcttta ttttcaatga
8400acaaggtgaa ttacttttac aacaaagagc cactgaaaaa ataactttcc
ctgatctttg 8460gactaacaca tgctgctctc atccactatg tattgatgac
gaattaggtt tgaagggtaa 8520gctagacgat aagattaagg gcgctattac
tgcggcggtg agaaaactag atcatgaatt 8580aggtattcca gaagatgaaa
ctaagacaag gggtaagttt cactttttaa acagaatcca 8640ttacatggca
ccaagcaatg aaccatgggg tgaacatgaa attgattaca tcctatttta
8700taagatcaac gctaaagaaa acttgactgt caacccaaac gtcaatgaag
ttagagactt 8760caaatgggtt tcaccaaatg atttgaaaac tatgtttgct
gacccaagtt acaagtttac 8820gccttggttt aagattattt gcgagaatta
cttattcaac tggtgggagc aattagatga 8880cctttctgaa gtggaaaatg
acaggcaaat tcatagaatg ctataacaac gcgtctacaa 8940ataaaaaagg
cacgtcagat gacgtgcctt ttttcttggg gcc 898318740DNAArtificial
SequenceSynthetic Construct 187aatcgtaggc cggataaggc gtttacgccg
catccggcaa 40188107DNAArtificial SequenceSynthetic Construct
188agattgctgc gaaatcgtag gccggataag gcgtttacgc cgcatccggc
aaaaatcctt 60aaatataaga gcaaacctgc aattaaccct cactaaaggg cggccgc
107189182DNAArtificial SequenceSynthetic Construct 189agcgtgaagt
gcaccaggac ggcgctattg ctctgtacag attcagacat gtttttacct 60cctttgcagt
gcgtcctgct gatgtgctca gtatcaccgc cagtggtatt tatgtcaaca
120ccgccagaga taatttatca ccgcagatgg ttatcttaat acgactcact
atagggctcg 180ag 18219021DNAArtificial SequenceSynthetic Construct
190acgcatctta tccggcctac a 2119121DNAArtificial SequenceSynthetic
Construct 191accgttgttg cgggtagact c 211926578DNAArtificial
SequenceSynthetic Construct 192ttcttgaaga cgaaagggcc tcgtgatacg
cctattttta taggttaatg tcatgataat 60aatggtttct tagacgtcag gtggcacttt
tcggggaaat gtgcgcggaa cccctatttg 120tttatttttc taaatacatt
caaatatgta tccgctcatg agacaataac cctgataaat 180gcttcaataa
tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
240tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
tggtgaaagt 300aaaagatgct gaagatcagt tgggtgcacg agtgggttac
atcgaactgg atctcaacag 360cggtaagatc cttgagagtt ttcgccccga
agaacgtttt ccaatgatga gcacttttaa 420agttctgcta tgtggcgcgg
tattatcccg tgttgacgcc gggcaagagc aactcggtcg 480ccgcatacac
tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
540tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
gtgataacac 600tgcggccaac ttacttctga caacgatcgg aggaccgaag
gagctaaccg cttttttgca 660caacatgggg gatcatgtaa ctcgccttga
tcgttgggaa ccggagctga atgaagccat 720accaaacgac gagcgtgaca
ccacgatgcc tgcagcaatg gcaacaacgt tgcgcaaact 780attaactggc
gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
840ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
ttattgctga 900taaatctgga gccggtgagc gtgggtctcg cggtatcatt
gcagcactgg ggccagatgg 960taagccctcc cgtatcgtag ttatctacac
gacggggagt caggcaacta tggatgaacg 1020aaatagacag atcgctgaga
taggtgcctc actgattaag cattggtaac tgtcagacca 1080agtttatcat
atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
1140gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
ttcgttccac 1200tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
gagatccttt ttttctgcgc 1260gtaatctgct gcttgcaaac aaaaaaacca
ccgctaccag cggtggtttg tttgccggat 1320caagagctac caactctttt
tccgaaggta actggcttca gcagagcgca gataccaaat 1380actgtccttc
tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
1440acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
taagtcgtgt 1500cttaccgggt tggactcaag acgatagtta ccggataagg
cgcagcggtc gggctgaacg 1560gggggttcgt gcacacagcc cagcttggag
cgaacgacct acaccgaact gagataccta 1620cagcgtgagc tatgagaaag
cgccacgctt cccgaaggga gaaaggcgga caggtatccg 1680gtaagcggca
gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
1740tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
tttgtgatgc 1800tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
cggccttttt acggttcctg 1860gccttttgct ggccttttgc tcacatgttc
tttcctgcgt tatcccctga ttctgtggat 1920aaccgtatta ccgcctttga
gtgagctgat accgctcgcc gcagccgaac gaccgagcgc 1980agcgagtcag
tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat
2040ctgtgcggta tttcacaccg catatatggt gcactctcag tacaatctgc
tctgatgccg 2100catagttaag ccagtataca ctccgctatc gctacgtgac
tgggtcatgg ctgcgccccg 2160acacccgcca acacccgctg acgcgccctg
acgggcttgt ctgctcccgg catccgctta 2220cagacaagct gtgaccgtct
ccgggagctg catgtgtcag aggttttcac cgtcatcacc 2280gaaacgcgcg
aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat
2340gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta
atgtctggct 2400tctgataaag cgggccatgt taagggcggt tttttcctgt
ttggtcactg atgcctccgt 2460gtaaggggga tttctgttca tgggggtaat
gataccgatg aaacgagaga ggatgctcac 2520gatacgggtt actgatgatg
aacatgcccg gttactggaa cgttgtgagg gtaaacaact 2580ggcggtatgg
atgcggcggg accagagaaa aatcactcag ggtcaatgcc agcgcttcgt
2640taatacagat gtaggtgttc cacagggtag ccagcagcat cctgcgatgc
agatccggaa 2700cataatggtg cagggcgctg acttccgcgt ttccagactt
tacgaaacac ggaaaccgaa 2760gaccattcat gttgttgctc aggtcgcaga
