U.S. patent application number 13/941299 was filed with the patent office on 2014-01-09 for genetically modified photoautotrophic ethanol producing host cells, method for producing the host cells, constructs for the transformation of the host cells, method for testing a photoautotrophic strain for a desired growth property and method of producing ethanol using the host cells.
This patent application is currently assigned to Algenol Biofuels Inc.. The applicant listed for this patent is Algenol Biofuels Inc.. Invention is credited to Kerstin Baier, Ulf Duhring, Heike Enke, Dan Kramer, Christine Oesterhelt, Craig Smith, Robert Paul Woods.
Application Number | 20140011264 13/941299 |
Document ID | / |
Family ID | 40844867 |
Filed Date | 2014-01-09 |
United States Patent
Application |
20140011264 |
Kind Code |
A1 |
Duhring; Ulf ; et
al. |
January 9, 2014 |
Genetically Modified Photoautotrophic Ethanol Producing Host Cells,
Method For Producing The Host Cells, Constructs For The
Transformation Of The Host Cells, Method For Testing A
Photoautotrophic Strain For A Desired Growth Property And Method Of
Producing Ethanol Using The Host Cells
Abstract
The invention provides genetically modified photoautotrophic
ethanol producing host cells. Ethanol production is controlled by
an inducible promoter. Promoters utilized may be endogenous or
heterologous, and genetic modification may be obtained by
extrachromosomal means or chromosomal insertion.
Inventors: |
Duhring; Ulf; (Berlin,
DE) ; Enke; Heike; (Berlin, DE) ; Kramer;
Dan; (Berlin, DE) ; Smith; Craig; (Naples,
FL) ; Woods; Robert Paul; (Naples, FL) ;
Baier; Kerstin; (Kleinmachnow, DE) ; Oesterhelt;
Christine; (Berlin, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Algenol Biofuels Inc. |
Bonita Springs |
FL |
US |
|
|
Assignee: |
Algenol Biofuels Inc.
Bonita Springs
FL
|
Family ID: |
40844867 |
Appl. No.: |
13/941299 |
Filed: |
July 12, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12851712 |
Aug 6, 2010 |
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13941299 |
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PCT/EP2009/000892 |
Feb 9, 2009 |
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12851712 |
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61065292 |
Feb 8, 2008 |
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Current U.S.
Class: |
435/257.2 |
Current CPC
Class: |
C12P 7/065 20130101;
C12N 15/74 20130101; Y02E 50/10 20130101; C12N 15/8243 20130101;
Y02E 50/17 20130101 |
Class at
Publication: |
435/257.2 |
International
Class: |
C12N 15/82 20060101
C12N015/82 |
Claims
1. A recombinant cyanobacterial host cell comprising an exogenous
polynucleotide sequence that binds to a polynucleotide sequence
encoding a component of pyruvate dehydrogenase (EC number
1.2.4.1).
2. The recombinant cyanobacterial host cell of claim 1 further
comprising exogenous genes selected from the group consisting of
adh and pdc.
3. A recombinant cyanobacterial host cell comprising a vector
comprising an exogenous polynucleotide sequence whose transcription
is operably linked to a promoter wherein a transcriptional product
of said exogenous polynucleotide sequence binds to a mRNA
polynucleotide produced from transcription of an endogenous
polynucleotide sequence encoding pyruvate dehydrogenase (EC number
1.2.4.1), and wherein said transcriptional product of said
exogenous polynucleotide sequence binds to said mRNA polynucleotide
and reduces translation of said mRNA polynucleotide in comparison
to the translation of said mRNA polynucleotide wherein said
exogenous polynucleotide sequence is not transcribed.
4. The recombinant cyanobacterial host cell of claim 3 wherein said
promoter is selected from the group consisting of ntcA, nblA, isiA,
petJ, petE, ggpS, psbA2, psaA, sigB, IrtA, htpG, nirA, hspA, clpB1,
hliB, and crhC.
5. The recombinant cyanobacterial host cell of claim 3 wherein said
host cell is a member of the genus Synechocystis.
6. The recombinant cyanobacterial host cell of claim 3 wherein said
exogenous polynucleotide sequence has an identity of greater than
70% to a polynucleotide sequence consisting of the sequence of
nucleotides 5 through 225 of SEQ ID NO: 24.
7. The recombinant cyanobacterial host cell of claim 3 wherein said
exogenous polynucleotide sequence has an identity of greater than
70% to a polynucleotide sequence consisting of the sequence of
nucleotides 8 through 90 of SEQ ID NO: 24.
8. The recombinant cyanobacterial host cell of claim 3 further
comprising an exogenous adh gene and an exogenous pdc gene wherein
transcription of said adh gene is operably linked to a promoter and
said pdc gene is operably linked to a promoter.
9. The recombinant cyanobacterial host cell of claim 8 wherein said
promoter of said exogenous adh gene and said promoter of said
exogenous pdc gene are the same promoter.
10. The recombinant cyanobacterial host cell of claim 9 wherein
said promoter is selected from the group consisting of ntcA, nblA,
isiA, petJ, petE, ggpS, psbA2, psaA, sigB. IrtA, htpG, nirA, hspA,
clpB1, hliB, crhC and rbcLS.
11. The recombinant cyanobacterial host cell of claim 8 wherein
said promoter of said exogenous adh gene and said promoter of said
exogenous pdc gene are different promoters.
12. The recombinant cyanobacterial host cell of claim 11 wherein
said promoters are selected from the group consisting of ntcA nblA,
isiA pet, petE, ggpS, psbA2, psaA, sigB, IrtA, htpG, nirA, hspA,
clpB1, hliB, crhC and rbcLS.
13. A recombinant cyanobacterial host cell comprising a disruption
of an endogenous polynucleotide sequence encoding pyruvate
dehydrogenase (EC number 1.2.4.1), and further comprising a vector
comprising an exogenous polynucleotide sequence encoding pyruvate
dehydrogenase (EC number 1.2.4.1), wherein transcription of said
exogenous polynucleotide sequence encoding pyruvate dehydrogenase
(EC number 1.2.4.1) is operably linked to an inducible
promoter.
14. The recombinant cyanobacterial host cell of claim 13 wherein
said inducible promoter is selected from the group consisting of
ntcA, nblA, isiA, petJ, petE, ggpS, psbA2, psaA, sigB, IrtA, htpG,
nirA, hspA, clpB1 hliB, and crhC.
15. The recombinant cyanobacterial host cell of claim 13 wherein
said host cell is a member of the genus Synechocystis.
16. The recombinant cyanobacterial host cell of claim 13 further
comprising an exogenous adh gene and an exogenous pdc gene wherein
transcription of said adh gene is operably linked to a promoter and
said pdc gene is operably linked to a promoter.
17. The recombinant cyanobacterial host cell of claim 16 wherein
said promoter of said exogenous adh gene and said promoter of said
exogenous pdc gene are the same promoter.
18. The recombinant cyanobacterial host cell of claim 17 wherein
said promoter is selected from the group consisting of ntcA, nblA,
isiA, pet, petE, ggpS, psbA2, psaA sigB, IrtA, htpG, nirA, hspA,
clpB1 hliB, crhC and rbcLS.
19. The recombinant cyanobacterial host cell of claim 16 wherein
said promoter of said exogenous adh gene and said promoter of said
exogenous pdc gene are different promoters.
20. The recombinant cyanobacterial host cell of claim 19 wherein
said promoters are selected from the group consisting of ntcA,
nblA, isiA, petJ, petE, ggpS, psbA2, psaA, sigB, IrtA, htpG, nirA,
hspA, clpB1, hliB, crhC and rbcLS.
21. The recombinant cyanobacterial host cell of claim 13 wherein
said exogenous polynucleotide sequence encodes for an enzyme that
has an amino acid sequence that has an identity of greater than 78%
to an amino acid sequence of pyruvate dehydrogenase from
Synechocystis PCC 6803, accession number NP.sub.--440765.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation of U.S. application Ser.
No. 12/851,712, filed on Aug. 6, 2010 which claims priority to
International Application No. PCT/EP2009/000892, filed Feb. 9,
2009, which claims priority to U.S. Provisional Application No.
61/065,292 filed on Feb. 8, 2008; all applications are incorporated
herein by reference.
REFERENCE TO SEQUENCE LISTING
[0002] This application contains a sequence listing submitted by
EFS-Web, thereby satisfying the requirements of 37 C.F.R.
.sctn.1.821-1.825.
FIELD OF THE INVENTION
[0003] This invention is related to the field of ethanol production
using genetically modified cells.
BACKGROUND OF THE INVENTION
[0004] Without new methods for biofuel production, the world will
continue to depend on fossil fuels for transportation. Accelerating
demand, diminishing reserves and geopolitic risks have in recent
years dramatically driven up the cost of fossil fuels. Use of
fossil fuels also releases carbon dioxide into the atmosphere,
which may cause deleterious environmental effects. Many governments
have prescribed a reduction in the use of fossil fuels in favor of
alternative renewable biofuels in an effort to stem the release of
carbon dioxide from transportation vehicles.
[0005] Ethanol can be used as renewable biofuel but methods do not
currently exist that can produce ethanol in sufficient quantities
and at a price that could lead to a widespread adoption of ethanol
as a major alternative to fossil fuels in the worldwide
transportation fuel market.
[0006] The patent and scientific literature cited herein
establishes the knowledge that is available to those with skill in
the art. The issued U.S. and foreign patents, published U.S. and
foreign patent applications, and all other publications cited
herein are hereby incorporated by reference. Additionally, all
amino acid and nucleic acid sequences with the respective amino
acid sequences encoded thereby identified by database accession
number are hereby incorporated by reference.
[0007] Aspects of the invention utilize techniques and methods
common to the fields of molecular biology, microbiology and cell
culture. Useful laboratory references for these types of
methodologies are readily available to those skilled in the art.
See, for example, Molecular Cloning: A Laboratory Manual (Third
Edition), Sambrook, J., et al. (2001) Cold Spring Harbor Laboratory
Press; Current Protocols in Microbiology (2007) Edited by Coico, R,
et al., John Wiley and Sons, Inc.; The Molecular Biology of
Cyanobacteria (1994) Donald Bryant (Ed.), Springer Netherlands;
Handbook Of Microalgal Culture Biotechnology And Applied Phycology
(2003) Richmond, A; (ed.), Blackwell Publishing; and "The
cyanobacteria, molecular Biology. Genomics and Evolution", Edited
by Antonia Herrero and Enrique Flores, Caister Academic Press,
Norfolk, UK, 2008.
SUMMARY OF THE INVENTION
Invention 1
[0008] It has been discovered that photoautotrophic cells having
increased metabolite production produce more ethanol. The inventors
have genetically modified photoautotrophic cells in order to
increase the activity or affinity of metabolic enzymes, resulting
in increased metabolite formation (e.g., pyruvate, acetaldehyde,
acetyl-CoA, or precursors thereof) compared to the respective
wildtype host cell. Moreover, the inventors have discovered that by
further genetically modifying these cells with the overexpression
of at least one enzyme of an ethanol pathway, an increased
production of ethanol is obtained compared to wildtype
photoautotrophic host cell.
[0009] These discoveries have been exploited to provide the present
invention, which includes compositions of matter directed to these
advantageous, genetically modified photoautotrophic ethanol
producing host cells, nucleic acid constructs and methods of making
the same.
[0010] In a first aspect, the invention provides a genetically
modified photoautotrophic, ethanol producing host cell comprising
at least one first genetic modification changing the enzymatic
activity or affinity of an endogenous host cell enzyme, the first
genetic modification resulting in an enhanced level of biosynthesis
of acetaldehyde, pyruvate, acetyl-CoA or precursors thereof
compared to the respective wild type host cell, and at least one
second genetic modification different from the first genetic
modification comprising an overexpressed enzyme for the formation
of ethanol.
[0011] In one embodiment, the at least one endogenous host cell
enzyme is selected from enzymes of the glycolysis pathway.
Calvin-cycle, intermediate steps of metabolism, amino acid
metabolism pathway, the fermentation pathway and the citric acid
cycle, wherein the activity of at least one of these enzymes is
enhanced compared to the respective wild type host cell. In a
further embodiment thereof, the genetically modified host cell has
at least one endogenous host cell enzyme that is overexpressed.
[0012] Various other embodiments of the invention provide a
genetically modified, photoautotrophic ethanol producing host cell
wherein the at least one endogenous host cell enzyme is selected
from a group consisting of phosphoglycerate mutase, enolase, and
pyruvate kinase. In a particular embodiment, the endogenous host
cell enzyme of the first genetic modification is malate
dehydrogenase.
[0013] Certain other embodiments of the invention are directed to a
genetically modified, photoautotrophic ethanol producing host cell
wherein the at least one endogenous host cell enzyme of the first
genetic modification comprises an NAD.sup.+/NADH-cofactor-specific
enzyme that has been genetically engineered to become
NADP.sup.+/NADPH-cofactor specific enzyme. In embodiments thereof,
the NAD.sup.+/NADH-cofactor specific enzyme is malate
dehydrogenase.
[0014] In another embodiment, the invention provides a genetically
modified, photoautotrophic ethanol producing host cell wherein the
at least one endogenous host cell enzyme is selected from the
glycolysis pathway or the citric acid cycle and is dependent upon a
cofactor, and the host cell further comprises an enhanced level of
biosynthesis of this cofactor compared to the respective wild type
host cell. In embodiments thereof, the invention provides a
genetically modified host cell wherein the at least one endogenous
host cell enzyme comprises a NAD
(P).sup.+/NAD(P)H-cofactor-specific enzyme, and the host cell
comprises an enhanced level of NAD (P).sup.+/NAD(P)H biosynthesis
compared to the respective wild type host cell. In a particular
embodiment thereof, the genetically modified, photoautotrophic host
cell comprises an NAD (P).sup.+ transhydrogenase that is
overexpressed and converts NADPH to NADH.
[0015] Alternative embodiments of this aspect of the invention also
provide for a genetically modified, photoautotrophic host cell
comprising a host cell NADH dehydrogenase converting NADH to
NAD.sup.+, wherein the activity of the NADH dehydrogenase is
reduced compared to its activity in the wild type host cell. In one
particular embodiment thereof, the gene coding for the NADH
dehydrogenase is disrupted by a heterologous nucleic acid
sequence.
[0016] Various other embodiments provide a genetically modified,
photoautotrophic host cell wherein the at least one endogenous host
cell enzyme is for the conversion of pyruvate or acetyl-CoA or for
the formation of reserve compounds and wherein its activity or
affinity is reduced. In an embodiment thereof, the genetically
modified host cell the reduction of activity is the result of a
disruption of the gene encoding the at least one endogenous host
cell enzyme. Another embodiment thereof provides genetically
modified, photoautotrophic host cell wherein the gene disruption is
caused by insertion of a biocide resistance gene into the
respective gene.
[0017] A different embodiment of the invention provides a
genetically modified, photoautotrophic host cell wherein the at
least one first genetic modification comprises the transcription of
an antisense mRNA molecule that binds to the mRNA encoding the at
least one endogenous host cell enzyme and wherein binding results
in a reduction of activity of the at least one endogenous host cell
enzyme.
[0018] In another embodiment, the activity of a metabolic enzyme of
the invention can be decreased or eliminated by RNA interference
technology. RNA interference (RNAi) is a post-transcriptional gene
silencing technique in which double-stranded RNAs (dsRNAs) are
introduced in an exogenous or transgenic fashion. RNAi molecules
that are complementary to known mRNA's specifically destroy that
particular mRNA, thereby diminishing or abolishing gene expression.
There are many teachings in the art known to one of ordinary skill.
For example, see RNAi: A Guide to Gene Silencing, edited by Gregoy
J. Hannon. 2003. Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y. ISBN: 0-87969-641-9; Current Protocols in Molecular
Biology, UNIT 26.6 RNAi in Transgenic Plants, (2005) Yin, Y.,
Chory, J., and Baukombe, D., John Wiley & Sons, Inc.;
Transgenic microalgae as green cell-factories (2004) Leon-Banares,
R., et al., Trends in Biotechnology 22(1): 45-52; ALGAL TRANSGENICS
IN THE GENOMIC ERA (2005) Walker, T. L., et al., Journal of
Phycology 41(6):1077-1093.
[0019] Various other embodiments of the invention provide a
genetically modified, photoautotrophic host cell wherein the at
least one endogenous host cell enzyme is selected from a group
consisting of: ADP-glucose-pyrophosphorylase, glycogen synthase,
alanine dehydrogenase, lactate dehydrogenase, pyruvate water
dikinase, phosphotransacetylase, pyruvate dehydrogenase and acetate
kinase. In an embodiment thereof, the at least one endogenous host
cell enzyme is glycogen synthase.
[0020] Different embodiments of the invention also provide a
genetically modified host cell wherein the reserve compounds are
selected from a group consisting of glycogen, polyhydroxyalkanoates
(e.g. poly-3-hydroxybutyrate or poly-4-hydroxybutyrate),
polyhydroxyvalerate, polyhydroxyhexanoate, polyhydroxyoctanoate,
amylopectin, starch, cyanophycin and their copolymers,
glucosylglycerol and extracellular polysaccharides.