cgttttgcag cagcagtcgc ttcacgttcg 2820ctcgcgtatc ggtgattcat
tctgctaacc agtaaggcaa ccccgccagc ctagccgggt 2880cctcaacgac
aggagcacga tcatgcgcac ccgtggccag gacccaacgc tgcccgagat
2940gcgccgcgtg cggctgctgg agatggcgga cgcgatggat atgttctgcc
aagggttggt 3000ttgcgcattc acagttctcc gcaagaattg attggctcca
attcttggag tggtgaatcc 3060gttagcgagg tgccgccggc ttccattcag
gtcgaggtgg cccggctcca tgcaccgcga 3120cgcaacgcgg ggaggcagac
aaggtatagg gcggcgccta caatccatgc caacccgttc 3180catgtgctcg
ccgaggcggc ataaatcgcc gtgacgatca gcggtccagt gatcgaagtt
3240aggctggtaa gagccgcgag cgatccttga agctgtccct gatggtcgtc
atctacctgc 3300ctggacagca tggcctgcaa cgcgggcatc ccgatgccgc
cggaagcgag aagaatcata 3360atggggaagg ccatccagcc tcgcgtcgcg
aacgccagca agacgtagcc cagcgcgtcg 3420gccgccatgc cggcgataat
ggcctgcttc tcgccgaaac gtttggtggc gggaccagtg 3480acgaaggctt
gagcgagggc gtgcaagatt ccgaataccg caagcgacag gccgatcatc
3540gtcgcgctcc agcgaaagcg gtcctcgccg aaaatgaccc agagcgctgc
cggcacctgt 3600cctacgagtt gcatgataaa gaagacagtc ataagtgcgg
cgacgatagt catgccccgc 3660gcccaccgga aggagctgac tgggttgaag
gctctcaagg gcatcggtcg agatcccggt 3720gcctaatgag tgagctaact
tacattaatt gcgttgcgct cactgcccgc tttccagtcg 3780ggaaacctgt
cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
3840cgtattgggc gccagggtgg tttttctttt caccagtgag acgggcaaca
gctgattgcc 3900cttcaccgcc tggccctgag agagttgcag caagcggtcc
acgctggttt gccccagcag 3960gcgaaaatcc tgtttgatgg tggttaacgg
cgggatataa catgagctgt cttcggtatc 4020gtcgtatccc actaccgaga
tatccgcacc aacgcgcagc ccggactcgg taatggcgcg 4080cattgcgccc
agcgccatct gatcgttggc aaccagcatc gcagtgggaa cgatgccctc
4140attcagcatt tgcatggttt gttgaaaacc ggacatggca ctccagtcgc
cttcccgttc 4200cgctatcggc tgaatttgat tgcgagtgag atatttatgc
cagccagcca gacgcagacg 4260cgccgagaca gaacttaatg ggcccgctaa
cagcgcgatt tgctggtgac ccaatgcgac 4320cagatgctcc acgcccagtc
gcgtaccgtc ttcatgggag aaaataatac tgttgatggg 4380tgtctggtca
gagacatcaa gaaataacgc cggaacatta gtgcaggcag cttccacagc
4440aatggcatcc tggtcatcca gcggatagtt aatgatcagc ccactgacgc
gttgcgcgag 4500aagattgtgc accgccgctt tacaggcttc gacgccgctt
cgttctacca tcgacaccac 4560cacgctggca cccagttgat cggcgcgaga
tttaatcgcc gcgacaattt gcgacggcgc 4620gtgcagggcc agactggagg
tggcaacgcc aatcagcaac gactgtttgc ccgccagttg 4680ttgtgccacg
cggttgggaa tgtaattcag ctccgccatc gccgcttcca ctttttcccg
4740cgttttcgca gaaacgtggc tggcctggtt caccacgcgg gaaacggtct
gataagagac 4800accggcatac tctgcgacat cgtataacgt tactggtttc
acattcacca ccctgaattg 4860actctcttcc gggcgctatc atgccatacc
gcgaaaggtt ttgcgccatt cgatggtgtc 4920cgggatctcg acgctctccc
ttatgcgact cctgcattag gaagcagccc agtagtaggt 4980tgaggccgtt
gagcaccgcc gccgcaagga atggtgcatg caaggagatg gcgcccaaca
5040gtcccccggc cacggggcct gccaccatac ccacgccgaa acaagcgctc
atgagcccga 5100agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat
ataggcgcca gcaaccgcac 5160ctgtggcgcc ggtgatgccg gccacgatgc
gtccggcgta gaggatcgag atctcgatcc 5220cgcgaaatta atacgactca
ctatagggga attgtgagcg gataacaatt cccctctaga 5280aataattttg
tttaacttta agaaggagat ataccatggg ccatcatcat catcatcatc
5340atcatcatca cagcagcggc catatcgaag gtcgtcatat gactgccgac
aacaatagta 5400tgccccatgg tgcagtatct agttacgcca aattagtgca
aaaccaaaca cctgaagaca 5460ttttggaaga gtttcctgaa attattccat
tacaacaaag acctaatacc cgatctagtg 5520agacgtcaaa tgacgaaagc
ggagaaacat gtttttctgg tcatgatgag gagcaaatta 5580agttaatgaa
tgaaaattgt attgttttgg attgggacga taatgctatt ggtgccggta
5640ccaagaaagt ttgtcattta atggaaaata ttgaaaaggg tttactacat
cgtgcattct 5700ccgtctttat tttcaatgaa caaggtgaat tacttttaca
acaaagagcc actgaaaaaa 5760taactttccc tgatctttgg actaacacat
gctgctctca tccactatgt attgatgacg 5820aattaggttt gaagggtaag
ctagacgata agattaaggg cgctattact gcggcggtga 5880gaaaactaga
tcatgaatta ggtattccag aagatgaaac taagacaagg ggtaagtttc
5940actttttaaa cagaatccat tacatggcac caagcaatga accatggggt
gaacatgaaa 6000ttgattacat cctattttat aagatcaacg ctaaagaaaa
cttgactgtc aacccaaacg 6060tcaatgaagt tagagacttc aaatgggttt
caccaaatga tttgaaaact atgtttgctg 6120acccaagtta caagtttacg
ccttggttta agattatttg cgagaattac ttattcaact 6180ggtgggagca
attagatgac ctttctgaag tggaaaatga caggcaaatt catagaatgc
6240tataacaacg cgtcggatcc ggctgctaac aaagcccgaa aggaagctga
gttggctgct 6300gccaccgctg agcaataact agcataaccc cttggggcct
ctaaacgggt cttgaggggt 6360tttttgctga aaggaggaac tatatccgga
tatcccgcaa gaggcccggc agtaccggca 6420taaccaagcc tatgcctaca
gcatccaggg tgacggtgcc gaggatgacg atgagcgcat 6480tgttagattt
catacacggt gcctgactgc gttagcaatt taactgtgat aaactaccgc
6540attaaagctt atcgatgata agctgtcaaa catgagaa
6578193323DNAArtificial SequenceSynthetic Construct 193agattgctgc
gaaatcgtag gccggataag gcgtttacgc cgcatccggc aaaaatcctt 60aaatataaga
gcaaacctgc aattaaccct cactaaaggg cggccgcgaa gttcctattc
120tctagaaagt ataggaactt cctcgagccc tatagtgagt cgtattaaga
taaccatctg 180cggtgataaa ttatctctgg cggtgttgac ataaatacca
ctggcggtga tactgagcac 240atcagcagga cgcactgcaa aggaggtaaa
aacatgtctg aatctgtaca gagcaatagc 300gccgtcctgg tgcacttcac gct
323194512PRTArtificial SequenceSynthetic Construct 194Met Ser Phe
Asp Ile Ala Lys Tyr Pro Thr Leu Ala Leu Val Asp Ser1 5 10 15 Thr
Gln Glu Leu Arg Leu Leu Pro Lys Glu Ser Leu Pro Lys Leu Cys 20 25
30 Asp Glu Leu Arg Arg Tyr Leu Leu Asp Ser Val Ser Arg Ser Ser Gly
35 40 45 His Phe Ala Ser Gly Leu Gly Thr Val Glu Leu Thr Val Ala
Leu His 50 55 60 Tyr Val Tyr Asn Thr Pro Phe Asp Gln Leu Ile Trp
Asp Val Gly His65 70 75 80 Gln Ala Tyr Pro His Lys Ile Leu Thr Gly
Arg Arg Asp Lys Ile Gly 85 90 95 Thr Ile Arg Gln Lys Gly Gly Leu
His Pro Phe Pro Trp Arg Gly Glu 100 105 110 Ser Glu Tyr Asp Val Leu
Ser Val Gly His Ser Ser Thr Ser Ile Ser 115 120 125 Ala Gly Ile Gly
Ile Ala Val Ala Ala Glu Lys Glu Gly Lys Asn Arg 130 135 140 Arg Thr
Val Cys Val Ile Gly Asp Gly Ala Ile Thr Ala Gly Met Ala145 150 155
160 Phe Glu Ala Met Asn His Ala Gly Asp Ile Arg Pro Asp Met Leu Val
165 170 175 Val Leu Asn Asp Asn Glu Met Ser Ile Ser Glu Asn Val Gly
Ala Leu 180 185 190 Asn Asn His Leu Ala Gln Leu Leu Ser Gly Lys Leu
Tyr Ser Ser Leu 195 200 205 Arg Glu Gly Gly Lys Lys Val Phe Ser Gly
Val Pro Pro Ile Lys Glu 210 215 220 Leu Leu Lys Arg Thr Glu Glu His
Ile Lys Gly Met Val Val Pro Gly225 230 235 240 Thr Leu Phe Glu Glu
Leu Gly Phe Asn Tyr Ile Gly Pro Val Asp Gly 245 250 255 His Asp Val
Leu Gly Leu Ile Thr Thr Leu Lys Asn Met Arg Asp Leu 260 265 270 Lys
Gly Pro Gln Phe Leu His Ile Met Thr Lys Lys Gly Arg Gly Tyr 275 280
285 Glu Pro Ala Glu Lys Asp Pro Ile Thr Phe His Ala Val Pro Lys Phe
290 295 300 Asp Pro Ser Ser Gly Cys Leu Pro Lys Ser Ser Gly Gly Leu
Pro Ser305 310 315 320 Tyr Ser Lys Ile Phe Gly Asp Trp Leu Cys Glu
Thr Ala Ala Lys Asp 325 330 335 Asn Lys Leu Met Ala Ile Thr Pro Ala
Met Arg Glu Gly Ser Gly Met 340 345 350 Val Glu Phe Ser Arg Lys Phe
Pro Asp Arg Tyr Phe Asp Val Ala Ile 355 360 365 Ala Glu Gln His Ala
Val Thr Phe Ala Ala Gly Leu Ala Ile Gly Gly 370 375 380 Tyr Lys Pro
Ile Val Ala Ile Tyr Ser Thr Phe Leu Gln Arg Ala Tyr385 390 395 400
Asp Gln Val Leu His Asp Val Ala Ile Gln Lys Leu Pro Val Leu Phe 405
410 415 Ala Ile Asp Arg Ala Gly Ile Val Gly Ala Asp Gly Gln Thr His
Gln 420 425 430 Gly Ala Phe Asp Leu Ser Tyr Leu Arg Cys Ile Pro Glu
Met Val Ile 435 440 445 Met Thr Pro Ser Asp Glu Asn Glu Cys Arg Gln
Met Leu Tyr Thr Gly 450 455 460 Tyr His Tyr Asn Asp Gly Pro Ser Ala
Val Arg Tyr Pro Arg Gly Asn465 470 475 480 Ala Val Gly Val Glu Leu
Thr Pro Leu Glu Lys Leu Pro Ile Gly Lys 485 490 495 Gly Ile Val Lys
Arg Arg Gly Glu Lys Leu Ala Ile Leu Asn Phe Gly 500 505 510
195471PRTArtificial SequenceSynthetic Construct 195Met Ser Glu Leu
Leu Ser Arg Ile Gly Ser Pro Ser Asp Val Arg Ala1 5 10 15 Leu Pro
Glu Glu Ala Leu Pro Leu Leu Cys Gln Glu Leu Arg Glu Asp 20 25 30
Ile Ile Ser Ile Cys Gly Arg Val Gly Gly His Leu Gly Ala Ser Leu 35
40 45 Gly Ala Val Glu Leu Ile Val Ala Leu His Arg Val Phe His Ser
Pro 50 55 60 Gln Asp Ala Leu Leu Phe Asp Val Gly His Gln Thr Tyr
Ala His Lys65 70 75 80 Leu Leu Thr Gly Arg Arg Asp Arg Met His Thr
Leu Arg His Ala Gly 85 90 95 Gly Ile Ala Pro Phe Leu Asp Pro Arg
Glu Ser Pro His Asp Ala Leu 100 105 110 Leu Ala Gly His Ser Cys Thr
Ala Val Ser Ala Ala Leu Gly Val Leu 115 120 125 Glu Gly Arg Arg Gln
Gln Gly His Arg Gly His Val Val Ala Val Leu 130 135 140 Gly Asp Gly
Gly Leu Thr Gly Gly Leu Thr Phe Glu Gly Leu Asn Asn145 150 155 160
Ala Gly Gly Ser Ser Leu Pro Leu Val Val Val Leu Asn Asp Asn Gln 165
170 175 Met Ser Ile Ser Ala Asn Val Gly Ala Ile Pro Ala Leu Leu Arg
Thr 180 185 190 Arg Glu Ala Arg Asp Phe Phe Glu Gly Leu Gly Phe Thr
Tyr Leu Gly 195 200 205 Pro Val Asp Gly His Asp Leu Pro Ala Leu Ile
Arg Ala Leu Arg Glu 210 215 220 Ala Arg Ala Ser Ser Arg Pro Val Val
Val His Ala Leu Thr Leu Lys225 230 235 240 Gly Lys Gly Phe Pro Pro
Ala Glu Ala Asp Thr Gln Thr Arg Gly His 245 250 255 Ala Met Gly Pro
Tyr Glu Trp Arg Asp Gly Lys Leu Val Arg Ser Arg 260 265 270 Gly Gly
Gln Arg Thr Phe Ser Glu Ala Phe Ala Ala Val Leu Glu Asp 275 280 285
Ala Met Ala Arg Asp Pro Arg Val Val Ala Val Thr Pro Ala Met Leu 290
295 300 Glu Gly Ser Ala Leu Asn Ala Leu Lys Ala Arg Phe Pro Asp Arg
Val305 310 315 320 His Asp Val Gly Ile Ala Glu Gln His Ala Val Thr
Phe Ser Ala Gly 325 330 335 Leu Ala Ser Ala Gly Ala Arg Pro Val Cys
Cys Ile Tyr Ser Thr Phe 340 345 350 Leu Gln Arg Ala Tyr Asp Gln Ile
Ile His Asp Val Cys Leu Pro Gly 355 360 365 Leu Pro Val Val Phe Ala
Val Asp Arg Ala Gly Leu Val Gly Ala Asp 370 375 380 Gly Ala Thr His
Gln Gly Thr Tyr Asp Val Ala Ser Leu Arg Pro Leu385 390 395 400 Pro
Asp Leu His Leu Trp Ser Pro Met Val Gly Glu Asp Leu Ala Pro 405 410