[0021] Further embodiments of the invention provide a genetically
modified, photoautotrophic host cell wherein the at least one
overexpressed enzyme for the formation of ethanol is an alcohol
dehydrogenase enzyme. In an embodiment thereof, the alcohol
dehydrogenase enzyme is a thermophilic alcohol dehydrogenase. Other
embodiments thereof provide a genetically modified,
photoautotrophic host cell wherein the alcohol dehydrogenase is
AdhE directly converting acetyl-CoA to ethanol. Another embodiment
thereof provides a genetically modified, photoautotrophic host cell
wherein the alcohol dehydrogenase comprises an amino acid sequence
at least 60% identical to AdhE from Thermosynechococcus elongatus
BP-1. A further embodiment is a genetically modified,
photoautotrophic host cell wherein the alcohol dehydrogenase is
AdhE from Thermosynechococcus elongatus BP-1. Different embodiments
thereof provide a genetically modified, photoautotrophic host cell
wherein the alcohol dehydrogenase enzyme is a Zn.sup.2+-dependent
dehydrogenase. In a preferred embodiment thereof, the invention
provides a genetically modified, photoautotrophic host cell wherein
the alcohol dehydrogenase enzyme is AdhI or AdhII from Zymomonas
mobilis or ADH from Synechocystis.
[0022] Certain embodiments of the invention provide a genetically
modified, photoautotrophic host cell comprising a pyruvate
decarboxylase enzyme converting pyruvate to acetaldehyde, and an
alcohol dehydrogenase enzyme converting the acetaldehyde to
ethanol. An embodiment thereof provides a genetically modified,
photoautotrophic host cell wherein the pyruvate decarboxylase
enzyme is from Zymomonas mobilis or Zymobacter palmae.
[0023] Other embodiments of the invention provide a genetically
modified, photoautotrophic host cell comprising a gene encoding the
at least one overexpressed enzyme for the formation of ethanol
which is integrated into the host cell genome. In an embodiment
thereof, the genetically modified, photoautotrophic host cell
further comprises a host gene encoding the at least one endogenous
host cell enzyme converting pyruvate, acetaldehyde or acetyl-CoA or
forming reserve compounds, wherein a gene encoding the at least one
overexpressed enzyme for the formation of ethanol is integrated
into said host gene thereby disrupting the host gene.
[0024] Various embodiments of the invention provide a genetically
modified, photoautotrophic host cell wherein the gene encoding the
at least one overexpressed enzyme for the formation of ethanol is
under the transcriptional control of a promoter endogenous to the
host cell.
[0025] Various embodiments of the invention provide a genetically
modified, photoautotrophic host wherein the gene encoding the at
least one overexpressed enzyme for the formation of ethanol is
under the transcriptional control of a heterologous promoter.
[0026] Various embodiments of the invention provide a genetically
modified, photoautotrophic host cell wherein the gene encoding the
at least one overexpressed enzyme for the formation of ethanol is
under the transcriptional control of an inducible promoter. In
embodiments thereof, the inducible promoter is induced under
conditions of nutrient starvation, by stationary growth phase, by
heat shock, by cold shock, by oxidative stress, by salt stress, by
light or by darkness. In a further embodiment thereof, the
inducible promoters are selected from a group consisting of ntcA,
nblA, isiA, petJ, petE, ggpS, psbA2, psaA, sigB, IrtA, htpG, nirA,
hspA, clpB1, hliB, and crhC. Other embodiments of the Invention
provide a genetically modified, photoautotrophic host cell wherein
the gene encoding the at least one overexpressed enzyme for the
formation of ethanol is under the transcriptional control of a
constitutive promoter, such as the rbcLS promoter.
[0027] In a second aspect, the Invention provides a genetically
modified, photoautotrophic ethanol producing host cell comprising
at least one first genetic modification changing the enzymatic
activity or affinity of an endogenous host cell enzyme, the first
genetic modification resulting in a level of biosynthesis of a
first metabolic intermediate for energy production or metabolism of
the host cell that is enhanced compared to level of biosynthesis in
the respective wild type host cell, and at least one second genetic
modification different from the first genetic modification
comprising an overexpressed first enzyme for the formation of
ethanol from the first metabolic intermediate.
[0028] An embodiment thereof provides a genetically modified host,
photoautotrophic cell further comprising at least one overexpressed
second enzyme, converting the first metabolic intermediate into a
second metabolic intermediate, wherein the at least one
overexpressed first enzyme converts the second metabolic
intermediate into ethanol.
[0029] Other embodiments of the second aspect provide a genetically
modified, photoautotrophic host cell wherein the endogenous host
cell enzyme is for conversion of the first metabolic intermediate
and wherein the activity of said host cell enzyme is reduced
compared to the respective wild type host cell. Certain other
embodiments herein provide a genetically modified, photoautotrophic
host cell wherein the endogenous host cell enzyme is for the
formation of the first metabolic intermediate and wherein the
activity of said host enzyme is enhanced compared to the respective
wild type host cell.
[0030] Another embodiment of the second aspect relates to a
genetically modified, photoautotrophic host cell wherein the first
metabolic intermediate comprises acetyl-CoA and the at least one
overexpressed first enzyme for ethanol formation comprises alcohol
dehydrogenase AdhE converting acetyl-CoA into ethanol.
[0031] Another embodiment of the second aspect relates to a
genetically modified, photoautotrophic host cell wherein the first
metabolic intermediate comprises pyruvate, the second metabolic
intermediate comprises acetaldehyde, and the at least one
overexpressed second enzyme for ethanol formation comprises
pyruvate decarboxylase, converting pyruvate into acetaldehyde, and
the at least one overexpressed first enzyme for ethanol formation
comprises alcohol dehydrogenase Adh, converting acetaldehyde into
ethanol. In another embodiment thereof, the invention provides a
genetically modified, photoautotrophic host cell wherein the host
gene encoding the at least one host cell enzyme is disrupted by a
heterologous nucleic acid sequence. In a further embodiment
thereof, the genetically modified, photoautotrophic host cell
further comprises a second gene encoding the at least one host cell
enzyme, wherein the second gene is under the transcriptional
control of an inducible promoter.
[0032] In a third aspect, the invention provides a genetically
modified, photoautotrophic, ethanol producing host cell comprising
at least one first genetic modification of at least one endogenous
host cell enzyme that is not pyruvate decarboxylase or alcohol
dehydrogenase, wherein the first genetic modification results in an
enhanced level of biosynthesis of acetaldehyde, pyruvate,
acetyl-CoA or precursors thereof compared to the respective wild
type host cell, and at least one second genetic modification
comprising at least one overexpressed enzyme for the formation of
ethanol.
[0033] In a fourth aspect, the invention provides a construct for
the transformation of a host cell by disrupting a host gene
encoding a host cell enzyme for conversion of a first metabolic
intermediate for energy production or metabolism of the host cell
or for formation of reserve compounds comprising a heterologous
nucleic acid sequence comprising a promoter and a biocide
resistance conferring gene under the transcriptional control of the
promoter, wherein the heterologous nucleic acid sequence is flanked
at its 5' and 3' end by nucleic acid sequences that bind said host
gene.
[0034] In a fifth aspect, the invention provides a construct for
the transformation of a host cell by disrupting a host gene
encoding a host cell enzyme for conversion of a first metabolic
intermediate for energy production or metabolism of the host cell
or for formation of reserve compounds comprising a coding nucleic
acid sequence comprising a promoter and a first gene encoding at
least one overexpressed first enzyme for the formation of ethanol
from the first metabolic intermediate under the transcriptional
control of the promoter, wherein the coding nucleic acid sequence
is flanked at its 5' and 3' end by nucleic acid sequences that bind
to said host gene.
[0035] In an embodiment of the fifth aspect, the construct
comprises a coding nucleic acid sequence further comprising a
second gene encoding at least one overexpressed second enzyme
converting the first metabolic intermediate into a second metabolic
intermediate, wherein the at least one overexpressed first enzyme
converts the second metabolic intermediate into ethanol. In an
embodiment thereof, the first metabolic intermediate comprises
pyruvate, and the second metabolic intermediate comprises
acetaldehyde, the second gene encodes pyruvate decarboxylase
enzyme, converting pyruvate into acetaldehyde, and the first gene
encodes alcohol dehydrogenase enzyme, converting acetaldehyde into
ethanol.
[0036] An embodiment of the fourth aspect provides a construct
wherein the first metabolic intermediate comprises acetyl-CoA and
the first gene encodes alcohol dehydrogenase AdhE, converting
acetyl-CoA into ethanol.
[0037] In a sixth aspect, the invention provides a construct for
the transformation of a host cell by disrupting a host gene
encoding a host cell enzyme for conversion of a first metabolic
intermediate for energy production or metabolism of the host cell
or for formation of reserve compounds comprising a coding nucleic
acid sequence comprising an inducible promoter and a gene encoding
said host cell enzyme under the transcriptional control of the
inducible promoter, wherein the coding nucleic acid sequence is
flanked at its 5' and 5' end by nucleic acid sequences that bind to
said host gene.
[0038] Various embodiments of the fourth, fifth and sixth aspect
provide a construct wherein the 5' and 3' flanking nucleic acid
sequences are at least 60% identical to at least a part of the host
gene. In an embodiment thereof, the 5' and 3' flanking nucleic acid
sequences are identical to the host gene, thereby enabling the
insertion of the coding nucleic acid sequence into the host gene by
homologous recombination.
[0039] In other embodiments of the fourth, fifth and sixth aspect,
the invention provides a construct wherein said host gene encodes
glycogen synthase. In further embodiments thereof, the construct
comprises a recombinant plasmid.
[0040] In a seventh aspect, the invention provides a method for
producing the genetically modified, photoautotrophic host cells
according to the first, second, third, fourth, fifth and sixth
aspect. The method comprises (A) providing a wild type host cell
showing a wildtype level of biosynthesis of a first metabolic
intermediate for energy production of the host cell, (B)
introducing at least one first genetic modification into the wild
type host cell enhancing the level of biosynthesis of the first
metabolic intermediate in comparison to the respective wild type
host cell, and (C) introducing at least one second genetic
modification into the wild type host cell resulting in at least one
overexpressed first enzyme for the formation of ethanol from the
first metabolic intermediate.
[0041] In an embodiment thereof, the method comprises that in step
(C) a further second genetic modification is introduced into the
host cell resulting in at least one overexpressed second enzyme for
the formation of ethanol, the at least one overexpressed second
enzyme converting the first metabolic intermediate into a second
metabolic intermediate, wherein the at least one overexpressed
first enzyme converts the second metabolic intermediate into
ethanol. In an embodiment thereof, the first metabolic intermediate
comprises pyruvate and the second metabolic intermediate comprises
acetaldehyde, and the overexpressed second enzyme comprises
pyruvate decarboxylase enzyme, converting pyruvate into
acetaldehyde, and the overexpressed first enzyme comprises alcohol
dehydrogenase enzyme Adh, converting acetaldehyde into ethanol.
[0042] The invention also provides an embodiment of the seventh
aspect wherein the first metabolic intermediate comprises
acetyl-CoA, and the overexpressed first enzyme comprises alcohol
dehydrogenase enzyme AdhE converting acetyl-CoA into ethanol.
[0043] Various embodiments of the seventh aspect provide a method
wherein in step (A) a wild type host cell is provided, which
further comprises a first host gene encoding at least one first
host cell enzyme for conversion of the first metabolic intermediate
or for forming reserve compounds, the first host cell gene is under
the transcriptional control of a first host promoter, and in step
(B) the activity or the affinity of the at least one first host
enzyme is reduced. In an embodiment thereof, the method comprises
that in step (B) the activity of the at least one host enzyme is
reduced by mutating either the first host promoter or the first
host gene or by disrupting the first host gene by introducing a
heterologous nucleic acid sequence into the first host gene.
[0044] Other embodiments of the seventh aspect related to a method
wherein in step (A) a wild type host cell is provided, which
further comprises a second host gene encoding at least one second
host cell enzyme for formation of the first metabolic intermediate
or precursors thereof, the second host gene is under the
transcriptional control of a second host promoter, and in step (B)
the activity or affinity of the at least one second host enzyme is
enhanced. In an embodiment thereof, the method provides that in
step (B) the activity of the at least one second host enzyme is
enhanced by mutating either the second host promoter or the second
host gene or by overexpressing the second host enzyme.
[0045] A further embodiment of the seventh aspect provides a method
wherein the first metabolic intermediate comprises pyruvate,
acetyl-CoA or acetaldehyde.
Invention 2
[0046] It has been discovered that there are advantages to
producing ethanol from genetically modified, photoautotrophic cells
having a Zn.sup.2+ dependent alcohol dehydrogenase enzyme. This
discovery has been exploited to provide the following invention,
which includes compositions of matter directed to these
advantageous, genetically modified, photoautotrophic ethanol
producing host cells, nucleic acid constructs and methods of making
the same.
[0047] In an eighth aspect, the invention provides genetically
modified photoautotrophic, ethanol producing host cell comprising
an overexpressed pyruvate decarboxylase enzyme converting pyruvate
to acetaldehyde and an overexpressed Zn.sup.2+ dependent alcohol
dehydrogenase enzyme converting acetaldehyde to ethanol.
[0048] In an embodiment thereof, the Zn.sup.2+ dependent alcohol
dehydrogenase enzyme comprises AdhI from Zymomonas mobilis.
[0049] In another embodiment thereof, the Zn.sup.2+ dependent
alcohol dehydrogenase enzyme comprises Synechocystis Adh.
[0050] In a ninth aspect, the invention provides a construct for
the transformation of a photoautotrophic host cell, the construct
comprising a heterologous nucleic acid sequence comprising a first
gene encoding a Zn.sup.2+ dependent alcohol dehydrogenase, wherein
the heterologous nucleic aid sequence is flanked at its 5' and 5'
end by nucleic acid sequences that bind to said host genome for
integration of the heterologous nucleic acid sequence into the host
genome.
[0051] In an embodiment thereof, the construct further comprises a
heterologous or endogenous promoter controlling the transcription
of the first gene.
[0052] In a tenth aspect, the invention provides a construct for
the transformation of a photoautotrophic host cell comprising a
heterologous nucleic acid sequence comprising a heterologous
promoter and a first gene encoding a Zn.sup.2+ dependent alcohol
dehydrogenase enzyme, wherein the first gene is under the
transcriptional control of the heterologous promoter.
[0053] Various embodiments of the ninth and tenth aspect related to
a construct further comprising a second gene encoding pyruvate
decarboxylase enzyme.
[0054] Other variant embodiments of the constructs of the ninth and
tenth aspect are directed to constructs that are a recombinant
circular plasmid.
Invention 3
[0055] It has been discovered that there are advantages to
producing ethanol from genetically modified, photoautotrophic cells
having a alcohol dehydrogenase enzyme that converts acetyl-CoA
directly to ethanol. This discovery has been exploited to provide
the following invention, which includes compositions of matter
directed to these advantageous, genetically modified,
photoautotrophic ethanol producing host cells, nucleic acid
constructs and methods of making the same.
[0056] In an eleventh aspect, the invention provides a genetically
modified, photoautotrophic ethanol producing host cell comprising
an overexpressed alcohol dehydrogenase enzyme directly converting
acetyl-CoA to ethanol.
[0057] In an embodiment thereof, the alcohol dehydrogenase
comprises AdhE. In a further embodiment thereof, the alcohol
dehydrogenase enzyme is a thermophilic alcohol dehydrogenase. In
another embodiment the AdhE-type alcohol dehydrogenase is from E.
coli.
[0058] In a twelfth aspect, the invention provides a construct for
the transformation of a photoautotrophic host cell comprising a
heterologous nucleic acid sequence comprising a gene encoding an
alcohol dehydrogenase, directly converting acetyl-CoA to ethanol,
wherein the heterologous nucleic acid sequence is flanked at its 5'
and 3' ends by nucleic acid sequences, that bind to said host
genome for integration of the heterologous nucleic acid sequence
into the host genome. In an embodiment thereof, the construct
further comprises a heterologous promoter controlling the
transcription of the gene.
[0059] In a thirteenth aspect, the invention provides a construct
for the transformation of a photoautotrophic host cell comprising a
heterologous nucleic acid sequence comprising a heterologous
promoter and a gene encoding an alcohol dehydrogenase, directly
converting acetyl-CoA to ethanol, wherein the gene is under the
transcriptional control of the heterologous promoter.
Invention 4
[0060] It has been discovered that there are advantages to
producing ethanol from genetically modified, photoautotrophic cells
comprising an enhanced level of enzyme cofactor biosynthesis. This
discovery has been exploited to provide the following invention,
which includes compositions of matter directed to these
advantageous, genetically modified, photoautotrophic ethanol
producing host cells.
[0061] In a fourteenth aspect, the invention provides a genetically
modified photoautotrophic, ethanol producing host cell comprising
an overexpressed NAD.sup.+/NADH-cofactor specific alcohol
dehydrogenase enzyme, wherein the host cell comprises an enhanced
level of NAD.sup.+/NADH biosynthesis compared to the respective
wild type host cell.
[0062] In an embodiment thereof, the genetically modified,
photoautotrophic host cell comprises a host NADH dehydrogenase
enzyme converting NADH to NAD.sup.+, wherein the activity of the
NADH dehydrogenase enzyme is reduced compared to the wild type host
cell.
[0063] In a further embodiment, the genetically modified,
photoautotrophic host cell comprises a NAD (P).sup.+
transhydrogenase converting NADPH to NADH, wherein this NAD
(P).sup.+ transhydrogenase is overexpressed.
Invention 5
[0064] It has been discovered that there are advantages to
producing ethanol from genetically modified, photoautotrophic cells
comprising the overexpression of enzyme(s) for ethanol production.
This discovery has been exploited to provide the following
invention, which includes compositions of matter directed to these
advantageous, genetically modified, photoautotrophic ethanol
producing host cells.
[0065] In a fifteenth aspect, the invention provides a genetically
modified photoautotrophic, ethanol producing host cell comprising a
coding nucleic acid sequence comprising a promoter and a gene
encoding at least one overexpressed enzyme for the formation of
ethanol under the transcriptional control of the promoter, wherein
the promoter can be induced by nutrient starvation, oxidative
stress, darkness, light, heat shock, salt stress, cold shock or
stationary growth of the host cell.
[0066] In an embodiment thereof, the inducible promoter is selected
from a group of promoters consisting of ntcA, nblA, isiA, petJ,
petE, sigB, IrtA, htpG, hspA, clpB1, hliB, ggpS, psbA2, psaA, nirA
and crhC.
[0067] In a sixteenth aspect, the invention provides a construct
for the transformation of a photoautotrophic host cell, comprising
a coding nucleic acid sequence comprising a promoter, which can be
induced by nutrient starvation of the host cell, and a gene
encoding at least one overexpressed enzyme for the formation of
ethanol under the transcriptional control of the promoter.
[0068] In an embodiment thereof, the coding nucleic acid sequence
is flanked at its 5' and 3' ends by nucleic acid sequences, that
bind to said host genome for integration of the coding sequence
into the host genome.
Invention 6
[0069] It has been discovered that photoautotrophic cell(s) can be
selected for certain advantageous growth properties. The invention
provides methods for selecting and identifying these cells having
wide ranging advantageous properties.
[0070] In a seventeenth aspect, the invention provides a method for
testing a photoautotrophic strain for a desired growth property
selected from a group of properties consisting of ethanol
tolerance, salt tolerance, above neutral pH tolerance, mechanical
stress tolerance, temperature tolerance and light tolerance. The
steps of this method comprise (a) providing a photoautotrophic
strain to be tested, (b) cultivating the photoautotrophic strain to
be tested in a liquid growth medium and subjecting the
photoautotrophic strain to a condition selected from a group of
conditions consisting of (i) adding ethanol to the growth medium,
(i) adding salt to the growth medium, (iii) increasing the pH of
the growth medium, (iv) agitating the growing culture, (v)
increasing the temperature of the growing culture, (vi) subjecting
the photoautotrophic strain to high light, and (c) determining the
viability of the cells of the photoautotrophic strain cultivated in
step (b).
[0071] In an embodiment thereof, determining the viability
comprises determining at least one parameter selected from a group
of parameters consisting of growth rate of the photoautotrophic
strain, ratio of living to dead cells, ability to be recultivable
in a liquid growth medium in the absence of the respective
conditions (i) to (vi), and microscopic analysis of the
photoautotrophic strain. In an embodiment thereof, the ratio of
living to dead cells is determined by detecting the presence of a
photopigment in the cells of the photoautotrophic strain. In a
further embodiment thereof, the presence of a photopigment is
detected by measuring the fluorescence of the photopigment.
[0072] In various embodiments of the seventeenth aspect, the growth
rate of the photoautotrophic strain is determined by measuring the
optical density of the cultivated cells.
[0073] In various embodiments of the seventeenth aspect, the steps
(b) and (c) are repeated alternatively and wherein in a subsequent
step (b2) the conditions are changed in comparison to the foregoing
step (b1) by at least one of increasing the amount of ethanol in
the growth medium, increasing the amount of salt in the growth
medium, increasing the pH in the growth medium, increasing the rate
of agitation during cultivation, and increasing the temperature
during cultivation. In an embodiment thereof the amount of ethanol
in the growth medium is increased stepwise. In a further embodiment
thereof the amount of ethanol in continuously increased during step
(b). Another embodiment thereof provides for that during method
step (b) adding the ethanol to the growth medium with a flow rate,
and the flow rate is increased between successive steps (b) until a
maximum flow rate is reached and then the flow rate is reduced
between successive steps (b).
[0074] Various embodiments of the seventeenth aspect comprise
method step (b) comprising the sub steps (b1) and (b2) and method
step (c) comprises the sub steps (c1) and (c2) and a plurality of
different photoautotrophic strains to be tested are first subjected
to a first condition including adding a first amount of ethanol to
the growth medium and cultivating the different photoautotrophic
strains for a first period of time during method step (b1) and
identifying the photoautotrophic strains found to be tolerant to
the first condition in method step (c1) are thereafter subjecting
the photoautotrophic strains identified in method step (c1) to a
second amount of ethanol for a second period of time in a
subsequent step (b2), and identifying the photoautotrophic strains
tolerant to the second condition in a method step (c2) the first
amount of ethanol being higher than the second amount of ethanol,
and the first period of time being smaller than the second period
of time.
[0075] In various embodiments of the seventeenth aspect during
method step (b) salt is added to the growth medium by adding a
salty growth medium. In an embodiment thereof a salty medium is
added selected from a group consisting of brackish water, salt
water, artificial sea water.
[0076] In various embodiments of the seventeenth aspect the
photoautotrophic strain is cultivated in a growth medium with a pH
of above 9, preferably above 10 to 12.
[0077] In various embodiments of the seventeenth aspect during
method step (b) the growth medium is stirred during the
cultivation.
[0078] In various embodiments of the seventeenth aspect during
method step (b) the photoautotrophic strain is cultivated in a
growth medium at temperatures of at least 42.degree. C.
[0079] In various embodiments of the seventeenth aspect during
method step (b) the photoautotrophic strain is subjected to a first
light intensity in the lag- and exponential growth phase and a
first CO.sub.2 concentration, and after having reached stationary
phase the light intensity is increased to a second light intensity
and the CO.sub.2 concentration is increased to a second CO.sub.2
concentration.
[0080] In various embodiments of the seventeenth aspect for
identifying toxin producing photoautotrophic strains the method
further comprises the method step of determining the presence and
amount of toxins produced by the photoautotrophic strain.
[0081] In various embodiments of the seventeenth aspect for
identifying genetically transformable photoautotrophic strains the
method further comprises the method step of subjecting the
photoautotrophic strain to a transforming factor, conferring a
marker property, and detecting the presence of the marker property
in the strain.
[0082] In various embodiments of the seventeenth aspect the method
further comprises the further method step of determining the
photosynthetic activity of the photoautotrophic strain to be
tested.
[0083] In various embodiments of the seventeenth aspect for
identifying a photoautotrophic strain with a tolerance for at least
a first and a second growth condition selected from the growth
conditions (i) to (vi) from a plurality of different
photoautotrophic strains the method comprises culturing the
plurality of different photoautotrophic strains under a first
growth condition in method step (b1), identifying the
photoautotrophic strains tolerant to the first growth condition in
method step (c1) and thereafter culturing the photoautotrophic
strains identified in method step (c1) under a second growth
condition in a further step (b2), the second growth condition being
different from the first growth condition, identifying the
photoautotrophic strains tolerant to the second growth condition in
method step (c2). In an embodiment thereof the method further
comprises identifying additionally at least one desired property,
selected from a group consisting of high photosynthetic activity,
lack of ability to produce toxins and ability to be genetically
transformable, from the plurality of different photoautotrophic
strains, wherein the method comprises at least one further method
step (d) selected from a group of method steps consisting of (vii)
determining the photosynthetic activity of the photoautotrophic
strain, (viii) subjecting the photoautotrophic strain to a
transforming factor, conferring a marker property, and detecting
the presence of the marker property in the strain, and (ix)
determining the presence and amount of toxins produced by the
photoautotrophic strain, and identifying the photoautotrophic
strain having any of the abilities (vii) to (ix) In a further
method step (e), wherein the methods steps (d) and (e) can be
performed before or after the method steps (b1) and (c1) or (b2)
and (c2).
[0084] In various embodiments of the seventeenth aspect the
photoautotrophic strains to be tested are from a collection of
different photoautotrophic strains. In an embodiment thereof the
photoautotrophic strains are obtained from a publicly available
strain database.
[0085] In various embodiments of the seventeenth aspect the
photoautotrophic strains to be tested are preselected from a group
of strains known to be fast growing, dominant strains with high
photosynthetic activity, known to be able to produce mass
populations in nature. In an embodiment thereof the
photoautotrophic strains to be tested are Cyanobacteria or algae
selected from Synechocystis, Synechococcus, Spirulina, Arthrospira,
Nostoc, Anabaena, Trichodesmium, Leptolyngbya, Plectonema,
Myxosarcina, Pleurocapsa, Oscillatoria, Phormidium,
Pseudanabaena.
Common Claims
[0086] In an eighteenth aspect, the inventions provided herein
relating to a genetically modified, photoautotrophic ethanol
producing host cell further comprise the cell to be tolerant to at
least one growth condition selected from a group consisting of
ethanol tolerance, salt tolerance, above neutral pH tolerance,
mechanical stress tolerance, temperature tolerance and light
tolerance, and additionally having at least one desired property,
selected from a group consisting of high photosynthetic activity,
lack of ability to produce toxins and ability to be genetically
transformable, wherein the host cell is identified by a method
according to the seventeenth aspect.
[0087] In an nineteenth aspect, the aspects of the disclosure
provided herewith relating to a genetically modified,
photoautotrophic ethanol producing host cell further comprise a
host cell that is an aquatic organism. In an embodiment thereof,
the host cell is selected from a group consisting of algae,
protists, and bacteria. In a further embodiment thereof the host
cell is a cyanobacterium. In a further embodiment thereof the
cyanobacterium is selected from a group consisting of
Synechococcus, Synechocystis, and Phormidium.
[0088] In an twentieth aspect, a method of producing ethanol is
provided, the method steps including (A) providing and culturing
genetically modified host cells according to any aspect of the
disclosure provided herein in a growth medium under the exposure of
light and CO.sub.2, the host cell(s) accumulating ethanol while
being cultured, and (B) isolating the ethanol from the host cell(s)
and/or the growth medium. In an embodiment thereof in method step
(A) host cells are provided, which comprise a genetically modified
gene encoding at least one enzyme for the formation of ethanol
under the transcriptional control of an inducible promoter, which
can be induced by exposure to an exogenous stimulus, the method
step (A) further comprising (A1) culturing the host cells under the
absence of the exogenous stimulus, and thereafter (A2) providing
the exogenous stimulus, thereby inducing ethanol production. In a
further embodiment thereof the exogenic stimulus can be provided by
changing the environmental conditions of the host cells. In an even
further embodiment thereof the exogenous stimulus can be provided
by subjecting the host cells to a stimulus selected from a group
consisting of darkness, nutrient starvation, oxidative stress, salt
stress, heat shock, cold shock, stationary growth and light. In an
embodiment thereof the exogenous stimulus comprises nutrient
starvation, and no new growth medium is added to the host cell
culture in method step (A), the host cell culture thereby growing
into nutrient starvation when reaching stationary growth phase.
[0089] A further embodiment of the twentieth aspect comprises in
method step (A) host cells are provided, which comprise a
genetically modified gene encoding at least one enzyme for the
formation of ethanol under the transcriptional control of a
constitutive promoter, the method step (A) comprising culturing the
host cells and producing ethanol.
[0090] In further embodiment of the twentieth aspect method step
(A) further comprises the method step of: (A3) adding a substrate
to the growth medium, the substrate used by the at least one
overexpressed enzyme for ethanol production to produce ethanol. In
an embodiment thereof the substrate is acetaldehyde.
[0091] In further embodiment of the twentieth aspect the method
further comprising the additional method step (C) using the host
cells after having isolated the ethanol in method step (B) as a
substrate for a heterotrophic organism. In an embodiment thereof a
heterotrophic fermentative organism is used to produce ethanol.
[0092] In further embodiment of the twentieth aspect during method
step (A) the genetically modified host cells produces a first
metabolic intermediate and at least partially secrete the first
metabolic intermediate into the growth medium, and during method
step (A) a microorganism is added to the growth medium, the
microorganism converting the first metabolic intermediate into
ethanol.
[0093] In a twenty-first aspect an isolated nucleic acid molecule
suitable to effect a change in gene expression of a target genome
in a photoautotrophic cell is provided, the isolated nucleic acid
comprising (a) a first polynucleotide sequence comprising a
promoter sequence operationally linked to a coding sequence,
wherein the coding sequence alters metabolism of the cell having
the target genome, (b) a second polynucleotide sequence, wherein
the second polynucleotide sequence is homologous to the target
genome and is located 5' to the first polynucleotide sequence; and
(c) a third polynucleotide sequence, wherein the third
polynucleotide sequence is homologous to the target genome and is
located 3' to the first polynucleotide sequence, and wherein the
second and third polynucleotide sequence are each at least about
500 bases and up to 1.5 to 2 kilobases in length, the sequence of
which is obtained from a single gene selected from the group
consisting of ADP-glucose-pyrophosphorylase, glycogen synthase,
alanine dehydrogenase, lactate dehydrogenase, pyruvate water
dikinase, phosphotransacetylase, pyruvate dehydrogenase and acetate
kinase, and wherein the second and third polynucleotide sequence
facilitate homologous recombination into the target genome of the
photoautotrophic cell.
[0094] A twenty-second aspect is directed to method of producing
the genetically modified, photoautotrophic ethanol producing host
cell of aspects 1, 2 or 3 described herein, the steps of the method
comprising (a) providing a wild type host cell; (b) measuring the
level of biosynthesis of acetaldehyde, pyruvate, acetyl-CoA or
precursors thereof (c) introducing at least one first genetic
modification into the wild type cell changing the enzymatic
activity or affinity of at least one endogenous host cell enzyme;
(d) introducing at least one second genetic modification, different
from the first genetic modification, comprising at least one
overexpressed enzyme for the formation of ethanol; and (e)
measuring and identifying an enhanced level of biosynthesis of
acetaldehyde, pyruvate, acetyl-CoA or precursors thereof compared
to the respective wild type host cell. An embodiment thereof
wherein step (e) identifies an enhanced level of biosynthesis for
pyruvate or acetyl-CoA. In an embodiment thereof an enhanced level
of biosynthesis of pyruvate is identified. An embodiment thereof
wherein an enhanced level of biosynthesis of acetyl-CoA is
identified.
[0095] In a further embodiment of the twenty-second aspect the
second genetic modification of step (d) comprises an overexpressed
pyruvate decarboxylase enzyme and an overexpressed alcohol
dehydrogenase enzyme. Another embodiment thereof comprises in step
(d) the at least one overexpressed enzyme for the formation of
ethanol is alcohol dehydrogenase E.
[0096] An embodiment of the twenty-second aspect wherein the first
genetic modification of step (c) comprises the overexpression of at
least one endogenous host cell enzyme.
[0097] An embodiment of the twenty-second aspect wherein the first
genetic modification of step (c) comprises two genetic alterations,
the first genetic alteration comprising an insertion into or
deletion of an endogenous host cell enzyme and the second genetic
alteration comprising the introduction of a metabolic enzyme gene
sequence that is overexpressed.
BRIEF DESCRIPTION OF THE DRAWINGS
[0098] FIG. 1, comprising 1A, 1, 1C, 1D, 1E, and 1F illustrates
some relevant metabolic pathways
[0099] FIG. 2 illustrates possible pathways leading to ethanol
production.
[0100] FIG. 3 illustrates possible pathways leading to ethanol
production
[0101] FIG. 4a presents the amino acid sequence of a glycogen
synthase gene of Synechocystis sp. PCC 6803 that is encoded by the
gene sll0945 (glgA1). (SEQ ID NO:1)
[0102] FIG. 4B presents the amino acid sequence of a second
glycogen synthase of Synechocystis sp. PCC 6803 that is encoded by
the gene sll1393 (glgA2). (SEQ ID NO:2)
[0103] FIG. 4C presents a schematic representation of restriction
sites used in the cloning strategy for pUC19-glgA1-Cm.
[0104] FIG. 4D is a schematic representation of gene organization
for the plasmid pUC19-glgA1-Cm.
[0105] FIG. 4E presents the nucleotide sequence of the construct
pUC19-glgA1-Cm. (SEQ ID NO:3)
[0106] FIG. 4F presents a schematic representation of restriction
sites used in the cloning strategy for pUC19-glgA2-Kan.
[0107] FIG. 4G is a schematic representation of gene organization
for the plasmid pUC19-glgA2-Kan.
[0108] FIG. 4H presents the nucleotide sequence of the construct
pUC19-glgA2-Kan. (SEQ ID NO:4)
[0109] FIG. 5A presents the amino acid sequence of the open reading
frame sll1682, which encodes alanine dehydrogenase (EC 1.4.1.1)
(Genbank No BAA16790) of Synechocystis sp. strain PCC6803. (SEQ ID
NO:5)
[0110] FIG. 5B presents a schematic representation of gene
organization for the plasmid pGEM-T/ald-KManti.
[0111] FIG. 5C presents the nucleotide sequence of the insert of
construct pGEM-T/ald-KManti. (SEQ ID NO:6)
[0112] FIG. 5A presents the amino acid sequence of the open reading
frame slr1176, which encodes ADP-glucose pyrophosphorylase (EC
2.7.7.27) (Genbank No BAA18822) of Synechocystis sp. strain
PCC6803. (SEQ ID NO:7)
[0113] FIG. 6B presents a schematic representation of gene
organization for the plasmid pGEM-T/glgC-KManti.
[0114] FIG. 6C presents the nucleotide sequence of the insert of
construct pGEM-T/glgC-KManti. (SEQ ID NO:8)
[0115] FIG. 6D presents a schematic representation of gene
organization for the plasmid pDrive/glgC-CMantisense.
[0116] FIG. 6E presents the nucleotide sequence of the insert of
construct pDrive/glgC-CMantisense. (SEQ ID NO:9)
[0117] FIG. 7A presents the amino acid sequence of the open reading
frame slr0301 that encodes pyruvate water dikinase/PEP synthase (EC
2.7.9.2) (Genbank No BAA10668) of Synechocystis sp. strain PCC6803.