415 Met Leu Asp Thr Ala Leu Ala Ala Pro His Ala Ser Val Ile Arg Phe
420 425 430 Pro Arg Gly Thr Leu Pro Pro Leu Pro Glu Gly Leu Gly Ala
Gly Glu 435 440 445 Ala Pro Leu Arg Gly Ala Arg Trp Leu Leu Arg Ala
Glu Gln Pro Arg 450 455 460 Leu Thr Leu Val Thr Leu Gly465 470
196479PRTArtificial SequenceSynthetic Construct 196Met Ala Lys Asn
Ile Leu Asn His Ile Asp Ser Pro Glu Asp Leu Arg1 5 10 15 Lys Leu
Asn Lys Lys Glu Leu Asp Leu Leu Ser Ile Glu Leu Arg Lys 20 25 30
Phe Ile Ile Asp Ile Val Ala Thr Lys Glu Gly His Leu Gly Ala Ser 35
40 45 Leu Gly Val Ile Glu Leu Thr Ile Ala Leu His Tyr Ile Phe Asn
Thr 50 55 60 Pro Glu Asp Leu Leu Val Trp Asp Val Gly His Gln Ala
Tyr Gly His65 70 75 80 Lys Ile Leu Thr Gly Arg Arg Asp Ile Phe Glu
Thr Asn Arg Gln Leu 85 90 95 Asn Gly Leu Ser Gly Phe Pro Lys Arg
Asp Glu Ser Glu Phe Asp Thr 100 105 110 Phe Gly Val Gly His Ser Ser
Thr Ser Ile Ser Ala Ala Leu Gly Met 115 120 125 Ala Ile Ala Ser Asn
Leu Lys Gly Glu Thr Glu Lys Gln His Ile Ala 130 135 140 Val Ile Gly
Asp Ala Ser Ile Ala Ser Gly Met Ala Phe Glu Gly Leu145 150 155 160
Asn His Ala Gly Val Thr Asn Ala Asn Leu Leu Val Ile Leu Asn Asp 165
170 175 Asn Ala Ile Gly Ile Asp Pro Ser Val Gly Ala Leu Lys Glu Tyr
Leu 180 185 190 Thr Lys Ala Arg Val Gly Tyr Lys Pro Ala Ser Asp Asn
Ile Ile Glu 195 200 205 Ala Leu Asn Phe Lys Tyr Phe Gly Pro Val Asp
Gly His Asp Leu Glu 210 215 220 Gly Leu Leu Lys Thr Leu Lys Glu Met
Lys Gln Ile Lys Gly Pro Lys225 230 235 240 Phe Leu His Val Ile Thr
Lys Lys Gly Lys Gly Leu Lys Lys Ala Glu 245 250 255 Glu Asp Gln Val
Lys Tyr His Ala Pro Gly Lys Phe Glu Pro Asn Thr 260 265 270 Gly Glu
Leu Leu Lys Tyr Asp Thr Asp Gly Leu Pro Leu Lys Tyr Gln 275 280 285
Asp Val Phe Gly Leu Thr Leu Val Glu Leu Ala Glu Lys Asn Glu Lys 290
295 300 Ile Ile Gly Ile Thr Pro Ala Met Pro Thr Gly Ser Ser Leu Lys
Tyr305 310 315 320 Met Met Lys Ala Phe Pro Glu Arg Ala Phe Asp Val
Gly Ile Ala Glu 325 330 335 Gln His Ala Val Thr Leu Ser Ala Gly Met
Ala Thr Gln Gly Phe Thr 340 345 350 Val Phe Cys Ala Ile Tyr Ser Thr
Phe Leu Gln Arg Ala Tyr Asp Gln 355 360 365 Leu Ile His Asp Val Ala
Ile Gln Asn Leu Pro Val Val Phe Cys Leu 370 375 380 Asp Arg Ala Gly
Leu Val Gly Glu Asp Gly Ala Thr His His Gly Val385 390 395 400 Phe
Asp Ile Ala Tyr Ala Arg Pro Ile Pro Asn Leu Met Ile Ala Ala 405 410
415 Pro Arg Asn Glu Val Glu Leu Arg Asn Leu Leu Tyr Thr Ala Gln Leu
420 425 430 Gly Leu Asp Ala Pro Met Ile Ile Arg Tyr Pro Arg Gly Arg
Gly Val 435 440 445 Leu Lys Glu Trp Lys Leu Pro Tyr Glu Lys Ala Glu
Ile Gly Lys Ala 450 455 460 Glu Cys Leu Lys Glu Gly Ser Glu Ile Ala
Ile Leu Ser Leu Gly465 470 475 197482PRTArtificial
SequenceSynthetic Construct 197Met Lys Ser Asp Leu Leu Ser Asn Ile
Tyr Asn Pro Ala Asp Leu Arg1 5 10 15 Leu Leu Lys Glu Glu Gln Leu
Thr Gln Val Ala Gln Glu Leu Arg Gln 20 25 30 Phe Ile Ile Asp Val
Val Ser Val Lys Glu Gly His Leu Gly Ala Ser 35 40 45 Leu Gly Val
Ile Glu Leu Thr Ile Ala Leu His Tyr Val Phe Asn Thr 50 55 60 Pro
Asp Asp Leu Leu Val Trp Asp Val Gly His Gln Ala Tyr Gly His65 70 75
80 Lys Ile Leu Thr Glu Arg Arg Glu Ile Phe His Thr Asn Arg Gln Ile
85 90 95 Asp Gly Ile Ser Gly Phe Pro Lys Arg Thr Glu Ser Val Tyr
Asp Thr 100 105 110 Phe Gly Val Gly His Ser Ser Thr Ser Ile Ala Ala
Leu Gly Met Ala 115 120 125 Ile Ala Ser Lys Leu Lys Gly Asp Leu Asp
Lys Gln His Ile Ala Val 130 135 140 Ile Gly Asp Ala Ser Ile Ala Ser
Gly Met Ala Phe Glu Gly Leu Asn145 150 155 160 His Ala Gly Val Thr
Asp Ala Asn Ile Leu Val Ile Leu Asn Asp Asn 165 170 175 Ala Ile Gly
Ile Asp Pro Ser Val Gly Ala Leu Lys Lys Tyr Leu Thr 180 185 190 Ala
Val Lys Asn Gly Lys Asn Pro Lys Gln Asn Asn Ile Ile Lys Ser 195 200
205 Leu Asn Phe Asp Tyr Ser Gly Pro Ile Asp Gly His Asp Phe Pro Thr
210 215 220 Leu Ile Lys Glu Leu Lys Arg Leu Lys Lys Ile Lys Gly Pro
Lys Phe225 230 235 240 Leu His Ile Val Thr Thr Lys Gly Lys Gly Leu
Gln Gln Ala Glu Glu 245 250 255 Asn Gln Val Lys Tyr His Ala Pro Gly
Lys Phe Asp Ala Ser Thr Gly 260 265 270 Glu Ile His Leu Lys Ser Glu
Glu Asn Leu Pro Pro Lys Tyr Gln Asp 275 280 285 Val Phe Gly Leu Thr
Val Leu Asp Leu Ala Arg Lys Asn Glu Lys Ile 290 295 300 Ile Gly Ile
Thr Pro Ala Met Pro Ser Gly Ser Ser Leu Lys Phe Met305 310 315 320
Met Asp Glu Leu Pro Asp Arg Ala Phe Asp Val Gly Ile Ala Glu Gln 325
330 335 His Ala Val Thr Leu Ala Ala Gly Met Thr Thr Gln Gly Met Met
Val 340 345 350 Tyr Cys Thr Ile Tyr Ser Thr Phe Leu Gln Arg Ala Tyr
Asp Gln Leu 355 360 365 Ile His Asp Val Ala Leu Gln Asn Leu Pro Val
Ile Phe Cys Leu Asp 370 375 380 Arg Ala Gly Leu Val Gly Glu Asp Gly
Ala Thr His His Gly Val Phe385 390 395 400 Asp Ile Ala Tyr Leu Arg
Ser Ile Pro Asn Met Ile Ile Tyr Ala Pro 405 410 415 Ile Asn Glu Ile
Ala Leu Gln Asn Ile Leu Tyr Thr Ala Gln Leu Gly 420 425 430 Leu Asn
Asn Pro Ile Ala Ile Arg Tyr Pro Arg Gly Arg Gly Val Ile 435 440 445
Lys Asn Trp Glu Val Glu Asn Phe Gly Gln Tyr Glu Lys Ile Lys Ile 450
455 460 Gly Glu Gly Asn Cys Leu Lys Asn Gly Thr Lys Thr Ala Val Leu
Ser465 470 475 480 Thr Gly198528PRTArtificial SequenceSynthetic
Construct 198Met Ala Asp Leu Val Ser Ala Pro Ala Met Ile Ser Gln
Ala Tyr Pro1 5 10 15 Leu Leu Ser Ser Ile His Ser Pro Ala Asp Leu
Lys Lys Leu Ser Leu 20 25 30 His Glu Leu Glu Leu Val Ala Ala Glu
Cys Arg Lys Lys Val Ile Glu 35 40 45 Leu Val Ser Gln Asn Gly Gly
His Phe Gly Ser Ser Leu Gly Val Val 50 55 60 Glu Leu Thr Val Ala
Leu His Tyr Val Tyr Gln Ser Pro Thr Asp Arg65 70 75 80 Ile Ile Trp
Asp Val Gly His Gln Ala Tyr Val His Lys Ile Leu Thr 85 90 95 Gly
Arg Leu Ala Gln Met Glu Thr Asn Arg Arg Tyr His Gly Leu Ala 100 105
110 Gly Phe Pro Lys Arg Ser Glu Ser Pro His Asp Ala Phe Gly Thr Gly
115 120 125 His Ala Ser Thr Ser Ile Ser Ala Ala Ala Gly Leu Ala Ala
Ala Arg 130 135 140 Asp Leu Ala Gly Arg Lys Glu Lys Val Val Ala Ile
Ile Gly Asp Gly145 150 155 160 Ser Leu Thr Gly Gly Met Ala Phe Glu
Ala Met Asn His Leu Gly Asp 165 170 175 Thr Lys Ser Asp Val Leu Val
Ile Leu Asn Asp Asn Gln Met Ala Ile 180 185 190 Ser Pro Ser Thr Gly
Gly Leu Lys Asn Tyr Leu Val Asn Leu Thr Leu 195 200 205 Asn Lys Thr
Tyr Asn Arg Leu Arg Lys Phe Val Trp Asp Ser Leu Ser 210 215 220 Leu
Leu His Asn Glu Ile Gly Glu Thr Ala Lys Thr Ala Val His Arg225 230
235 240 Ile Glu Asp Gly Ile Lys Ala Ala Phe Thr Pro Gly Ala Tyr Phe
Glu 245 250 255 Ala Leu Gly Phe Arg Tyr Phe Gly Pro Ile Asp Gly His
Asn Met Glu 260 265 270 Gln Leu Ile Lys Ala Leu Arg Glu Met Arg Gln
Leu His His Pro Lys 275 280 285 Leu Leu His Val Ile Thr Thr Lys Gly
Lys Gly Phe Lys Pro Ala Glu 290 295 300 Glu Asn Gln Pro Lys Trp His
Ala Ser Val Gly Gly Phe Asp Ile Glu305 310 315 320 Thr Gly Lys Asn
Val Lys Ala Pro Gly Lys Pro Ala Lys Pro Lys Tyr 325 330 335 Gln Glu
Val Phe Gly Glu Ala Leu Val Glu Leu Ala Leu Lys Asp Pro 340 345 350
Thr Ile Thr Ala Ile Thr Ala Ala Met Pro Ser Gly Thr Ser Leu Asp 355
360 365 Leu Phe Gln Gln Ala Ile Pro Ser Arg Cys Phe Asp Val Gly Ile
Ala 370 375 380 Glu Gln His Ala Val Thr Phe Ala Ala Gly Leu Ala Cys
Gly Gly Phe385 390 395 400 Lys Pro Val Phe Ala Val Tyr Ser Thr Phe
Leu Gln Arg Ala Tyr Asp 405 410 415 Gln Leu Ile His Asp Val Ala Leu
Gln Asn Leu His Val Val Phe Ala 420 425 430 Ile Asp Arg Ala Gly Leu
Val Gly Glu Asp Gly Pro Thr His His Gly 435 440 445 Ala Phe Asp Leu
Ser Tyr Leu Asn Val Val Pro Asn Leu Thr Ile Met 450 455 460 Ala Pro
Gly Asp Glu Gln Glu Leu Arg Asn Met Leu Tyr Thr Ala Leu465 470 475
480 Tyr Asp Ile Lys Gly Pro Val Ala Ile Arg Tyr Pro Arg Gly Ser Gly
485 490 495 Ser Gly Ala Thr Leu His Lys Glu Phe Thr Pro Val Pro Val
Gly Arg 500 505 510 Gly Arg Ile Leu Arg Asp Gly Lys Ser Val Ala Leu
Leu Gly Ile Gly 515 520 525 199514PRTArtificial SequenceSynthetic
Construct 199Met His Ile Ser Glu Leu Thr His Pro Asn Glu Leu Lys
Gly Leu Ser1 5 10 15 Ile Arg Glu Leu Glu Glu Val Ser Arg Gln Ile
Arg Glu Lys His Leu 20 25 30 Gln Thr Val Ala Thr Ser Gly Gly His
Leu Gly Pro Gly Leu Gly Val 35 40 45 Val Glu Leu Thr Val Ala Leu
Tyr Ser Thr Leu Asp Leu Asp Lys Asp 50 55 60 Arg Val Ile Trp Asp
Val Gly His Gln Ala Tyr Pro His Lys Met Leu65 70 75 80 Thr Gly Arg
Tyr His Asp Phe His Thr Leu Arg Gln Lys Asp Gly Val 85 90 95 Ala
Gly Tyr Leu Lys Arg Ser Glu Ser Arg Phe Asp His Phe Gly Ala 100 105
110 Gly His Ala Ser Thr Ser Ile Ser Ala Gly Leu Gly Met Ala Leu Ala
115 120 125 Arg Asp Ala Lys Gly Glu Asp Phe Lys Val Val Ser Ile Ile
Gly Asp 130 135 140 Gly Ala Leu Thr Gly Gly Met Ala Leu Glu Ala Ile
Asn His Ala Gly145 150 155 160 His Leu Pro His Thr Arg Leu Met Val
Ile Leu Asn Asp Asn Glu Met 165 170 175 Ser Ile Ser Pro Asn Val Gly
Ala Ile Ser Arg Tyr Leu Asn Lys Val 180 185 190 Arg Leu Ser Ser Pro
Met Gln Phe Leu Thr Asp Asn Leu Glu Glu Gln 195 200 205 Ile Lys His
Leu Pro Phe Val Gly Asp Ser Leu Thr Pro Glu Met Glu 210 215 220 Arg
Val Lys Glu Gly Met Lys Arg Leu Val Val Pro Lys Val Gly Ala225 230
235 240 Val Ile Glu Glu Leu Gly Phe Lys Tyr Phe Gly Pro Ile Asp Gly
His 245 250 255 Ser Leu Gln Glu Leu Ile Asp Thr Phe Lys Gln Ala Glu
Lys Val Pro 260 265 270 Gly Pro Val Phe Val His Val Ser Thr Thr Lys
Gly Lys Gly Tyr Asp 275 280 285 Leu Ala Glu Lys Asp Gln Val Gly Tyr
His Ala Gln Ser Pro Phe Asn 290 295 300 Leu Ser Thr Gly Lys Ala Tyr
Pro Ser Ser Lys Pro Lys Pro Pro Ser305 310 315 320 Tyr Ser Lys Val
Phe Ala His Thr Leu Thr Thr Leu Ala Lys Glu Asn 325 330 335 Pro Asn
Ile Val Gly Ile Thr Ala Ala Met Ala Thr Gly Thr Gly Leu 340 345 350
Asp Lys Leu Gln Ala Lys Leu Pro Lys Gln Tyr Val Asp Val Gly Ile 355
360 365 Ala Glu Gln His Ala Val Thr Leu Ala Ala Gly Met Ala Cys Glu
Gly 370 375 380 Ile Arg Pro Val Val Ala Ile Tyr Ser Thr Phe Leu Gln