(SEQ ID NO:10)
[0118] FIG. 7B presents a schematic of gene organization for the
plasmid pGEM-T/ppsA-anti.
[0119] FIG. 7C presents the nucleotide sequence of the insert of
construct pGEM-T/ppsA-anti. (SEQ ID NO:11)
[0120] FIG. 8A presents the amino acid sequence of open reading
frame slr 1556 that encodes a putative lactate dehydrogense (EC
1.1.1.28), (annotated as 2-hydroxyaciddehydrogenase homolog)
(GenBank No. P74586) of Synechocystis sp. strain PCC6803. (SEQ ID
NO:12)
[0121] FIG. 8B presents a schematic representation of restriction
sites used in the cloning strategy for pBlue Idh-Kan-a.
[0122] FIG. 8C presents a schematic for the gene organization of
the plasmid pBlue Idh-Kan-a.
[0123] FIG. 8D presents the nucleotide sequence of the insert
contained in the construct pBlue Idh-Kan-a. (SEQ ID NO:13)
[0124] FIG. 9A presents the amino acid sequence of the open reading
frame sll 1299 that encodes a putative acetate kinase (EC 2.7.2.1)
(Genbank No. P73162). (SEQ ID NO:14)
[0125] FIG. 9B presents a schematic representation of restriction
sites used in the cloning strategy for pBlue-ack-Kan-b.
[0126] FIG. 9C presents a schematic for the gene organization of
the plasmid pBlue-ack-Kan-b.
[0127] FIG. 9D presents the nucleotide sequence of the insert of
construct pBlue-ack-Kan-b. (SEQ ID NO:15)
[0128] FIG. 10A presents the amino acid sequence of the open
reading frame slr2132 that encodes a phosphoacetyltransacetylase
(EC 2.3.1.8) (Genbank No. P75662) of Synechocystis sp. strain
PCC6803. (SEQ ID NO:16)
[0129] FIG. 10B presents a schematic representation of restriction
sites used in the cloning strategy for pUC pta-Cm.
[0130] FIG. 10C presents a schematic for the gene organization of
the plasmid pUC pta-Cm.
[0131] FIG. 10D presents the nucleotide sequence of the insert of
construct pUC pta-Cm. (SEQ ID NO:17)
[0132] FIG. 11A presents the amino acid sequence of open reading
frame slr1830 that encodes poly(3-hydroxyalkanoate) synthase
[EC:2.3.1.](Genbank No BAA17430) of Synechocystis sp. strain
PCC6803. (SEQ ID NO:18)
[0133] FIG. 11B presents a schematic representation of gene
structure for the plasmid as plC20H/AphaC-KM.
[0134] FIG. 11C presents the nucleotide sequence of the insert of
construct as plC20H/AphaC-KM. (SEQ ID NO:19)
[0135] FIG. 11D presents the amino acid sequence of ORF a114645 for
PCC 7120 (SEQ ID NO:20).
[0136] FIG. 11E presents a schematic representation of restriction
sites and gene organization for the PCC 7120 glgC knockout.
[0137] FIG. 11F presents the sequence of the insert of pRL271 agp
(a114645)::C.K3-PpetE-pdc-adhII (SEQ ID NO:21).
[0138] FIG. 11G presents the amino acid sequence of
Glucose-1-phosphate adenylytransferase
(ADP-glucose-pyrophosphorylase, agp, glgC), EC 2.7.7.27, of
Anabaena variabilis ATCC29314. (SEQ ID NO:22)
[0139] FIG. 12A presents the amino acid sequence of open reading
frame sll1721 that encodes the .beta.-subunit of the E1 component
of the pyruvate dehydrogenase (EC 1.2.4.1) (Genbank No BAA17445) of
Synechocystis sp. strain PCC6803. (SEQ ID NO:23)
[0140] FIG. 12B presents a schematic of gene organization for the
plasmid pSK9/pdhBanti.
[0141] FIG. 12C presents the nucleotide sequence of the insert for
pSK9/pdhBanti. (SEQ ID NO:24)
[0142] FIG. 12D presents a schematic representation of gene
organization for the plasmid pSK9/pdhB.
[0143] FIG. 12E presents the nucleotide sequence of the insert for
the construct pSK9/pdhB. (SEQ ID NO:25)
[0144] FIG. 12F presents a schematic of gene organization for the
plasmid pGEM-T/ApdhB-KMantisense.
[0145] FIG. 12G presents the nucleotide sequence of the insert of
construct pGEM-T/ApdhB-KMantisense. (SEQ ID NO:26)
[0146] FIG. 13A presents a schematic representation of the cloning
vector pGEM-T.
[0147] FIG. 13B presents the nucleotide sequence of vector pGEM-T.
(SEQ ID NO:27)
[0148] FIG. 14A presents a schematic representation of the cloning
vector pDrive.
[0149] FIG. 14B presents the nucleotide sequence of vector pDrive.
(SEQ ID NO:28)
[0150] FIG. 15A presents a schematic representation of the cloning
vector pBluescript II SK (+).
[0151] FIG. 15B presents the nucleotide sequence of the vector
pBluescript II SK (+). (SEQ ID NO:29)
[0152] FIG. 16A presents a schematic representation of the cloning
vector pUC19.
[0153] FIG. 168 presents the nucleotide sequence of the vector
pUC19. (SEQ ID NO:30)
[0154] FIG. 17A presents a schematic representation of genes
organized in the vector pSK9.
[0155] FIG. 17B presents the nucleotide sequence of the vector
pSK9. (SEQ ID NO:31)
[0156] FIG. 18A presents the amino acid sequence of open reading
frame slr0721 that encodes malic enzyme 1 (EC 1.1.1.38) (Genbank No
P72661) of Synechocystis sp. strain PCC6803. (SEQ ID NO:32)
[0157] FIG. 18B presents a schematic of genes organized in the
construct of Synechocystis sp. strain PCC6803.
[0158] FIG. 18C presents the nucleotide sequence of the insert of
construct pSK9/me-long. (SEQ ID NO:33)
[0159] FIG. 19A presents the amino acid sequence of open reading
frame sll0891 that encodes malate dehydrogenase (EC 1.1.1.37)
(Genbank No Q55383) of Synechocystis sp. strain PCC6803. (SEQ ID
NO:34)
[0160] FIG. 19B presents a schematic representation of gene
organization for the construct pSK9-mdh.
[0161] FIG. 19C presents the nucleotide sequence of the insert of
construct pSK9-mdh. (SEQ ID NO:35)
[0162] FIG. 19D presents a schematic representation of gene
organization for the construct pSK9/me-mdh.
[0163] FIG. 19E presents the nucleotide sequence of the insert of
construct pSK9/me-mdh. (SEQ ID NO:36)
[0164] FIG. 20A presents the amino acid sequence of open reading
frame sll0587 that encodes a pyruvate kinase 1 (EC 2.7.1.40 (PK1))
(Genbank No 055863) of Synechocystis sp. strain PCC6803. (SEQ ID
NO:37)
[0165] FIG. 20B presents a schematic representation of gene
organization for the construct pVZ321-pyk1.
[0166] FIG. 20C presents the nucleotide sequence of the insert of
construct pVZ321-pyk1. (SEQ ID NO:38)
[0167] FIG. 20D presents a schematic representation of gene
organization for the construct pVZ321 PpetJ pyk1.
[0168] FIG. 20E presents the nucleotide sequence of the insert
found in construct pVZ321 PpetJ pyk1. (SEQ ID NO:39)
[0169] FIG. 21A presents the amino acid sequence of open reading
frame sll1275 that encodes pyruvate kinase 2 (EC 2.7.1.40 (PK2))
(Genbank No P73534) of Synechocystis sp. strain PCC6803. (SEQ ID
NO:40)
[0170] FIG. 21B presents a schematic representation of gene
organization for the construct pVZ321pyk2.
[0171] FIG. 21C presents the nucleotide sequence of the insert of
pVZ321pyk2. (SEQ ID NO:41)
[0172] FIG. 21C presents a schematic representation of gene
organization for the construct. pVZ321 PpetJ pyk2.
[0173] FIG. 21E presents the nucleotide sequence for the insert of
the construct pVZ321 PpetJ pyk2. (SEQ ID NO:42)
[0174] FIG. 22A presents a schematic representation of the gene
organization for the p67 insert.
[0175] FIG. 22B presents the amino acid sequence of pyruvate kinase
I (E. coli K12). (SEQ ID NO:43)
[0176] FIG. 22C presents the amino acid sequence of enolase
(Zymomonas mobilis). (SEQ ID NO:44)
[0177] FIG. 22D presents the amino acid sequence of
phosphoglycerate mutase (Zymomonas mobilis). (SEQ ID NO:45)
[0178] FIG. 22E presents the nucleotide sequence of the insert of
plasmid #67. (SEQ ID NO:46)
[0179] FIG. 22F presents a schematic representation of gene
organization for the construct pVZ321-p67.
[0180] FIG. 22G presents a schematic representation of gene
organization for construct pVZ322-p67.
[0181] FIG. 23A presents the amino acid sequence of open reading
frame slr0752 that encodes the enolase (eno, 2-phosphoglycerate
dehydratase) (EC 4.2.1.11) (Genbank No. BAA18749) of Synechocystis
sp. strain PCC6803. (SEQ ID NO:47)
[0182] FIG. 23B presents a schematic representation of gene
organization for construct pVZ321 PpetJ eno.
[0183] FIG. 23C presents the nucleotide sequence of the insert of
construct pVZ321-PpetJ-eno. (SEQ ID NO:48)
[0184] FIG. 24A presents the amino acid sequence of open reading
frame slr1124 that encodes the phosphoglycerate mutase (pgm or
gpmB) (EC 5.4.2.1) (Genbank No. BAA16651) of Synechocystis sp.
strain PCC6803. (SEQ ID NO:49)
[0185] FIG. 24B presents a schematic representation of the gene
organization of construct pVZ321-PpetJ-pgm.
[0186] FIG. 24C presents the nucleotide sequence of the insert of
construct pVZ321-PpetJ-pgm. (SEQ ID NO:50)
[0187] FIG. 24D presents a schematic representation of gene
organization for the construct pVZ322-PpetJ-pyk1-eno-pgm.
[0188] FIG. 24E presents a schematic representation of gene
organization for the construct pVZ322-PpetJ-pyk2-eno-pgm.
[0189] FIG. 24F presents the nucleotide sequence of the insert of
construct pVZ322-PpetJ-pyk1-eno-pgm. (SEQ ID NO:51)
[0190] FIG. 24G presents the nucleotide sequence of the insert of
Construct pVZ322-PpetJ-pyk2-eno-pgm. (SEQ 10 NO:52)
[0191] FIG. 25A presents the amino acid sequence for open reading
frame slr0453 that encodes the probable phosphoketolase (phk), (EC
4.1.2.-) (Genbank No. P74690) of Synechocystis sp. strain PCC6803.
(SEQ ID NO:53)
[0192] FIG. 25B presents a schematic representation of the gene
organization for the construct pVZ322 PpetJ-phk.
[0193] FIG. 25C presents the nucleotide sequence of the insert of
the construct pVZ322 PpetJ-phk (SEQ ID NO:54)
[0194] FIG. 26A presents the amino acid sequence of open reading
frame slr2132 that encodes a phosphoacetyltransacetylase (pta) (EC
2.3.1.8) (Genbank No. P73662) of Synechocystis sp. strain PCC6803.
(SEQ ID NO:55)
[0195] FIG. 26B presents a schematic representation of gene
organization in the construct pVZ322 PpetJ pta.
[0196] FIG. 26C presents the nucleotide sequence of the insert of
construct pVZ322 PpetJ pta. (SEQ ID NO:56)
[0197] FIG. 26D presents a schematic representation of gene
organization in construct pVZ322 PpetJ phK pta.
[0198] FIG. 26E presents the nucleotide sequence of the insert of
construct pVZ322 PpetJ phK pta. (SEQ ID NO:57)
[0199] FIG. 27A presents the amino acid sequence of open reading
frame slr0091 encodes a aldehyde dehydrogenase (aldh) (EC 1.2.1.3)
(Genbank No. BAA10564) a Synechocystis sp. strain PCC6803. (SEQ ID
NO:58)
[0200] FIG. 27B is a schematic representation of gene organization
in construct pVZ 322 PpetJ aldh.
[0201] FIG. 27C presents the nucleotide sequence of the insert of
construct pVZ 322 PpetJ aldh. (SEQ ID NO:59)
[0202] FIG. 28A presents the amino acid sequence of open reading
frame sll0920 that encodes phosphoenolpyruvate carboxylase (EC
4.1.1.31) (Genbank No. BAA18393) of Synechocystis sp. strain
PCC6803. (SEQ ID NO:60)
[0203] FIG. 28B is a schematic representation of gene organization
in pVZ321-PpetJ-ppc.
[0204] FIG. 28C presents the nucleotide sequence of the insert of
construct pVZ321-PpetJ-ppc. (SEQ ID NO:61)
[0205] FIG. 28D presents the nucleotide sequence of primer
SynRbc-BglII-fw (SEQ ID NO:62).
[0206] FIG. 28E presents the nucleotide sequence of primer
SynRbc-PstI-rev (SEQ ID NO:63).
[0207] FIG. 28F presents the nucleotide sequence of primer
SynRbc-SacI-fw (SEQ ID NO:64).
[0208] FIG. 28G presents the nucleotide sequence of the rbcLXS
operon of Synechocystis PCC 6803 (SEQ ID NO:65).
[0209] FIG. 28H presents the amino acid sequence of the rbcL large
subunit of Synechocystis PCC 6803 (SEQ ID NO:66).
[0210] FIG. 28I presents the amino acid sequence of the rbcX
Rubisco chaperonin protein of Synechocystis PCC 6803 (SEQ ID
NO:67).
[0211] FIG. 28J presents the amino acid sequence of the ribulose
bisphosphate carboxylase small subunit (rbcS) of Synechocystis PCC
6803 (SEQ ID NO:68).
[0212] FIG. 28K is a schematic presentation of gene organization
for plasmid pVZ321b-Prbc-SynRbcLXS.
[0213] FIG. 29A is a schematic representation of the structure of
the vector pSK9.
[0214] FIG. 29B presents the nucleotide sequence of the vector
pSK9. (SEQ ID NO:69)
[0215] FIG. 30A is a schematic representation of gene organization
in construct pVZ321. (GenBank No. AF100176).
[0216] FIG. 30B presents the nucleotide sequence of the pVZ321
vector. (SEQ ID NO:70)
[0217] FIG. 31A is a schematic representation of gene organization
for construct pVZ322.
[0218] FIG. 31B presents the nucleotide sequence of the pVZ322
vector (SEQ ID NO:71).
[0219] FIG. 32A is a schematic representation of gene organization
of construct plC PpetJ.
[0220] FIG. 32B presents the nucleotide sequence of the construct
plC PpetJ. (SEQ ID NO:72)
[0221] FIG. 32C is a graphic presentation demonstrating growth
properties and extracellular pyruvate levels of the
.DELTA.glgA1/.DELTA.glgA2 double mutant (M8) under nitrogen replete
and nitrogen starved conditions.
[0222] FIG. 32D is a graphic presentation of pyruvate levels in
wildtype and mutant (.DELTA.glgA/.DELTA.glgA2) media/cells as
determined enzymatically and by ion chromatography.
[0223] FIG. 32E is a graphic presentation of the conductimetric
detection of pyruvate in methanol extracts (snapshot) of cultures
of wildtype and a glycogen synthase deficient mutant after 24 h
under N-deficient conditions.
[0224] FIG. 32F is a graphic depiction showing the that the
pyruvate concentration in the growth medium is higher for the M8
mutant without Adh and Pdc enzymes than for the M8 mutant including
both ethanol forming enzymes under the conditions of nitrogen
starvation.
[0225] FIG. 32G is a graphic depiction of the ethanol concentration
determined in the growth medium for the M8 mutant with the Adh and
Pdc enzymes under the conditions of nitrogen starvation and without
nitrogen starvation.
[0226] FIG. 32H is a graphic depiction of ethanol generation in
glycogen deficient Synechocystis pVC mutants with ZmPDC and ZmADHII
under the control of the Iron-dependent isiA promoter.
[0227] FIG. 32I is a graphic presentation of ethanol production in
wildtype, ack and ack/pta double mutant cells.
[0228] FIG. 32J is a graphic presentation of ethanol production in
wildtype, ack and ack/pta double mutant cells when normalized for
optical density.
[0229] FIG. 32K is a graphic presentation of demonstrating that
pVZ321b-Prbc-SynRbcLXS grows as fast as the Synechocystis wild type
and shows no phenotypical differences except for the chlorophyll
content that is reduced by 20-30% compared to wild type.
[0230] FIG. 32L is a graphic presentation of demonstrating the
growth parameter (OD at 750 nm and Chlorophyll content) of
Synechocystis wild type and a mutant that over-express the
endogenous RuBisCO operon.
[0231] FIG. 32M is a graphic presentation of ethanol production for
the mutant Synechocystis PCC6803 harboring the
pSK10-PisiA-PDC/ADHII plasmid and the mutant additionally
containing the vector pVZ321b-Prbc-SynRbc.
[0232] FIG. 32N is a graphic presentation of ethanol production
normalized to the OD.sub.750 for the mutant Synechocystis PCC6803
harboring the pSK10-PisiA-PDC/ADHII plasmid and the mutant
additionally containing the vector pVZ321b-Prbc-SynRbc
[0233] FIG. 33A is a schematic representation of gene organization
for the construct pVZ-PisiA-pdc/adh.
[0234] FIG. 33B is a schematic representation of gene organization
for the construct pVZ-PntcA-pdc/adh.