Arg Gly Tyr385 390 395 400 Asp Gln Ile Ile His Asp Val Cys Ile Gln
Lys Leu Pro Val Phe Phe 405 410 415 Cys Leu Asp Arg Ala Gly Ile Val
Gly Ala Asp Gly Pro Thr His Gln 420 425 430 Gly Met Tyr Asp Ile Ala
Tyr Leu Arg Cys Ile Pro Asn Leu Val Leu 435 440 445 Met Ala Pro Lys
Asp Glu Ala Glu Leu Gln Gln Met Leu Val Thr Gly 450 455 460 Val Asn
Tyr Thr Gly Gly Ala Ile Ala Met Arg Tyr Pro Arg Gly Asn465 470 475
480 Gly Ile Gly Val Pro Leu Met Glu Glu Gly Trp Glu Pro Leu Glu Ile
485 490 495 Gly Lys Ala Glu Ile Leu Arg Ser Gly Asp Asp Val Leu Leu
Leu Gly 500 505 510 Tyr Gly200516PRTArtificial SequenceSynthetic
Construct 200Met Asn Leu Ser Asp Leu Ser His Pro Asn Gln Leu Arg
Asp Leu Ser1 5 10 15 Val Ser Gln Leu Gly Arg Leu Ala Gln Gln Ile
Arg Asp Lys His Leu 20 25 30 Gln Thr Val Ala Ala Thr Gly Gly His
Leu Gly Pro Gly Leu Gly Val 35 40 45 Val Glu Leu Thr Leu Ala Leu
Tyr Lys Thr Leu Asp Leu Asp Arg Asp 50 55 60 Arg Val Val Trp Asp
Val Gly His Gln Ala Tyr Pro His Lys Met Leu65 70 75 80 Thr Gly Arg
Tyr Ala Asn Phe His Thr Leu Arg Gln Lys Asp Gly Leu 85 90 95 Ala
Gly Tyr Leu Lys Arg Ala Glu Ser Pro Phe Asp Cys Trp Gly Ala 100 105
110 Gly His Ala Ser Thr Ser Ile Ser Ala Ala Leu Gly Met Ala Leu Ala
115 120 125 Arg Asp Phe Gln Gly Leu Asn Arg Lys Val Val Ala Ile Ile
Gly Asp 130 135 140 Gly Ala Leu Thr Gly Gly Met Ala Leu Glu Ala Leu
Asn His Ala Gly145 150 155 160 His Leu Ser Lys Thr Asn Leu Met Val
Ile Leu Asn Asp Asn Glu Met 165 170 175 Ser Ile Ser Glu Asn Val Gly
Gly Leu Ser Leu Tyr Leu Asn Arg Leu 180 185 190 Arg Thr Asp Pro Ala
Leu Arg Gln Ile Arg Ser Asn Leu Glu Thr Gln 195 200 205 Leu Arg Asn
Ile Pro Leu Val Gly Pro Thr Phe Ser Pro Glu Phe Glu 210 215 220 Arg
Phe Lys Asp Thr Val Lys Tyr Met Thr Met Thr Arg Ser Lys Ala225 230
235 240 Gly Val Ile Phe Glu Glu Leu Gly Phe Thr Tyr Leu Gly Pro Ile
Asp 245 250 255 Gly His Asn Leu Gly Asp Leu Ile Glu Thr Phe Glu Phe
Ala His Ser 260 265 270 Leu Pro Gly Pro Val Phe Leu His Ala Ile Thr
Val Lys Gly Lys
Gly 275 280 285 Tyr Glu Val Ala Glu Gln Asn Gln Ile Lys Tyr His Ala
Gln Ser Ala 290 295 300 Phe Asp Leu Ala Thr Gly Lys Ala Lys Pro Ala
Ser Lys Pro Thr Pro305 310 315 320 Pro Ala Tyr Thr Ser Val Phe Ala
Gln Thr Leu Val Lys Leu Ala Glu 325 330 335 Gln Asn Glu Lys Ile Val
Gly Ile Thr Ala Ala Met Pro Thr Gly Thr 340 345 350 Gly Leu Asp Lys
Phe Lys Glu Arg Phe Ala Asp Arg Tyr Phe Asp Val 355 360 365 Gly Ile
Ala Glu Gln His Ala Val Thr Met Ala Ala Gly Leu Ala Ala 370 375 380
Asp Gly Met Arg Pro Val Ala Ala Ile Tyr Ser Thr Phe Leu Gln Arg385
390 395 400 Ala Phe Asp Gly Ile Ile His Asp Val Ala Ile Gln Asp Leu
Pro Val 405 410 415 Phe Phe Cys Leu Asp Arg Ala Gly Val Val Gly Glu
Asp Gly Pro Thr 420 425 430 His His Gly Val Phe Asp Leu Ala Tyr Leu
Arg Gln Ile Pro Gly Leu 435 440 445 Val Val Met Ala Pro Lys Asp Glu
Ala Glu Leu Gln Arg Met Met Val 450 455 460 Thr Gly Ile Gln Tyr Thr
Lys Gly Pro Ile Ala Val Arg Tyr Pro Arg465 470 475 480 Gly Ser Gly
Ser Gly Ala Pro Leu Met Ala Glu Gly Trp Asp Pro Val 485 490 495 Pro
Ile Gly Lys Ala Glu Val Leu Arg Ser Gly Asp Asp Leu Leu Ile 500 505
510 Val Ala Ile Gly 515 201510PRTArtificial SequenceSynthetic
Construct 201Met Lys Met Lys Asn Ile Leu Asp Lys Tyr Gln Asp Phe
Asp Ser Ile1 5 10 15 Lys Ser Met Ser Ile Asn Glu Leu Asn Gln Phe
Ser Tyr Glu Ile Arg 20 25 30 Glu Phe Leu Ile Asp Asn Val Ser Lys
Thr Gly Gly His Leu Ala Ser 35 40 45 Asn Leu Gly Val Val Glu Leu
Thr Leu Ser Ile Phe Asn Val Phe Asp 50 55 60 Leu Asn Lys Asp Lys
Val Ile Trp Asp Val Gly His Gln Ala Tyr Val65 70 75 80 His Lys Ile
Leu Thr Gly Arg Lys Asp Lys Phe Asn Thr Leu Arg Gln 85 90 95 Tyr
Gly Gly Leu Ser Gly Phe Pro Lys Ile Cys Glu Ser Pro Tyr Asp 100 105
110 Val Phe Glu Thr Gly His Ser Ser Thr Ser Ile Ser Ala Ala Leu Gly
115 120 125 Met Ala Arg Ala Arg Asp Ile Lys Gly Glu Asn Asn Lys Val
Ile Ala 130 135 140 Val Ile Gly Asp Gly Ala Leu Thr Gly Gly Met Ala
Leu Glu Ala Leu145 150 155 160 Asn Asp Leu Gly Phe Asn Lys Thr Asp
Leu Ile Ile Ile Leu Asn Asp 165 170 175 Asn Gln Met Ser Ile Ala Glu
Asn Val Gly Gly Met Ser Ser Tyr Leu 180 185 190 Ser Lys Val Arg Leu
Asp Pro Thr Tyr Asn Lys Leu Lys Lys Glu Val 195 200 205 Asn Asn Thr
Leu Asn Lys Ile Pro Asn Val Gly Lys Gly Met Ala Arg 210 215 220 Ser
Leu Glu Lys Val Lys Asn Gly Ile Lys Gln Met Ile Val Pro Gly225 230
235 240 Met Leu Phe Glu Asn Leu Gly Ile Lys Tyr Leu Gly Pro Ile Asp
Gly 245 250 255 His Asp Ile Lys Glu Leu Ser Lys Val Met Lys Met Ala
Lys Asn Ile 260 265 270 Asn Gly Pro Val Leu Ile His Thr Ile Thr Lys
Lys Gly Lys Gly Tyr 275 280 285 Ala Tyr Ala Glu Lys Lys Pro Asp Lys
Phe His Gly Ile Gly Pro Phe 290 295 300 Asp Cys Asp Ser Gly Glu Val
Asn Ser Lys Thr Cys Leu Thr Tyr Ser305 310 315 320 Lys Val Phe Gly
Glu Glu Leu Thr Lys Ile Ala Lys Glu Asp Lys Lys 325 330 335 Val Val
Ala Ile Thr Ala Ala Met Lys Asp Gly Thr Gly Leu Arg Lys 340 345 350
Phe Gly Glu Thr Phe Pro Lys Arg Phe Phe Asp Val Gly Ile Ala Glu 355
360 365 Gln His Ala Val Thr Leu Ala Ala Gly Ile Ala Thr Glu Gly Leu
Lys 370 375 380 Pro Val Phe Ala Val Tyr Ser Thr Phe Leu Gln Arg Ala
Tyr Asp Gly385 390 395 400 Ile Leu His Asp Ile Cys Ile Gln Asn Leu
Pro Val Val Leu Gly Ile 405 410 415 Asp Arg Ala Gly Ile Val Gly Ser
Asp Gly Glu Thr His Gln Gly Ile 420 425 430 Phe Asp Leu Ser Tyr Leu
Ser Ser Leu Pro Asn Met Thr Ile Ile Ala 435 440 445 Pro Lys Cys Leu
Glu Glu Met Gly Ile Met Leu Arg Trp Ala Leu Asn 450 455 460 Gln Asn
Ser Pro Val Ala Ile Arg Tyr Pro Arg Gly Gly Asp Ile Lys465 470 475
480 Ser Leu Glu Met Thr Pro Ile Lys Asn Met Lys Lys Gly Lys Trp Glu
485 490 495 Val Ile Cys Glu Glu Gly Asp Ile Ala Ile Ile Ala Thr Gly
500 505 510 202509PRTArtificial SequenceSynthetic Construct 202Met
Leu Gln Gln Ile Arg Gly Pro Ala Asp Leu Gln His Leu Ser Gln1 5 10
15 Ala Gln Leu Arg Glu Leu Ala Ala Glu Ile Arg Glu Phe Leu Ile His
20 25 30 Lys Val Ala Ala Thr Gly Gly His Leu Gly Pro Asn Leu Gly
Val Val 35 40 45 Glu Leu Thr Leu Ala Leu His Arg Val Phe Asp Ser
Pro His Asp Pro 50 55 60 Ile Ile Phe Asp Thr Gly His Gln Ala Tyr
Val His Lys Met Leu Thr65 70 75 80 Gly Arg Ser Gln Asp Phe Ala Thr
Leu Arg Lys Lys Gly Gly Leu Ser 85 90 95 Gly Tyr Pro Ser Arg Ala
Glu Ser Glu His Asp Trp Val Glu Ser Ser 100 105 110 His Ala Ser Ala
Ala Leu Ser Tyr Ala Asp Gly Leu Ala Lys Ala Phe 115 120 125 Glu Leu
Thr Gly His Arg Asn Arg His Val Val Ala Val Val Gly Asp 130 135 140
Gly Ala Leu Thr Gly Gly Met Cys Trp Glu Ala Leu Asn Asn Ile Ala145
150 155 160 Ala Ser Arg Arg Pro Val Ile Ile Val Val Asn Asp Asn Gly
Arg Ser 165 170 175 Tyr Ala Pro Thr Ile Gly Gly Val Ala Asp His Leu
Ala Thr Leu Arg 180 185 190 Leu Gln Pro Ala Tyr Glu Gln Ala Leu Glu
Thr Gly Arg Asp Leu Val 195 200 205 Arg Ala Val Pro Leu Val Gly Gly
Leu Trp Phe Arg Phe Leu His Ser 210 215 220 Val Lys Ala Gly Ile Lys
Asp Ser Leu Ser Pro Gln Leu Leu Phe Thr225 230 235 240 Asp Leu Gly
Leu Lys Tyr Val Gly Pro Val Asp Gly His Asp Glu Arg 245 250 255 Ala
Val Glu Val Ala Leu Arg Ser Ala Arg Arg Phe Gly Ala Pro Val 260 265
270 Ile Val His Val Val Thr Arg Lys Gly Met Gly Tyr Pro Pro Ala Glu
275 280 285 Ala Asp Gln Ala Glu Gln Met His Ser Thr Val Pro Ile Asp
Pro Ala 290 295 300 Thr Gly Gln Ala Thr Lys Val Ala Gly Pro Gly Trp
Thr Ala Thr Phe305 310 315 320 Ser Asp Ala Leu Ile Gly Tyr Ala Gln
Lys Arg Arg Asp Ile Val Ala 325 330 335 Ile Thr Ala Ala Met Pro Gly
Pro Thr Gly Leu Thr Ala Phe Gly Gln 340 345 350 Arg Phe Pro Asp Arg
Leu Phe Asp Val Gly Ile Ala Glu Gln His Ala 355 360 365 Met Thr Ser
Ala Ala Gly Leu Ala Met Gly Gly Leu His Pro Val Val 370 375 380 Ala
Ile Tyr Ser Thr Phe Leu Asn Arg Ala Phe Asp Gln Ile Met Met385 390
395 400 Asp Val Ala Leu His Lys Leu Pro Val Thr Met Val Leu Asp Arg
Ala 405 410 415 Gly Ile Thr Gly Ser Asp Gly Ala Ser His Asn Gly Met
Trp Asp Leu 420 425 430 Ser Met Leu Gly Ile Val Pro Gly Ile Arg Val
Ala Ala Pro Arg Asp 435 440 445 Ala Thr Arg Leu Arg Glu Glu Leu Gly
Glu Ala Leu Asp Val Asp Asp 450 455 460 Gly Pro Thr Ala Leu Arg Phe
Pro Lys Gly Asp Val Gly Glu Asp Ile465 470 475 480 Ser Ala Leu Glu
Arg Arg Gly Gly Val Asp Val Leu Ala Ala Pro Ala 485 490 495 Asp Gly
Leu Asn His Asp Val Leu Leu Val Ala Ile Gly 500 505
203475PRTArtificial SequenceSynthetic Construct 203Met Asn Lys His
Pro Glu Phe Leu Leu Asn Lys Ile Lys Ser Pro Lys1 5 10 15 Asp Leu
Lys Lys Leu Asp Leu Lys Glu Leu Glu Gln Leu Ala Ser Glu 20 25 30
Ile Arg Thr Leu Ile Leu Glu Lys Asp Ala Ala Lys Gly Gly His Leu 35
40 45 Gly Pro Asp Leu Gly Ile Val Glu Ala Thr Ile Ala Tyr His Tyr
Val 50 55 60 Phe Asp Ala Pro Lys Asp Lys Ile Val Trp Asp Val Ser
His Gln Thr65 70 75 80 Tyr Pro His Lys Met Leu Thr Gly Arg Ala Tyr
Gly Trp Leu Asp Pro 85 90 95 Asp Lys Tyr Glu Asp Val Thr Pro Tyr
Ser Asn Pro Glu Glu Ser Pro 100 105 110 Tyr Asp Tyr Phe Ala Val Gly
His Thr Ser Thr Ser Ile Ala Leu Ala 115 120 125 Thr Gly Met Ala Arg
Ala Arg Asp Met Leu Gly Glu His Glu Asn Ile 130 135 140 Thr Ala Leu
Ile Gly Asp Gly Ser Leu Thr Gly Gly Leu Ala Phe Glu145 150 155 160
Gly Phe Asn Asn Ala Ala Asp Glu Lys His Asn Leu Ile Ile Val Val 165
170 175 Asn Asp Asn Gln Met Ser Ile Asp Asp Asn Val Gly Gly Val Val
Thr 180 185 190 Ala Leu Lys Lys Leu Arg Glu Ser Asn Gly Gln Thr Ala
Asp Asn Pro 195 200 205 Phe Thr Ala Met Gly Leu Asp Tyr Lys Tyr Val
Asp Gln Gly Asn Asp 210 215 220 Leu Lys Ala Met Ile Asp Ala Phe Lys
Ser Ile Lys Asp Ile Asp His225 230 235 240 Pro Ile Val Leu His Ile
Asn Thr Leu Lys Gly Lys Gly Tyr Glu Pro 245 250 255 Ala Ile Glu Asn
Glu Ala Ser His His Trp Val Leu Pro Phe Asp Leu 260 265 270 Lys Thr
Asp Lys Thr Thr Val Pro Ala Pro Lys Thr Pro Asn Pro Thr 275 280 285