[0235] FIG. 33C is a schematic representation of gene organization
for the construct pVZ-PnblA-pdc/adh.
[0236] FIG. 33D presents the nucleotide sequence of the insert of
the vector pCB4-LR(TF)pa that encodes Z. mobilis adhII and pdc
genes. (SEQ ID NO:73)
[0237] FIG. 33E is a schematic representation of restriction sites
present in the Z. mobilis adhII and pdc fragment.
[0238] FIG. 33F presents the amino acid sequence of Z. mobilis pdc
protein. (SEQ ID NO:74)
[0239] FIG. 33G presents the amino acid sequence of the Z. mobilis
AdhII protein. (SEQ ID NO:75)
[0240] FIG. 34A presents the nucleotide sequence for the isiA
promoter (Synechocystis sp. PCC6803) (isiA gene: sll0247), which is
induced under iron starvation conditions. (SEQ ID NO:76)
[0241] FIG. 34B presents the nucleotide sequence for the nblA
promoter (Synechocystis sp. PCC6803) (nblA gene: ss10452), which is
induced under nitrogen starvation conditions. (SEQ ID NO:77)
[0242] FIG. 34C presents the nucleotide sequence for the ntcA
promoter (Synechocystis sp. PCC6803) (ntcA gene: sll1423), which is
induced under nitrogen starvation. (SEQ ID NO:78)
[0243] FIG. 35A presents the nucleotide sequence of the cloning
vector pVZ321b, a derivative of pVZ321. (SEQ ID NO:79)
[0244] FIG. 35B is a schematic representation of gene organization
for the cloning vector pVZ321b.
[0245] FIG. 36A presents the nucleotide sequence for the petJ
promoter (Synechocystis sp. PCC 6803) (petJ gene: sll1796)
(encoding for cytochrome c553), which is induced under copper
starvation conditions. (SEQ ID NO:80)
[0246] FIG. 36B is a schematic representation of gene organization
for the construct pVZ321b-PpetJ-PDC-ADHII.
[0247] FIG. 36C presents the nucleotide sequence of the sigB
promoter (Synechocystis sp. PCC 6803) (sigB gene: sll0306)
(encoding for RNA polymerase group 2 sigma factor), which is
induced after heat shock, in stationary growth phase/nitrogen
starvation and darkness. (SEQ ID NO:81)
[0248] FIG. 36D is a schematic representation of gene organization
for the construct pVZ321b-PsigB-PDC-ADHII.
[0249] FIG. 36E presents the nucleotide sequence of the htpG
promoter (Synechocystis sp. PCC 6803) (htpG gene: sll0430)
(encoding for heat shock protein 90, molecular chaperone), which is
induced after heat shock. (SEQ ID NO:82)
[0250] FIG. 36F is a schematic representation of gene organization
for the construct pVZ321b-PhtpG-PDC-ADHII.
[0251] FIG. 36G presents the nucleotide sequence of the IrtA
promoter (Synechocystis sp. PCC 6803) (1rtA gene: sll0947)
(encoding the light repressed protein A homology, which is induced
after light to dark transition. (SEQ ID NO:83)
[0252] FIG. 36H is a schematic representation of gene organization
in the construct pVZ321b-PlrtA-PDC-ADHII.
[0253] FIG. 36I presents the nucleotide sequence of the psbA2
promoter (Synechocystis sp. PCC 6803) (psbA2 gene: slr1311)
(encoding the photosystem II D1 protein), which is induced after
dark to light transition. (SEQ ID NO:24)
[0254] FIG. 36J is a schematic representation of gene organization
for the construct pVZ31b-PpsbA2-PDC-ADHII.
[0255] FIG. 36K presents the nucleotide sequence of the rbcL
promoter (Synechocystis sp. PCC 6803) (rbcL gene: slr0009)
(encoding the ribulose biphosphate carboxylase/oxygenase large
subunit), which is a constitutive and strong promoter under
continuous light conditions. (SEQ ID NO:85)
[0256] FIG. 36L is a schematic representation of gene organization
for the construct pVZ321b-PrbcL-PDC-ADHII.
[0257] FIG. 36M presents the nucleotide sequence for the psaA
promoter (Synechocystis sp. PCC6803) (PsaA gene: slr1834) (encoding
P700 apoprotein subunit Ia), which is induced under low white light
and orange light, low expression level under high light and red
light, and repressed in darkness. (SEQ ID NO:86)
[0258] FIG. 36N is a schematic representation of the gene
organization of the construct pVZ321b-PpsaA-PDC-ADHII.
[0259] FIG. 36O presents the nucleotide sequence of the ggpS
promoter (Synechocystis sp. PCC6803) (ggpS gene: sll1566) (encoding
glucosylglycerolphosphate synthase), which is induced after salt
stress. (SEQ ID NO:87)
[0260] FIG. 36P is a schematic representation of the gene
organization of the construct pVZ321b-PggpS-PDC-ADHII.
[0261] FIG. 36Q presents the nucleotide sequence of the nirA
promoter (Synechocystis sp. PCC6803) (nirA gene: slr0898) (encoding
ferredoxin-nitrite reductase), which is induced after transition
from ammonia to nitrate. (SEQ ID NO:88)
[0262] FIG. 36R is a schematic representation of the gene
organization of the construct pVZ321c-PnirA-PDC-ADHII.
[0263] FIG. 36S presents the nucleotide sequence of the petE
promoter (Anabaena sp. PCC7120) (petE gene: a110258) (encoding
plastocyanin precursor), which is induced at elevated copper
concentrations. (SEQ ID NO:89)
[0264] FIG. 36T is a schematic representation of gene organization
for the construct pVZ321c-PpetE-PDC-ADHII.
[0265] FIG. 36U presents the nucleotide sequence of the hspA
promoter (Synechocystis sp. PCC6803) (hspA gene: sll1514) 16.6 kDa
small heat shock protein, molecular chaperone multi-stress
responsive promoter (heat, cold, salt and oxidative stress). (SEQ
ID NO:90)
[0266] FIG. 36V is s schematic representation of gene organization
for the construct pVZ321c-PhspA-PDC-ADHII.
[0267] FIG. 36W presents the nucleotide sequence for the hliB
promoter (Synechocystis sp. PCC6803) (hliB gene: ssr2595) high
light-inducible polypeptide HliB, CAB/ELIP/HLIP superfamily)
(multi-stress responsible promoter (heat, cold, salt and oxidative
stress). (SEQ ID NO:91)
[0268] FIG. 36X is a schematic representation of gene organization
of the construct pVZ321-PhliB-PDC-ADHII.
[0269] FIG. 36Y presents the nucleotide sequence of the clpB1
promoter (Synechocystis sp. PCC6803) (clpB1 gene: slr1641)
ATP-dependent Clp protease, Hsp 100, ATP-binding subunit ClpB
multi-stress responsible promoter (heat, cold, salt and oxidative
stress). (SEQ ID NO:92)
[0270] FIG. 36Z is a schematic representation of the gene
organization for the construct pVZ321c-PclpB1-PDC-ADHII.
[0271] FIG. 37A presents the nucleotide sequence of the adhA gene
from Zymomonas mobilis ZM4. (SEQ ID NO:93)
[0272] FIG. 37B presents the amino acid sequence for the ZmAdhI
protein sequence (AAV89860). (SEQ ID NO:4)
[0273] FIG. 37C is a schematic presentation of the gene
organization for construct pVZ321b-PisiA-PDC-ADHI.
[0274] FIG. 37D is a schematic presentation of the gene
organization for construct pVZ321b-PntcA-PDC-ZmADHI.
[0275] FIG. 37E is a schematic presentation of the gene
organization for construct pVZ321b-PnblA PDC-ZmADHI.
[0276] FIG. 38A presents the nucleotide sequence of SynAdh, the adh
gene (slr1192) of Synechocystis sp. PCC 6803. (SEQ ID NO:95)
[0277] FIG. 38B presents the amino acid sequence of SynAdh (protein
sequence BAA18840) of Synechocystis sp. PCC 6803. (SEQ ID
NO:96)
[0278] FIG. 38C is a schematic representation of the gene
organization for construct pVZ321b-PisiA-PDC-SynADH.
[0279] FIG. 38D is a schematic representation of the gene
organization for construct pVZ321b-PntcA-PDC-SynADH.
[0280] FIG. 38E is a schematic representation of the gene
organization for construct pVZ321b-PnblA-PDC-SynADH.
[0281] FIG. 39A presents the nucleotide sequence of EcAdhE, the
AdhE gene from E. coli K12. (SEQ ID NO:97)
[0282] FIG. 39B presents the amino acid sequence of EcAdhE (protein
sequence NP 415757). (SEQ ID NO:98)
[0283] FIG. 39C is a schematic representation of the gene
organization for construct pVZ321b-PisiA-PDC-EcAdhE.
[0284] FIG. 39D is a schematic representation of the gene
organization for construct pVZ321b-PntcA-PDC-EcAdhE.
[0285] FIG. 39E is a schematic representation of the gene
organization for construct pVZ321b-PnblA-PDC-EcAdhE.
[0286] FIG. 40A presents the nucleotide sequence of ThAdhE, the adh
E gene (tlr0227) from Thermosynechococcus elongatus BP-1. (SEQ ID
NO:99)
[0287] FIG. 40B presents the amino acid sequence of ThAdhE (protein
sequence BAC07780). (SEQ ID NO:100)
[0288] FIG. 40C is a schematic representation of the gene
organization for the construct pVZ321b-PisiA-ThAdhE.
[0289] FIG. 40D is a schematic representation of the gene
organization for the construct pVZ321b-PntcA-ThAdhE.
[0290] FIG. 40E is a schematic representation of the gene
organization for the construct pVZ321b-PnblA-ThAdhE.
[0291] FIG. 41A presents the nucleotide sequence of ZpPdcpdc gene
from Zymobacter palmae ATCC 51623 (SEQ ID NO:101)
[0292] FIG. 41B presents the amino acid sequence of ZpPdc (protein
sequence AAM49566). (SEQ ID NO:102)
[0293] FIG. 42A presents the nucleotide sequence of pSK10 cloning
vector (derivate of pSK9 [V. V. Zinchenko, Moscow, Russia;
unpublished]). (SEQ ID NO:103)
[0294] FIG. 42B is a schematic representation of the gene
organization for the plasmid pSK10.
[0295] FIG. 42C is a schematic representation of the gene
organization of the construct pSK10-PisiA-PDC-ADHII.
[0296] FIG. 42D is a schematic representation of the gene
organization of the construct pSK10-PnblA-PDC-ADHII.
[0297] FIG. 42E is a schematic representation of the gene
organization of the construct pSK10-PntcA-PDC-ADHII.
[0298] FIG. 42F is a schematic representation of the gene
organization of the construct pSK10-PisiA-PDC-ADHI.
[0299] FIG. 42G is a schematic representation of the gene
organization of the construct pSK1-PnblA-PDC-ADHI.
[0300] FIG. 42H is a schematic representation of the gene
organization of the construct pSK10-PntcA-PDC-ADHI.
[0301] FIG. 42I is a schematic representation of the gene
organization of the construct pSK10-PisiA-PDC-SynADH.
[0302] FIG. 42J is a schematic representation of the gene
organization of the construct pSK11-PnblA-PDC-SynADH.
[0303] FIG. 42K is a schematic representation of the gene
organization of the construct pSK10-PntcA-PDC-SynADH.
[0304] FIG. 42L is a schematic representation of the gene
organization of the construct pSK10-PisiA-PDC-EcAdhE.
[0305] FIG. 42M is a schematic representation of the gene
organization of the construct pSK10-PnblA-PDC-EcAdhE.
[0306] FIG. 42N is a schematic representation of the gene
organization of the construct pSK10-PntcA-PDC-EcAdhE.
[0307] FIG. 42O is a schematic representation of the gene
organization of the construct pSK10-PisiA-PDC-ThAdhE.
[0308] FIG. 42P is a schematic representation of the gene
organization of the construct pSK10-PnblA-PDC-ThAdhE.
[0309] FIG. 42Q is a schematic representation of the gene
organization of the construct pSK10-PntcA-PDC-ThAdhE.
[0310] FIG. 42R presents the nucleotide sequence of the crhC
promoter (Anabaena sp. PCC7120) (crhC gene: alr4718, RNA helicase
crhC cold shock inducible) (SEQ ID NO:104).
[0311] FIG. 42S presents the nucleotide sequence of the petE
promoter (Anabaena sp. PCC7120) petE gene: a110258, plastocyanin
precursor (petE) induced by addition of Cu. (SEQ ID NO:105)
[0312] FIG. 42T presents the gene organization of plasmid
pRL1049-PpetE-PDC-ADHII.
[0313] FIG. 42U presents the nucleotide sequence of plasmid
pRL1049-PpetE-PDC-ADHII (SEQ ID NO:106).
[0314] FIG. 42V depicts the gene organization of plasmid
pRL593-PisiA-PDC-ADHII.
[0315] FIG. 42W presents the nucleotide sequence of plasmid
pRL593-PisiA-PDC-ADHII (SEQ ID NO:107).
[0316] FIG. 42X is a graphic depiction of ethanol production rate
in Anabaena PCC7120 harboring pRL593-PisiA-PDC-ADHII following
induction by iron starvation was measured in BG11 medium (+N) and
in medium lacking combined nitrogen(-N) in day (12 h)/night (12 h)
cycle.
[0317] FIG. 42Y is a graphic depiction of ethanol production rate
in Anabaena PCC7120 harboring pRL593-PisiA-PDC-ADHII following
induction by iron starvation was measured in BG11 medium (+N) and
in medium lacking combined nitrogen(-N) in day (12 h)/night (12 h)
cycle, wherein values are normalized for optical density.
[0318] FIG. 43A is a photographic depiction of a Western Blot that
was used to quantify the induction rate of the used promoters by
determining the relative abundance of the Z. mobilis ADHII and PDC
enzymes expressed in Synechocystis with and without nutrient
starvation.
[0319] FIG. 43B is a photograph of a Western Blot that was used to
determine the relative abundance of the Z. mobilis ADHII and PDC
enzymes expressed in Synechocystis with and without nutrient
starvation.
[0320] FIG. 44A is a graphic representation of ethanol production
rates of genetically modified photoautotrophic host cells
containing Zymomonas mobilis PDC and ADHII as a second genetic
modification.
[0321] FIG. 44B is a graphic representation of ethanol production
in Synechocystis pVZ mutants having ZmPdC and ZmADHII under the
control of isiA, and iron-dependent promoter.
[0322] FIG. 44C is a graphic presentation of ethanol production in
glycogen deficient Synechocystis pVZ mutants having ZmPdc and
ZmAdhII under the control of isiA, an iron-dependent promoter.
[0323] FIG. 44D is a graphic presentation of ethanol production in
Synechocystis pVZ mutants having ZmPdc and SynAdh under the control
of rbcLS, a constitutive promoter.
[0324] FIG. 45 is a graphic presentation of ethanol production in
Synechocystis expressing different 3 variants of E. coli AdhE
compared to wild-type.
[0325] FIG. 46A is a graphic representation of growth over time for
the captioned mutant strains.
[0326] FIG. 46B is a graphic representation of ethanol production
over time (% v/v) for the captioned mutant strains.
[0327] FIG. 46C is a graphic representation of ethanol production
per growth for the captioned mutant strains.
[0328] FIG. 46D is a graphic representation of measurements on
outgas samples of Synechocystis mutants that express ZmPdc/ZmAdhI
(dashed line), ZmPdc/ZmAdhII (solid line) and ZmPdc/SynAdh (dotted
line) analyzed by gas chromatography. The grey arrow indicates the
acetaldehyde, and the black arrow indicates the ethanol peak.
[0329] FIG. 46E is a graphic depiction of acetaldehyde production
after addition of ethanol in different concentrations. Wild type
and ethanol producing transgenic cells are presented.
[0330] FIG. 46F is a graphic depiction of the pH-dependency of
acetaldehyde reduction by crude extracts containing the
Synechocystis Adh.
[0331] FIG. 46G is a graphic depiction summarizing the acetaldehyde
reduction rates at different cosubstrate concentrations.
Measurements were performed at pH 6.1
[0332] FIG. 46H is a graphic depiction of Lineweaver-Burk plots,
which depict the reciprocal of the rate of acetaldehyde reduction
versus the reciprocal of the concentration of NADH (squares) or
NADPH (rhombi), respectively. K.sub.m and v.sub.max values are
discussed in the text.
[0333] FIG. 46-I is a photographic depiction of SDS/PAGE analysis
of recombinantly expressed SynAdh showing that SynAdh was enriched,
but not purified to homogeneity.
[0334] FIG. 47A presents a phylogenetic analysis examining
different zinc binding ADH proteins.
[0335] FIG. 47B presents in tabular form all genes identified by
the Zn-binding SynAdh clade.
[0336] FIG. 47C presents the amino acid sequence of a
zinc-containing alcohol dehydrogenase family protein of
Synechocystis sp. PCC 6803, identified by Genbank Accession No. NP
443028.1. (SEQ ID NO:108)
[0337] FIG. 47D presents the amino acid sequence of a
zinc-containing alcohol dehydrogenase family protein of
Oceanobacter sp. RED65, identified by Genbank Accession No.
ZP_-01306627.1. (SEQ ID NO:109)
[0338] FIG. 47E presents the amino acid sequence of an alcohol
dehydrogenase, zinc-binding protein of Limnobacter sp. MED105,
identified by Genbank Accession No. ZP_-01914609.1 (SEQ ID
NO:110)
[0339] FIG. 47F presents the amino acid sequence of an alcohol
dehydrogenase GroES-like protein of Psychrobacter cryohalolentis
KS, identified by Genbank Accession No. YP_-581659.1. (SEQ ID
NO:111)
[0340] FIG. 47G presents the amino acid sequence of an alcohol
dehydrogenase GroES-like domain family of Verrucomicrobiae
bacterium DG1235. Identified by Genbank Accession No. EDY84203.1.