Thr Val Val Leu Asp Phe Leu Lys Lys His Ile Glu Asn Gln Glu Asn 290
295 300 Ile Leu Ala Ile Asn Ala Ala Ile Pro Gly Val Phe Gly Leu Gly
Glu305 310 315 320 Ile Lys Asn Lys Tyr Pro Lys Asn Tyr Lys Asp Val
Gly Ile Ala Glu 325 330 335 Gln Glu Ser Val Ala Phe Ala Ala Gly Ala
Val Lys Glu Gly Ile Thr 340 345 350 Pro Val Leu Phe Glu Asn Ser Thr
Phe Leu Gln Arg Ala Tyr Asp Gln 355 360 365 Leu Ser His Asp Val Ala
Ala Asn Asp Leu Pro Val Val Met Val Val 370 375 380 Ala Gly Gly Gly
Ile Thr Gly Thr Ser Lys Thr His Leu Gly Ile Phe385 390 395 400 Asp
Asn Ala Met Val Ala Asn Trp Pro Asn Trp Glu Tyr Leu Ala Pro 405 410
415 Thr Thr Leu Asn Glu Leu Lys Ser Met Leu Glu Trp Ala Val Lys Gln
420 425 430 Arg Lys His Pro Ile Ala Ile Lys Ser Pro Val Asn Pro Ile
Pro Glu 435 440 445 Gly Glu Pro Val Asp Glu Asp Tyr Ser Thr Ile Lys
Tyr Asp Val Lys 450 455 460 Pro Gly Ser Lys Val Ala Ile Ile Gly Leu
Gly465 470 475 204431PRTArtificial SequenceSynthetic Construct
204Met Asn Lys His Pro Glu Phe Leu Leu Asn Lys Ile Glu Asn Pro Lys1
5 10 15 Asp Leu Lys Lys Leu Asp Leu Lys Glu Leu Glu Gln Leu Ala Ser
Glu 20 25 30 Ile Arg Thr Leu Ile Leu Glu Lys Asp Ala Ala Glu Gly
Gly His Leu 35 40 45 Gly Pro Asp Leu Gly Ile Val Glu Ala Thr Ile
Ala Tyr His Tyr Val 50 55 60 Phe Asp Ala Pro Lys Asp Lys Ile Ile
Trp Asp Val Ser His Gln Thr65 70 75 80 Tyr Pro His Lys Met Leu Thr
Gly Arg Ala Tyr Ala Trp Leu Asp Pro 85 90 95 Asp Lys Tyr Glu Asp
Val Thr Pro Tyr Ser Asn Pro Asp Glu Ser Pro 100 105 110 Tyr Asp Tyr
Tyr Ala Val Gly His Thr Ser Thr Ser Val Ala Leu Ala 115 120 125 Thr
Gly Met Ala Arg Ala Arg Asp Met Met Gly Asn His Glu Asn Ile 130 135
140 Thr Ala Leu Ile Gly Asp Gly Ser Leu Thr Gly Gly Leu Ala Phe
Glu145 150 155 160 Gly Phe Asn Asn Ala Ala Asp Glu Lys His Asn Leu
Ile Ile Val Val 165 170 175 Asn Asp Asn Gln Met Ser Ile Asp Asp Asn
Val Gly Gly Val Val Thr 180 185 190 Ala Leu Lys Lys Leu Arg Glu Ser
Asn Gly Gln Ala Glu Asp Asn Pro 195 200 205 Phe Thr Ala Met Gly Leu
Asp Tyr Lys Tyr Val Gly Gln Gly Asn Asp 210 215 220 Ile Lys Ser Met
Ile Asp Ala Phe Lys Ala Val Lys Asp Ile Asp His225 230 235 240 Pro
Ile Val Leu His Ile Asn Thr Leu Lys Gly Lys Gly Tyr Glu Pro 245 250
255 Ala Ile Glu Asn Glu Ala Ser His His Trp Val Leu Pro Phe Asp Leu
260 265 270 Lys Thr Asp Lys Pro Thr Val Pro Ala Pro Lys Thr Pro Asn
Ala Ser 275 280 285 Thr Val Ile Leu Asp Phe Leu Lys Lys His Ile Glu
Asn Gln Glu Asn 290 295 300 Ile Leu Val Ile Asn Ala Ala Ile Pro Gly
Val Phe Gly Leu Gly Glu305 310 315 320 Ile Lys Asn Asn Tyr Pro Lys
Asn Tyr Lys Asp Val Gly Ile Ala Glu 325 330 335 Gln Glu Ser Val Ala
Phe Ala Ala Gly Ala Val Lys Glu Gly Ile Thr 340 345 350 Pro Ile Leu
Phe Glu Asn Ser Thr Phe Leu Gln Arg Ala Tyr Asp Gln 355 360 365 Leu
Ser His Asp Val Ala Ala Asn Asp Leu Pro Val Val Met Ile Val 370 375
380 Ala Gly Gly Gly Ile Thr Ala Ala Ser Lys Thr His Leu Gly Ile
Phe385 390 395 400 Asp Asn Thr Met Val Ala Asn Trp Pro Asn Trp Glu
Tyr Leu Ala Pro 405 410 415 Thr Thr Leu Asn Glu Leu Lys Ser Met Leu
Glu Trp Ala Val Lys 420 425 430 205343PRTArtificial
SequenceSynthetic Construct 205Met Ser Val Leu Glu Lys Ile Asp Ser
Pro Ala Asp Leu Lys Lys Val1 5 10 15 Ser Asn Gln Glu Leu Glu Glu
Leu Ala Ser Glu Ile Arg Thr Ala Val 20 25 30 Leu His Lys Val Ser
Asn Ile Gly Gly His Val Gly Pro Asn Leu Gly 35 40 45 Val Thr Glu
Leu Thr Ile Ala Leu His Lys Val Phe Asn Ser Pro Ile 50 55 60 Asp
Lys Phe Ile Trp Asp Val Ser His Gln Thr Tyr Pro His Lys Ile65 70 75
80 Leu Thr Gly Arg Lys Asn Gly Phe Thr Asp Gly His Phe His Asp Ile
85 90 95 Thr Pro Tyr Thr Ser Gln Arg Glu Ser Glu His Asp Phe Phe
Thr Val 100 105 110 Gly His Thr Ser Thr Ser Ile Ala Asn Ala
Leu Gly Tyr Ala Lys Ala 115 120 125 Arg Asp Leu Thr Asn Asp Lys Gly
Asn Ile Val Ala Val Ile Gly Asp 130 135 140 Gly Ser Leu Ser Gly Gly
Leu Ala Met Glu Ala Leu Asn Asn Ala Gly145 150 155 160 Asp Phe Lys
Gly Asn Leu Ile Ile Leu Val Asn Asp Asn Gln Met Ser 165 170 175 Ile
Ala Glu Asn His Gly Gly Leu Tyr Arg Asn Leu Ala Glu Leu Arg 180 185
190 Ala Thr Asn Gly Gln Ala Glu Asn Asn Phe Phe Lys Thr Phe Gly Leu
195 200 205 Asp Tyr Lys Tyr Leu Glu Asn Gly Asn Asp Ile Glu Ser Leu
Ile His 210 215 220 Leu Phe Glu Glu Val Lys Asp Ile Asp His Pro Ile
Val Leu His Ile225 230 235 240 His Thr Glu Lys Gly Arg Gly Tyr Gln
Pro Ala Leu Glu Asn Lys Glu 245 250 255 Ala Phe His Trp His Met Pro
Phe Asp Leu Glu Thr Gly Gln Ser Lys 260 265 270 Val Ile Asp Ser Gly
Lys Ser Tyr Ser Ser Val Met Leu Asp Tyr Met 275 280 285 Asp Lys Lys
Val Ser Glu Gly Leu Pro Leu Val Ala Ile Asn Ala Ala 290 295 300 Ile
Pro Gly Ile Phe Gly Leu Lys Gln Phe Ala Ala Lys Tyr Pro Asp305 310
315 320 Arg Tyr Ile Asp Ala Gly Ile Ala Glu Gln Phe Thr Ile Thr Phe
Gly 325 330 335 Gly Ala Met Ala Ala Ala Gly 340
* * * * *
References