(SEQ ID NO:112)
[0341] FIG. 47H presents the amino acid sequence of a
zinc-containing alcohol dehydrogenase family protein of
Saccharophagus degradans 2-40, identified by Genbank
Accession No. YP_-529423.1. (SEQ ID NO:113)
[0342] FIG. 47I presents the amino acid sequence of a
zinc-containing alcohol dehydrogenase family protein of Alteromonas
macleodii `Deep ecotype`, Identified by Genbank Accession No.
YP_-002126870.1. (SEQ ID NO:114)
[0343] FIG. 47J presents the amino acid sequence of a
zinc-containing alcohol dehydrogenase family protein of
Acaryochloris marina MBIC11017, identified by Genbank Accession No.
YP_-001519107.1 (SEQ ID NO:115)
[0344] FIG. 47K presents the amino acid sequence of an alcohol
dehydrogenase GroES domain protein of Cyanothece sp. PCC 7424,
identified by Genbank Accession No. YP_-002380432.1. (SEQ ID
NO:116)
[0345] FIG. 47L presents the amino acid sequence of an alcohol
dehydrogenase GroES domain protein of Cyanothece sp. PCC 7424,
identified by Genbank Accession No. ZP_-02976085.1. (SEQ ID
NO:117)
[0346] FIG. 47M presents the amino acid sequence of an alcohol
dehydrogenase GroES domain protein of Cyanothece sp. PCC 7822,
identified by Genbank Accession No. ZP_-03154326.1 (SEQ ID
NO:118)
[0347] FIG. 47N presents the amino acid sequence of an alcohol
dehydrogenase GroES domain protein of Cyanothece sp. PCC 8801,
identified by Genbank Accession No. YP_-002371662.1. (SEQ ID
NO:119)
[0348] FIG. 47O presents the amino acid sequence of an alcohol
dehydrogenase GroES domain protein of Cyanothece sp. PCC 8801,
identified by Genbank Accession No. ZP_-02941996.1 (SEQ ID
NO:120)
[0349] FIG. 47P presents the amino acid sequence of an alcohol
dehydrogenase GroES domain protein of Cyanothece sp. PCC 8802,
identified by Genbank Accession No. ZP_-03143898.1. (SEQ ID
NO:121)
[0350] FIG. 47Q presents the amino acid sequence of an alcohol
dehydrogenase GroES-like domain family of Microcoleus chtonoplastes
PCC 7420, identified by Genbank Accession No. EDX77810.1. (SEQ ID
NO:122)
[0351] FIG. 47R presents the amino acid sequence of an
uncharacterized zinc-type alcohol dehydrogenase-like protein of
Microcystis aeruginosa NIES-843, identified by Genbank Accession
No. YP_-001659961.1. (SEQ ID NO:123)
[0352] FIG. 47S presents the amino acid sequence of an unnamed
protein product of Microcystis aeruginosa PCC 7806, identified by
Genbank Accession No. CA090817.1. (SEQ ID NO:124)
[0353] FIG. 47T presents the amino acid sequence of a
zinc-containing alcohol dehydrogenase superfamily protein of
Synechococcus sp. WH 5701, identified by Genbank Accession No.
ZP_-01085101.1 (SEQ ID NO:125)
[0354] FIG. 47U presents the amino acid sequence of a
zinc-containing alcohol dehydrogenase superfamily protein of
Synechococcus sp. RS9917, identified by Genbank Accession No.
ZP_-01079933.1. (SEQ ID NO:126)
[0355] FIG. 47V presents the amino acid sequence of a
zinc-containing alcohol dehydrogenase superfamily protein of
Synechococcus sp. WH 5701, identified by Genbank Accession No.
ZP_-01085101.1. (SEQ ID NO:127)
[0356] FIG. 47W presents the amino acid sequence of a zn-dependent
alcohol dehydrogenase of Synechococcus sp. WH 7803, identified by
Genbank Accession No. YP_-001224538.1. (SEQ ID NO:128)
[0357] FIG. 47X presents the amino acid sequence of a
zinc-containing alcohol dehydrogenase superfamily protein of
Synechococcus sp. WH 7805, identified by Genbank Accession No.
ZP_-01125148.1. (SEQ ID NO:129)
[0358] FIG. 48A is a graphic depiction of the OD.sub.750 growth
properties of Synechocystis wild type and mutants that express
Pdc/Adh enzyme and Pdc enzyme alone.
[0359] FIG. 48B is a graphic depiction of ethanol production for
Synechocystis wild type and mutants that express Pdc/Adh enzyme and
Pdc enzyme alone.
[0360] FIG. 48C is a graphical presentation of data for an ethanol
concentration time course under limiting CO.sub.2 conditions; these
data are presented in tabular form in FIG. 48C.
[0361] FIG. 48D is a graphical presentation of data for an ethanol
concentration time course under limiting CO.sub.2 conditions; these
data are presented in tabular form in FIG. 48E.
[0362] FIG. 48E is a graphical presentation of data for an ethanol
concentration time course under limiting CO.sub.2 conditions; these
data are presented in tabular form in FIG. 48G.
[0363] FIG. 48F is a graphical presentation of data for an ethanol
concentration time course under limiting CO.sub.2 conditions; these
data are presented in tabular form in FIG. 48I.
[0364] FIG. 49A is a tabular presentation of cyanobacterial
promoters used to express ethanologenic enzymes in Synechocystis
6803.
[0365] FIG. 49B is a graphic depiction of growth properties of 6803
transformed with pVZ321b-PisiA-PDC/ADH as monitored by determining
the OD.sub.750.
[0366] FIG. 49C is a graphic depiction of iron-induced ethanol
production of 6803 transformed with pVZ321b-PisiA-PDC/ADH.
[0367] FIG. 49D is a graphic depiction of ethanol production of
Synechocystis 6803 pVZ321b-PnblA-PDC/ADH that express Pdc/Adh
enzymes under the control of the nitrogen dependent
nblA-promoter.
[0368] FIG. 49E is a graphic depiction of the growth properties of
cells with PnirA-PDC when nitrogen is provided by ammonia or
nitrate.
[0369] FIG. 49F is a graphic depiction of ethanol production of
cells with PnirA-PDC when nitrogen is provided by ammonia or
nitrate.
[0370] FIG. 49G is a graphic depiction of ethanol production
normalized for culture optical density of cells with PnirA-PDC when
nitrogen is provided by ammonia or nitrate.
[0371] FIG. 49H is a graphic depiction of growth of Synechocystis
6803 pVZ321b-PpetJ-PDC/ADH.
[0372] FIG. 49I is a graphic depiction of ethanol production of
Synechocystis 6803 pVZ321b-PpetJ-PDC/ADH.
[0373] FIG. 49J is a graphic depiction ethanol productivity per
growth of Synechocystis 6803 pVZ321b-PpetJ-PDC/ADH.
[0374] FIG. 49K is a graphic depiction of the growth of
Synechocystis 6803 pVZ321b-PpetE-PDC/ADH.
[0375] FIG. 49L is a graphic depiction ethanol production of
Synechocystis 6803 pVZ321b-PpetE-PDC/ADH.
[0376] FIG. 49M is a graphic depiction of ethanol production of
Synechocystis 6803 pVZ321b-PcrhC-PDC/ADH.
[0377] FIG. 49N is a graphic depiction of growth properties of
Synechocystis 6805 pVZ321b-PhspA-PDC, pVZ321b-PhtpG-PDC,
pVZ321b-PhliB-PDC and pVZ321b-PclpB1-PDC.
[0378] FIG. 49O is a graphic depiction of ethanol production of
Synechocystis 6803 pVZ321b-PhspA-PDC, pVZ321b-PhtpG-PDC,
pVZ321b-PhliB-PDC and pVZ321b-PclpB1-PDC.
[0379] FIG. 49P is a graphic presentation of growth properties
under different conditions of cells containing
pVZ321b-PpetJ-PDC/SynADH.
[0380] FIG. 49Q is a graphic presentation of ethanol production
under different growth conditions of cells containing
pVZ321b-PpetJ-PDC/SynADH.
[0381] FIG. 49R is a graphic presentation of ethanol production per
OD under different growth conditions of cells containing
pVZ321b-PpetJ-PDC/SynADH.
[0382] FIG. 50A presents the nucleotide sequence of ScPDC1. (SEQ ID
NO:130)
[0383] FIG. 50B presents the amino acid sequence of ScPDC1. (SEQ ID
NO:131)
[0384] FIG. 50C presents the nucleotide sequence of ScADH1. (SEQ ID
NO:132)
[0385] FIG. 50D presents the amino acid sequence of ScADH1. (SEQ ID
NO:133)
[0386] FIG. 50E presents the nucleotide sequence of Chlamydomonas
Pcyc6. (SEQ ID NO:134)
[0387] FIG. 50F presents the nucleotide sequence of Chlamydomonas
FEA1. (SEQ ID NO:135)
[0388] FIG. 50G presents the nucleotide sequence of a synthetic ble
marker gene. (SEQ ID NO:136)
[0389] FIG. 50H presents the nucleotide sequence of ARG7 gene of
Chlamydomonas. (SEQ ID NO:137)
[0390] FIG. 50I is a schematic presentation of gene organization of
pSP124S.
[0391] FIG. 50J presents the nucleotide sequence of pSP124S. (SEQ
ID NO:158)
[0392] FIG. 50K is a schematic presentation of gene organization of
pXX311.
[0393] FIG. 50L presents the nucleotide sequence of pXX311. (SEQ ID
NO:139)
[0394] FIG. 50M is a schematic presentation of gene organization of
ARG7_pKS.
[0395] FIG. 50N is a schematic presentation illustrating the
construction of ScPDC1 3'UTR pKS.
[0396] FIG. 50O is a schematic presentation illustrating the
construction of Pcyc6 ScPDC1 3'UTR pKS.
[0397] FIG. 50P is a schematic presentation Illustrating the
construction of Pcyc6ScPDC1 3'UTR Pcyc6 ScADH1 3'UTR pKS.
[0398] FIG. 50Q is a schematic presentation of gene organization
for pCYC6-PDC1-ADH1 pSP124S.
[0399] FIG. 50R is a schematic presentation of gene organization
for pFEA1-PDC1-ADH1 pSP124S.
[0400] FIG. 50S is a schematic presentation of gene organization
for pCYC6-PDC1-ADH1 ARG7.
[0401] FIG. 50T is a schematic presentation of gene organization
for pFEA1-PDC1-ADH1 ARG7.
[0402] FIG. 50U is a schematic presentation of gene organization
for ScPDC1-pXX311.
[0403] FIG. 50V is a graphic presentation of Chlamydomonas ethanol
production.
[0404] FIG. 50W presents in tabular format results for all high
priority tests (photosynthetic activity, the long and short term
ethanol tolerance test and the salt and thermo tolerance test).
[0405] FIG. 51A is a graphic representation of ethanol production
after the addition of acetaldehyde. Different acetaldehyde
concentrations were added to a culture of strain 6803pVZPisiA, and
the ethanol content in the medium was measured for 60 minutes.
[0406] FIG. 51B is a graphic representation of the correlation of
ethanol production rate and acetaldehyde concentration. Given are
the initial ethanol rates (calculated with FIG. 51A) in correlation
to the initial acetaldehyde concentrations.
[0407] FIG. 51C (Lineweaver-Burk-Plot) is a graphic representation
of the reciprocal of the initial velocity versus the reciprocal of
the acetaldehyde concentration. Intact cells were used.
[0408] FIG. 51D (Lineweaver-Burk-Plot) is a graphic representation
of the reciprocal of the initial velocity versus the reciprocal of
the acetaldehyde concentration. The results shown are from a repeat
of the experiment with intact cells.
[0409] FIG. 51E (Lineweaver-Burk-Plot) is a graphic representation
of the Adh activities of a crude extract of strain 6803PVZPisiA
were measured in presence of different concentration of
acetaldehyde. In contrast to the experiments with intact cells in
this experiment NADH was added in excess. Shown is the reciprocal
of the initial velocity versus reciprocal of the concentration of
acetaldehyde.
[0410] FIG. 51F (Lineweaver-Burk-Plot) Similar to the experiment
summarized in FIG. 51E, Adh activities of a crude extract of strain
6803PVZPisiA were measured in the presence of different
concentrations of acetaldehyde. The assays contained an over excess
either of NADH or of NADPH. Substantial differences between NADH
(squares) and NADPH (diamonds) were not observed.
DETAILED DESCRIPTION OF EMBODIMENTS
Definitions
[0411] As used herein, the term "genetically modified" refers to
any change in the endogenous genome of a wild type cell or to the
addition of non-endogenous genetic code to a wild type cell, e.g.,
the introduction of a heterologous gene. More specifically, such
changes are made by the hand of man through the use of recombinant
DNA technology or mutagenesis. The changes can involve protein
coding sequences or non-protein coding sequences such as regulatory
sequences as promoters or enhancers.
[0412] The term "nucleic acid" is intended to include nucleic acid
molecules, e.g., polynucleotides which include an open reading
frame encoding a polypeptide, and can further include non-coding
regulatory sequences, and introns. In addition, the terms are
intended to include one or more genes that map to a functional
locus. In addition, the terms are intended to include a specific
gene for a selected purpose. The gene can be endogenous to the host
cell or can be recombinantly introduced into the host cell.
[0413] The phrase "operably linked" means that the nucleotide
sequence of the nucleic acid molecule or gene of interest is linked
to the regulatory sequence(s) in a manner which allows for
expression (e.g., enhanced, increased, constitutive, basal,
attenuated, decreased or repressed expression) of the nucleotide
sequence and expression of a gene product encoded by the nucleotide
sequence (e.g., when the recombinant nucleic acid molecule is
included in a recombinant vector, as defined herein, and is
introduced into a microorganism)
[0414] The term "recombinant nucleic acid molecule" includes a
nucleic acid molecule (e.g., a DNA molecule) that has been altered,
modified or engineered such that it differs in nucleotide sequence
from the native or natural nucleic acid molecule from which the
recombinant nucleic acid molecule was derived (e.g., by addition,
deletion or substitution of one or more nucleotides).
Advantageously, a recombinant nucleic acid molecule (e.g., a
recombinant DNA molecule) includes an isolated nucleic acid
molecule or gene of the present invention
[0415] The terms "host cell" and "recombinant host cell" are
intended to include a cell suitable for genetic manipulation, e.g.,
which can incorporate heterologous polynucleotide sequences, e.g.,
which can be transfected. The cell can be a prokaryotic or a
eukaryotic cell. The term is intended to include progeny of the
cell originally transfected. In particular embodiments, the cell is
a prokaryotic cell, e.g., a cyanobacterial cell. Particularly, the
term recombinant host cell is intended to include a cell that has
already been selected or engineered to have certain desirable
properties and suitable for further modification using the
compositions and methods of the invention.
[0416] The term "promoter" is intended to include a polynucleotide
segment that can transcriptionally control a gene-of-interest,
e.g., a pyruvate decarboxylase gene, that it does or does not
transcriptionally control in nature. In one embodiment, the
transcriptional control of a promoter results in an increase in
expression of the gene-of-interest. In another embodiment, a
promoter is placed 5' to the gene-of-interest. A promoter can be
used to replace the natural promoter, or can be used in addition to
the natural promoter. A promoter can be endogenous with regard to
the host cell in which it is used or it can be a heterologous
polynucleotide sequence introduced into the host cell, e.g.,
exogenous with regard to the host cell in which it is used.
Promoters of the invention may also be inducible, meaning that
certain exogenous stimuli (e.g., nutrient starvation, heat shock,
mechanical stress, light exposure, etc.).
[0417] The term "about" is used herein to mean approximately, in
the region of, roughly, or around. When the term "about" is used in
conjunction with a numerical value/range, it modifies that
value/range by extending the boundaries above and below the
numerical value(s) set forth. In general, the term "about" is used
herein to modify a numerical value(s) above and below the stated
value(s) by a variance of 20%.
[0418] As used herein, the phrase "increased activity" refers to
any genetic modification resulting in increased levels of enzyme in
a host cell. As known to one of ordinary skill in the art, enzyme
activity may be increased by increasing the level of transcription,
either by modifying promoter function or by increasing gene copy
number, increasing translational efficiency of an enzyme messenger
RNA, e.g., by modifying ribosomal binding, or by increasing the
stability of a enzyme protein, which because the half-life of the
protein is increased, will lead to more enzyme molecules in the
cell. All of these represent non-limiting examples of increasing
the activity of an enzyme. (mRNA Processing and Metabolism: Methods
and Protocols, Edited by Daniel R. Schoenberg, Humana Press Inc.,
Totowa, N.J.; 2004; ISBN 1-59259-750-5; Prokaryotic Gene Expression
(1999) Baumberg, S., Oxford University Press, ISBN 0199636036; The
Structure and Function of Plastids (2006) Wise, R. R. and Hoober J.
K., Springer, ISBN 140203217X; The Biomedical Engineering Handbook
(2000) Bronzino, J. D., Springer, ISBN 354066808X).
[0419] In one aspect the invention also provides nucleic acids,
which are at least 60%, 70%, 80% 90% or 95% identical to the
promoter nucleic acids disclosed therein and to the nucleic adds,
which encode proteins, for example enzymes for ethanol formation or
host cell enzymes involved in the conversion or formation of acetyl
CoA acetaldehyde or pyruvate or for formation of reserve compounds.
The invention also provides amino acid sequences for enzymes for
ethanol formation or host cell enzymes involved in the conversion
or formation of acetyl-CoA, acetaldehyde or pyruvate or for
formation of reserve compounds, which are at least 60%, 70%, 80%
90% or 95% identical to the amino acid sequences disclosed
therein.
[0420] The percentage of identity of two nucleic acid sequences or
two amino acid sequences can be determined using the algorithm of
Thompson et al. (CLUSTALW, 1994 Nucleic Acid Research 22: 4673-4,
680). A nucleotide sequence or an amino acid sequence can also be
used as a so-called "query sequence" to perform against public
nucleic acid or protein sequence databases in order, for example,
to identify further unknown homologous promoters, which can also be
used in embodiments of this invention. In addition, any nucleic
acid sequences or protein sequences disclosed in this patent
application can also be used as a "query sequence" in order to
identify yet unknown sequences in public databases, which can
encode for example new enzymes, which could be useful in this
invention. Such searches can be performed using the algorithm of
Karlin and Altschul (1999 Proceedings of the National Academy of
Sciences U.S.A. 87: 2,264 to 2,268), modified as in Karlin and
Altschul (1993 Proceedings of the National Academy of Sciences
U.S.A. 90: 5,873 to 5,877). Such an algorithm is incorporated in
the NBLAST and XBLAST programs of Altschul et al. (1999 Journal of
Molecular Biology 215: 403 to 410). Suitable parameters for these
database searches with these programs are, for example, a score of
100 and a word length of 12 for BLAST nucleotide searches as
performed with the NBLAST program. BLAST protein searches are
performed with the XBLAST program with a score of 50 and a word
length of 3. Where gaps exist between two sequences, gapped BLAST
is utilized as described in Altschul et al. (1997 Nucleic Acid
Research, 25: 3,389 to 3,402).
[0421] Database entry numbers given in the following are for the
CyanoBase, the genome database for cyanobacteria (available on the
world wide web at bacteria.kazusa.or.jp/cyanobase/ndex.html);
Yazukazu et al. "CyanoBase, the genome database for Synechocystis
sp. Strain PCC6803: status for the year 2000", Nucleic Acid
Research, 2000, Vol. 18, page 72.
Embodiments
[0422] It is one object of embodiments of the invention to provide
a genetically modified host cell, which can be used for production
of ethanol.
[0423] This object is reached by providing a genetically modified
host cell according to base claim 1. Further embodiments of the
genetically modified host cell, as well as constructs for producing
the genetically modified host cells and a method for producing
ethanol using the genetically modified host cells are subject
matters of further claims.
[0424] Embodiment of genetic knockout and/or overexpression of
metabolic pathway enzymes
[0425] In a first aspect the invention provides a genetically
modified photoautotrophic, ethanol producing host cell
comprising:
[0426] at least one first genetic modification changing the
enzymatic activity or affinity of an endogenous host cell
enzyme,
[0427] the first genetic modification resulting in an enhanced
level of biosynthesis of acetaldehyde, pyruvate, acetyl-CoA or
precursors thereof compared to the respective wild type host
cell,
[0428] at least one second genetic modification different from the
first genetic modification comprising an overexpressed enzyme for
the formation of ethanol.
[0429] Acetaldehyde, pyruvate and acetyl-coA or their precursors
are important metabolic intermediates for energy production in
cells. In photoautotrophic cells, which use light, COs, and water
as a source of energy to produce carbohydrates via photosynthesis,
acetaldehyde, pyruvate, acetyl-CoA and their precursors can be
formed by conversion of organic molecules obtained via CO.sub.2
fixation in the Calvin-cycle, for example 3-phosphoglycerate.
Pyruvate, acetyl-CoA and their precursors are important metabolic
intermediates obtained e.g. by photosynthetic CO.sub.2 fixation in
photoautotrophic cells. Acetaldehyde is a metabolic intermediate of
the anoxygenic fermentation pathway in many photoautotrophic
cells.
[0430] Precursors of pyruvate and acetyl-CoA are organic compounds,
which can be converted into these important metabolic intermediates
via the enzymatic action of enzymes of the photoautotrophic cell.
For example the organic compounds 2-phosphoglycerate,
3-phosphoglycerate or phosphoenolpyruvate can be converted into
pyruvate by enzymes of the glycolytic pathway in photoautotrophic
cells.
[0431] The genetically modified photoautotrophic ethanol producing
host cell comprises at least two different genetic modifications, a
first and a second genetic modification. The first genetic
modification changes the enzymatic activity or affinity of an
endogenous host enzyme, resulting in a higher level of biosynthesis
of acetyl-CoA, acetaldehyde, pyruvate or precursors thereof. The
endogenous host enzyme is already present in an unmodified wild
type host cell and its activity or affinity is changed by the first
genetic modification in order to increase the level of biosynthesis
of metabolic intermediates, which are also present in the wild type
host cell and which can be used to form ethanol.
[0432] Furthermore the genetically modified photoautotrophic
ethanol producing host cell comprises a second genetic modification
in the form of at least one overexpressed enzyme, which can form
ethanol, for example from the above-mentioned important metabolic
intermediates. In a further embodiment the overexpressed enzyme for
ethanol formation can catalyze the last step of ethanol formation
leading to the final product ethanol. The overexpressed enzyme for
ethanol formation can also catalyze the penultimate step of ethanol
formation resulting in a metabolic intermediate, which can further
be converted by another enzyme for ethanol formation into the final
product ethanol.
[0433] The enzyme for ethanol formation can, for example, be an
endogenous enzyme already present in a wild type photoautotrophic
host cell, which is not genetically modified. In this case the
activity or affinity of the enzyme for ethanol formation can be
enhanced by the second genetic modification, for example by genetic
engineering or random mutagenesis. This can, for example, be done
by genetically modifying the amino acid sequence of the enzyme by
site directed or random mutagenesis of the gene encoding this
endogenous enzyme, thereby enhancing its activity for formation of
ethanol. Another possibility is to increase the number of gene
copies encoding for the enzyme in the host cell or simply by
enhancing the rate of transcription of the gene already present in
the wild type cell to increase the abundance of its messenger RNA
in the second genetic modification. This can be done for example by
replacing or mutating the endogenous promoter controlling the
transcription of the endogenous gene encoding the enzyme for
ethanol formation.
[0434] Alternatively or additionally a heterologous enzyme for
ethanol formation can be introduced into the host cell by the
second genetic modification, if that enzyme is not present in an
genetically unmodified wild type host cell. This can be done, for
example, by introducing a construct, for example a DNA vector into
the host cell including a heterologous gene encoding the
overexpressed enzyme for ethanol formation. In the case that an
endogenous enzyme for ethanol formation is already present in a
photoautotrophic wild type host cell, the heterologous enzyme for
ethanol formation can enhance the activity of the endogenous enzyme
resulting in a higher rate of ethanol formation.
[0435] The enzymatic activity and the affinity of an enzyme for its
substrate are important kinetic constants. The enzymatic activity
is given by the parameter V.sub.max, which reflects the maximal
velocity of an enzymatic reaction occurring at high substrate
concentrations when the enzyme is saturated with its substrate. The
affinity is given by the Michaelis-Menten constant K.sub.m which is
the substrate concentration required for an enzyme to reach
one-half its maximum velocity. In order to increase the enzymatic
activity V.sub.max has to be increased, whereas for increasing the
affinity K.sub.m has to be reduced. Regarding a further explanation
of enzyme kinetics we refer to the chapter "enzyme kinetics" in the
textbook "Biochemistry" by Donald Voet and Judith Voet (John Wiley
& Sons, 1990. pages 335 to 340).
[0436] The higher level of biosynthesis of acetyl-CoA,
acetaldehyde, pyruvate or precursors thereof results in a change of
the flux of the acetyl-CoA, acetaldehyde, pyruvate or precursors
thereof in the direction of the at least one overexpressed enzyme
for ethanol formation so that formation of ethanol can be increased
in comparison to a photoautotrophic ethanol producing host cell
harboring only the second genetic modification, but lacking the
first genetic modification. Acetyl-CoA, acetaldehyde, pyruvate or
precursors thereof are transient metabolic intermediates, which are
often rapidly processed into other metabolites by the
photoautotrophic host cell and therefore a change in the level of
biosynthesis of these metabolic intermediates can be hard to detect
in photoautotrophic host cells featuring the first genetic
modification but lacking the second genetic modification.
[0437] A first genetic modification therefore results in a higher
level of biosynthesis of acetyl-CoA, acetaldehyde, pyruvate or
precursors thereof compared to the respective wild type host cell,
if after introduction of the second genetic modification a higher
level of ethanol formation can be detected in a cell harboring the
first and second genetic modification than in a cell only harboring
the second genetic modification but lacking the first genetic
modification. This even applies if a change in the level of
biosynthesis of these metabolic intermediates could not be detected
in the photoautotrophic host cell harboring the first genetic
modification but lacking the second genetic modification in
comparison to the respective wild-type photoautotrophic host cell,
which does not harbor the first and second genetic
modification.
[0438] The genetically modified photoautotrophic host cell can
comprise more than one first genetic modification and also more
than one second genetic modification. For example the first genetic
modification can comprise at least two genetic modifications, one
first genetic modification (a), which is a down-regulation or a
knock out of gene expression of a metabolic enzyme and at least one
further first genetic modification (b), which is an increase in
metabolic enzyme activity and/or substrate affinity for a
endogenous enzyme for formation of acetyl-CoA, pyruvate or
acetaldehyde or precursors thereof.
[0439] In a further embodiment thereof, the total number of
possible one first genetic modifications (a) is represented by N,
wherein N is a number from 0 to 50, and N indicates the number of
genetic modifications resulting in the down-regulation or knockout
of metabolic enzyme activity and/or substrate affinity, and the
number of further first genetic modifications (b) is represented by
P, wherein P is a number from 0 to 50, resulting in an increase in
metabolic enzyme activity and/or substrate affinity for a
endogenous enzyme for formation of acetyl-CoA, pyruvate or
acetaldehyde or precursors thereof. The numerical values for
genetic modification (a) N and genetic modification (b) P are
selected independently from one another as long as the sum of P+N
is at least one. By way of non-limiting example, (a) N may have a
numerical value of 1, indicating a single genetic modification, and
(b) P may have a numerical value of 2, indicating two genetic
modifications. Alternatively (a) N may have numerical value of 2,
indicating two genetic modifications, and (b) P may have a
numerical value of 1. indicating a single genetic modification.
Thus, as will be understood to those skilled in the art, the
invention provides herein for a wide variety of genetically
modified, photoautotrophic ethanol producing host cells comprising
a multitude of genetic modifications, the combination of which
result in an enhanced level of biosynthesis of acetaldehyde,
pyruvate, acetyl-CoA or precursors thereof.
[0440] The genetically modified photoautotrophic host cell shows a
high production of ethanol due to the fact that the ethanol forming
enzyme is overexpressed due to the second genetic modification
leading to a high enzymatic activity or activity for ethanol
formation and that at the same time a higher level of biosynthesis
of acetaldehyde, pyruvate, acetyl-CoA or their precursors is formed
in the cells compared to the respective wild type cells due to the
first genetic modification. Acetaldehyde, pyruvate, acetyl-CoA or
their precursors serve as substrates for the ethanol production.
These metabolic intermediates can either be a direct substrate for
the overexpressed enzyme for the formation of ethanol or for
another second overexpressed enzyme for ethanol formation, which
then catalyzes the formation of a substrate for the first
overexpressed enzyme for ethanol formation.
[0441] In yet another embodiment of the genetically modified host
cell
[0442] the at least one endogenous host cell enzyme is selected
from enzymes of the glycolysis pathway, Calvin-cycle, intermediate
steps of metabolism, amino acid metabolism, the fermentation
pathway and the citric acid cycle, wherein the activity of at least
one of these enzymes is enhanced compared to the respective wild
type host cell.
[0443] Enzymes of intermediate steps of metabolism are enzymes
which connect different metabolic pathways. For example the
glycolysis and the citric acid cycle are connected via the enzyme
malic enzyme converting malate into pyruvate.
[0444] In particular the endogenous host cell enzyme can be
selected from only one of the above pathways or in the case that
more than one endogenous host cell enzyme is mutated in a first
genetic modification can be selected from any possible combination
of the above pathways.
[0445] The Calvin-cycle is an important part of photosynthesis and
includes the light-independent reactions, where CO.sub.2 is
captured from the environment of the cell and converted into
organic compounds, for example three-carbon compounds such as
3-phosphoglycerate. CO.sub.2 may also be captured by alternative
routes into four-carbon compounds such as oxaloacetate. These
processes are also referred to as C3-carbon fixation and C4-carbon
fixation.
[0446] Photosynthetic CO.sub.2 fixation can lead to the production
of carbon storage compounds such as reserve carbohydrates like
glycogen, starch or sucrose.
[0447] The glycolysis pathway is normally the first step of
carbohydrate catabolism in order to generate adenosine triphosphate
(ATP) and reductants such as nicotinamide adenine dinucleotide
(NADH). Glycolysis furthermore can produce pyruvate which is an
important compound for the citric acid cycle that generates
reductant for aerobic respiration and Intermediates for
biosynthesis. Furthermore, glycolysis serves to synthesize various
6- and 3-carbon intermediate compounds which can be used for other
cellular processes such as amino acid biosynthesis.
[0448] Pyruvate produced via glycolysis is one of the major sources
for the citric acid cycle, which is an important part of a
metabolic pathway for the chemical conversion of carbohydrates, fat
and proteins into carbon dioxide and water to generate energy for
the host cell. Pyruvate can, for example, be fed into the citric
acid cycle via acetyl-CoA (acetyl-CoA). Furthermore, pyruvate can
also be metabolized to acetaldehyde via other enzymes. Therefore,
enhancing the activity or affinity of at least one of the
endogenous host cell enzymes of the Calvin-cycle or glycolysis
pathway or the citric acid cycle in a first genetic modification
can result in a higher level of biosynthesis of pyruvate, or
acetyl-CoA or their precursors, respectively. This in turn can
result in a higher ethanol production due to the fact that these
metabolic intermediates can be ultimately converted to ethanol via
the at least one overexpressed enzyme for the formation of ethanol
provided by the second genetic modification.
[0449] In certain aspects and embodiments of the invention the
enzymatic activity or affinity of any of these enzymes can be
enhanced, for example, by increasing the activity or affinity of
the enzymes present in the wild type host cell. Non-limiting
examples contemplated by the invention include site directed
mutagenesis or random mutagenesis and by increasing the amount of
enzymes in the host cell. The latter is achieved, for example by
introducing mutations in the promoter regions controlling the
transcriptional activity of the genes encoding the enzymes or by
introducing additional gene copies coding for these enzymes into
the host cell.
[0450] In a further embodiment at least one enzyme of the
glycolysis pathway, the citric acid cycle, the intermediate steps
of metabolism, the amino acid metabolism, the fermentation pathway
or the Calvin-cycle of the host cell is overexpressed.
Overexpression of an enzyme already present in a wild type host
cell is an effective method to enhance the enzymatic activity of
enzymes in a cell. Overexpression can also be achieved by
introducing a heterologous enzyme into the host cell, which
exhibits the same enzymatic activity as the host cell enzyme, which
should be overexpressed. For example if 3-phosphoglycerate mutase
should be overexpressed in the cyanobacterium Synechocystis a
plasmid comprising a heterologous gene encoding 3-phosphoglycerate
mutase from Zymomonas mobilis can be introduced into the host cell.
Another non-limiting example is the overexpression of pyruvate
kinase from E. coli in Synechocystis, thereby raising the enzymatic
activity of the endogenous host cell enzyme pyruvate kinase in
Synechocystis.
[0451] In the case that the enzymatic activity of malate
dehydrogenase, an enzyme of the citric acid cycle and malic enzyme,
an enzyme of the intermediate steps of metabolism is enhanced,
malate dehydrogenase can stimulate the conversion of oxaloacetate
to pyruvate via malate. Malate dehydrogenase catalyzes the
conversion of oxaloacetate to malate using NADH:
Oxaloacetate+NADH+H.sup.+.fwdarw.malate+NAD.sup.+
Malic enzyme catalyzes the conversion of malate into pyruvate using
NADP.sup.+:
malate+NADP.sup.+.fwdarw.pyruvate+CO.sub.2+NADPH
[0452] In C4-plants the released CO.sub.2 can be fixed by
ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) and NADPH
can be used for CO.sub.2-fixation in the Calvin-cycle. The
enzymatic activity or affinity of RubisCO can be enhanced in a
first genetic modification in order to increase the
CO.sub.2-fixation and direct more carbon towards ethanol formation.
This can be done for example by overexpressing only the small and
the large subunits of RubisCO or a complete RubisCO operon also
including a RubisCO Chaperonin in the photoautotrophic host cells
such as prokaryotic cells. The RubisCO Chaperonin can assist in the
folding of the RubisCO enzyme, which is a complex of eight large
and eight small subunits in cyanobacteria and algae. The binding
sites for the substrate ribulose 1,5-bisphosphate are located on
the large subunits, whereas the small subunits have regulatory
functions. RubisCO catalyzes bifunctional the initial step in the
carbon dioxide assimilatory pathway and photorespiratory pathway in
photosynthetic organisms. The enzyme catalyzes the carboxylation of
ribulose-1,5-bisphosphate into two molecules of 3-phosphoglycerate
(3-PGA) in the carbon dioxide assimilatory pathway, but also the
oxygenation of ribulose-1,5-bisphosphate resulting in 3-PGA and
2-Phosphoglycolate (2-PG) in the photorespiratory pathway. In order
to direct the carbon provided by the CO.sub.2-fixation into ethanol
formation, the carbon dioxide assimilatory pathway has to be
enhanced and the activity of the photorespiratory pathway has to be
reduced. Some photoautotrophic cells such as cyanobacteria have
mechanisms to actively uptake CO.sub.2 and HCO3- and to raise the
CO.sub.2-concentration in the proximity of RubisCO (Badger M. R.,
and Price, G. D. (2003) J. Exp. Bot. 54, 609-622). This reduces the
oxygenase activity of the enzyme. Nevertheless the cyanobacterial
photosynthesis is not efficient enough to completely abolish the
formation of 2-PG. Cyanobacteria produce significant amounts of
2-PG, particularly at elevated oxygen concentrations or after a
change to low CO.sub.2-concentrations.
[0453] In order to enhance the carbon dioxide fixating activity of
RubisCO random or side directed mutagenesis can be performed to
achieve higher COs fixation according to some embodiments of the
Invention. Efforts to select RubisCO enzymes with improved activity
using random mutagenesis were successful when the large subunit of
RubisCO from Synechococcus PCC 7942 was mutagenized and
co-expressed with the small subunit of RubisCO and
phosphoribulokinase (prkA) in E. coli (Directed evolution of
RubisCO hypermorphs through genetic selection in engineered E.
coli, Parikh et al, Protein Engineering, Design & Selection
vol. 19 no. 3 pp. 113-119, 2006). This strategy was also successful
in the case of the similar enzymes from Synechococcus PCC 6301 in
E. coli (Artificially evolved Synechococcus PCC 6301 RubisCO
variants exhibit improvements in folding and catalytic efficiency,
Greene et al., Biochem J. 404 (3): 517-24, 2007).
[0454] Another way of increasing the enzymatic activity of RubisCO
according to the invention involves overexpressing heterologous
RubisCO in order to increase the CO.sub.2 fixation as it was shown
in case of the heterologous expression of RubisCO from
Allochromatium vinosum in Synechococcus PCC 7942 (Expression of
foreign type I ribulose-1,5-bisphosphate carboxylase/oxygenase (EC
4.1.1.39) stimulates photosynthesis in cyanobacterium Synechococcus
PCC 7942 cells, Iwaki et al, Photosynthesis Research 88: 287-297,
2006).
[0455] Overexpression of RubisCO in photoautotrophic host cells
such as cyanobacteria also harboring at least one overexpressed
enzyme for the formation of ethanol surprisingly not just results
in an increased activity of RubisCO, but also leads to an increased
biomass of the cells and a higher growth rate accompanied by a
slight increase in the rate of ethanol production.
[0456] In addition the photorespiration activity of RubisCO can be
reduced or eliminated by random or side directed mutagenesis.
Certain embodiments of the invention relate to the overexpression
of at least one enzyme from the glycolysis pathway. Non-limiting
examples are phosphoglycerate mutase, enolase and pyruvate
kinase.
[0457] Phosphoglycerate mutase catalyzes the reversible reaction
leading from 3-phosphoglycerate formed in the Calvin-cycle to
2-phosphoglycerate. 2-phosphoglycerate in turn can then, in a
reversible reaction catalyzed by the enzyme enolase, be converted
to phosphoenolpyruvate. Phosphoenolpyruvate can further be
converted to pyruvate via the enzymatic action of pyruvate kinase.
Therefore, enhancing the activity of any or all of these enzymes
enhances the pyruvate pool in the host cell by enhancing the
conversion of 3-phosphogylcerate formed in the Calvin-cycle to
pyruvate. Pyruvate itself can then either be a direct substrate for
the at least one overexpressed enzyme for ethanol formation or it
can further be converted into another intermediate, which then can
be further metabolized by the enzyme for ethanol formation in order
to form high amounts of ethanol.
[0458] An enzyme of the fermentation pathway, which can be
overexpressed is for example the acetaldehyde dehydrogenase enzyme,
which can convert acetyl-CoA to acetaldehyde, thereby increasing
the level of biosynthesis of acetaldehyde in the host cell.
Alternatively other aldehyde dehydrogenases enzymes could be
expressed in order to increase the level of biosynthesis of
acetaldehyde in the host cell.
[0459] Enzymes of the intermediate steps of metabolism, which can
be overexpressed are for example pyruvate dehydrogenase enzyme
converting pyruvate into acetyl-CoA, increasing the level of
biosynthesis of acetyl-CoA in the host cell. In addition or
alternatively phosphotransacetylase converting acetyl-CoA to
acetylphosphate can be overexpressed in the host cell, thereby
increasing the level of biosynthesis of acetaldehyde in the host
cell.
[0460] Another non-limiting example of an enzyme, whose activity or
affinity can be increased is the enzyme PEP-carboxylase
(phosphoenolpyruvate carboxylase). This enzyme catalyzes the
addition of CO.sub.2 to phosphoenolpyruvate (PEP) to form the
four-carbon compound oxaloacetate (OAA). This PEP-carboxylase
catalyzed reaction is used for CO.sub.2 fixation and can enhance
the photosynthetic activity leading to higher CO.sub.2 fixation,
which can be used for ethanol formation.
[0461] In particular the enzymatic activity or affinity of
PEP-carboxylase, malate dehydrogenase and malic enzyme can be
enhanced concomitantly. This leads to a higher CO.sub.2 fixation
and an enhanced level of biosynthesis of pyruvate. In addition the
decarboxylation of malate to pyruvate catalyzed by the enzyme malic
enzyme, enhances the CO.sub.2 partial pressure leading to an
increased efficiency of the Calvin cycle. PEP-carboxylase is used
for CO.sub.2 fixation in C4-plants and can also be found in
cyanobacteria.
[0462] Furthermore it is possible to overexpress enzymes of the
amino acid metabolism of the host cell, which for example convert
certain amino acids into pyruvate leading to an enhanced
biosynthesis of pyruvate in the host cell. For example serine can
directly be converted to pyruvate in the cyanobacterium
Synechocystis PCC 6803. The open reading frame sir 2072, which is
annotated as ilvA (threonine dehydratase), EC 4.3.1.19, can
catalyze the deamination of serine to pyruvate.
[0463] According to a further aspect of the invention the enzymatic
activity or affinity of the enzyme phosphoketolase (EC 4.1.2-,
putative phosphoketolase in Synechocystis PCC 6803 slr 0453) is
enhanced in a first genetic modification in order to increase the
level of biosynthesis of precursor molecules for the generation of
acetyl-CoA and acetaldehyde. Phosphoketolase catalyses the
formation of acetyl phosphate and glyceraldehyde 3-phosphate, a
precursor of 3-phosphoglycerate from xylulose-5-phosphate which is
an intermediate of the Calvin cycle.
[0464] According to another embodiment of the invention in
combination with enhancing the enzymatic activity or affinity of
phosphoketolase enzyme, the polyhydroybutyrate (PHB) pathway is
knocked out in order to avoid PHB accumulation due to an increased
level of acetyl-CoA biosynthesis (Control of
Poly-.beta.-Hydroxybutyrate Synthase Mediated by Acetyl Phosphate
in Cyanobacteria, Miyake et al., Journal of Bacteriology, p.
5009-5013, 1997). Additionally AdhE can be overexpressed at the
same time to convert the acetyl-CoA to ethanol.
[0465] Endogenous host enzymes of the glycolysis pathway, the
Calvin-cycle, the intermediate steps of metabolism, the amino acid
metabolism pathways, the fermentation pathways or the citric acid
cycle, can be dependent upon a cofactor. The invention also
provides an enhanced level of biosynthesis of this cofactor
compared to the respective wild type host cell, thereby increasing
the activity of these enzymes. Such an enhanced level of
biosynthesis of this cofactor can be provided in a first genetic
modification.
[0466] An enhanced level of the cofactor biosynthesis also results
in an enhanced enzymatic activity or affinity of these above
mentioned enzymes and therefore in an enhanced level of
biosynthesis of pyruvate, acetyl-CoA, acetaldehyde or their
precursors in the cell.
[0467] For example, alcohol dehydrogenase enzymes are often
NAD.sup.+/NADH cofactor dependent enzymes. In this case, their
enzymatic activity can be enhanced by raising the level of NADH
biosynthesis in the host cell. This can, for example, be done by
overexpressing NAD (P).sup.+ transhydrogenases, which transfer
reduction equivalents between NADP(H) to NAD(H). These NAD
(P).sup.+ transhydrogenases are oxidoreductases.
[0468] Furthermore the host cell can comprise a host NADH
dehydrogenase converting NADH to NAD.sup.+ wherein the activity of
the NADH dehydrogenase is reduced compared to the wild type host
cell.
[0469] For example, point mutations can be introduced into the gene
encoding the NADH dehydrogenase in order to reduce the activity or
affinity of this enzyme or alternatively the gene encoding the NADH
dehydrogenase can be knocked-out by inserting for example
heterologous nucleic acid sequences into the gene, thereby
disrupting it.
[0470] Alternatively, in order to enhance the enzymatic activity of
an enzyme, which is NADP.sup.+/NADPH cofactor dependent as, for
example the malic enzyme, the level of NADP.sup.+/NADPH in the host
cell also can be increased.
[0471] In many of photoautotrophic cells the level of NAD.sup.+
plus NADH to NADP.sup.+ plus NADPH is around 1:10. Due to this high
imbalance of NADH to NADPH, the conversion of an NAD.sup.+/NADH
cofactor specific enzyme via site directed mutagenesis or random
mutagenesis of the enzyme into an NADP.sup.+/NADPH dependent enzyme
can increase its activity. The changing of the cofactor specificity
of alcohol dehydrogenase via in vitro random mutagenesis is for
example described in the publication "Alteration of Substrate
Specificity of Zymomonas mobilis Alcohol Dehydrogenase-2 Using in
Vitro Random Mutagenesis" (Protein Expression and Purification
Volume 9, issue 1, February 1997, Pages 83-90).
[0472] A further embodiment of the invention provides a genetically
modified host cell [0472] wherein the at least one endogenous host
cell enzyme is for the conversion of pyruvate or acetyl-CoA or for
the formation of reserve compounds, wherein its activity or
affinity is reduced.
[0473] Alternatively or in addition to enhancing the activity of
enzymes forming pyruvate, acetaldehyde, acetyl-CoA or precursors
thereof, the activity of the enzymes converting the above-mentioned
important intermediate metabolic compounds into other compounds can
be reduced by the way of the first genetic modification. The
inventors found out that by reducing the activity of at least one
of these enzymes the level of biosynthesis of pyruvate, acetyl-CoA,
acetaldehyde or their precursors can be risen compared to a wild
type host cell. In addition, the inventors made the observation
that by reducing the activity of host enzymes forming reserve
compounds, for example glycogen, more carbohydrates formed via
photosynthesis in the photoautotrophic host cells are shuffled into
the glycolysis pathway and the citric acid cycle, thereby enhancing
the level of biosynthesis of pyruvate, acetaldehyde, acetyl-CoA or
their precursors. Due to the fact that these metabolic
intermediates are used by at least one overexpressed enzyme for the
formation of ethanol, a higher ethanol production of such a
genetically modified host cell can be observed.
[0474] The enzymatic activity of at least one of these enzymes can
be reduced, for example by introducing point mutations into the
genes encoding these enzymes, thereby reducing the activity of
these enzymes. Alternatively or in addition, the promoter regions
controlling the transcriptional activity of these genes can be
mutated, resulting in a lower transcriptional activity and
therefore a reduced level of protein translation in the genetically
modified host cell.
[0475] A point mutation, or single base substitution, is a type of
mutation that causes the replacement of a single base nucleotide
with another nucleotide.
[0476] A "promoter" is an array of nucleic acid control sequences
that direct transcription of an associated nucleic acid sequence,
which may be a heterologous or endogenous nucleic acid sequence. A
promoter includes nucleic acid sequences near the start site of
transcription, such as a polymerase binding site for a RNA
polymerase used for the synthesis of messenger RNA. The promoter
also optionally includes distal enhancer or repressor elements
which can be located as much as several thousand base pairs from
the start site of transcription.
[0477] Furthermore, it is possible that the host cell comprises
disruptions in the host gene encoding at least one of the enzymes
of the host cell converting pyruvate, acetyl-CoA, the precursors
thereof or for forming reserve compounds. In this case, the
enzymatic activity of the enzymes can be eliminated to a full
extent due to the fact that the disrupted gene does not encode for
a functional protein anymore.
[0478] The disruption of the gene can be furthermore caused by an
insertion of a biocide resistance gene into the respective gene.
This has the advantage that so-called "knockout mutants" containing
the insertions in the respective genes can easily be selected by
culturing the genetically modified host cells in selective medium
containing the biocide to which the genetically modified host cell
is resistant.
[0479] The term "biocide" refers to a chemical substance, which is
able to inhibit the growth of cells or even kill cells, which are
not resistant to this biocide. Biocides can include herbicides,
algaecides and antibiotics, which can inhibit the growth of plants,
algae or microorganisms such as bacteria, for example
cyanobacteria.
[0480] Alternatively or in addition for disrupting the gene
encoding one of the enzymes converting pyruvate acetyl-CoA or
acetaldehyde or forming reserve compounds, the enzymatic activity
of one of these enzymes can also be reduced by using the antisense
messenger RNA concept.
[0481] A wild type cell normally comprises at least one host gene
encoding for the host enzyme or protein, wherein transcription of
this gene results in a sense messenger RNA (mRNA), which codes for
the functional protein and is translated into the protein via
translation mediated by the ribosomes, ribonucleoprotein complexes
present in cells. The messenger RNA is normally a single stranded
RNA molecule encoding the amino acid sequence of the enzyme in the
form of the genetic code. Specifically, the genetic code defines a
mapping between tri-nucleotide sequences called codons in the
messenger RNA and the amino acids of the amino acid sequence; every
triplet of nucleotides in a nucleic acid sequence of the mRNA
specifies a single amino acid. This messenger RNA molecule is
normally called sense RNA. In order to reduce or even eliminate the
enzymatic activity of the enzyme encoded by this gene a nucleic
acid sequence can be introduced into the host cell, which upon
transcription results in a RNA strand complementary to the sense
messenger RNA strand, the so-called antisense RNA. This antisense
RNA can then interact with the sense RNA, forming a double-stranded
RNA species which cannot be translated by the ribosomes into a
functional protein anymore. Depending on the ratio of the sense RNA
to the antisense RNA in the host cell, the level of enzymatic
activity of the enzyme can be reduced or even eliminated. Different
antisense RNA approaches for the regulation of gene expression are
described in the following publications:
[0482] Duhring U, Axmann I M, Hess W R, Wilde A. "An Internal
antisense RNA regulates expression of the photosynthesis gene isiA"
(Proc Natl Acad Sci USA. 2006 May 2; 103(18):7054-8).
[0483] Udekwu K I, Darfeuille F, Vogel J, Reimegard J, Holmqvist E,
Wagner E G. "Hfq-dependent regulation of OmpA synthesis is mediated
by an antisense RNA" (Genes Dev. 2005 Oct. 1; 19(19):2355-66)
[0484] Prime enzyme targets for down regulation of enzymatic
activity or for elimination of enzymatic activity are
ADP-glucose-pyrophosphorylase, glycogen synthase, alanine
dehydrogenase, lactate dehydrogenase, pyruvate water dikinase,
phosphotransacetylase, and acetate kinase as well as pyruvate
dehydrogenase.
[0485] ADP-glucose-pyrophosphorylase catalyzes the conversion of
glucose-1-phosphate into ADP-glucose, which is a precursor for the
reserve polysaccharide glycogen in many photoautotrophic host
cells. The enzyme glycogen synthase catalyzes the addition of
further glucose monomers donated by ADP glucose to the ends of
glycogen primers.
[0486] The inventors found out that by reducing or even eliminating
the formation of reserve carbohydrates such as starch or glycogen,
the level of biosynthesis of pyruvate, acetyl-CoA or acetaldehyde
can be raised compared to the level of biosynthesis a wild type
host cell. This finding was particularly true for the reduction of
the enzymatic affinity and activity of glycogen synthase and
ADP-glucose-pyrophosphorylase. A knock out of both enzymes in
photoautotrophic host cells lacking at least one overexpressed
enzyme for ethanol production as a second genetic modification
resulted in a big increase of pyruvate secreted into the growth
medium. Further introducing a second genetic modification into
these photoautotrophic host cells resulted in an increased fraction
of fixed carbon being diverted to ethanol production.
[0487] Alanine dehydrogenase catalyzes the reversible reductive
amination of pyruvate to alanine using NADH as a reductant A
reduction of activity of alanine dehydrogenase can result in a
higher level of pyruvate.
[0488] The enzyme lactate dehydrogenase catalyzes the
inter-conversion of pyruvate to the fermentative end product
lactate using NADH as a reductant. Reducing or inhibiting the
enzymatic action of lactate dehydrogenase can result in an increase
of the level of biosynthesis of pyruvate in the genetically
modified host cell.
[0489] The enzyme pyruvate water dikinase catalyzes the
ATP-dependent conversion of pyruvate, ATP and water to adenosine
monophosphate (AMP), phosphoenolpyruvate and phosphate. Due to that
a reduction of the enzymatic activity of pyruvate water dikinase
can also result in an increased level of pyruvate in the host
cell.
[0490] The enzyme phosphotransacetylase catalyzes the reversible
transfer of an acetyl group from acetyl-CoA to a phosphate thereby
forming acetylphosphate. A reduction of the enzymatic activity of
this enzyme can also result in an increased level of acetyl-CoA as
well as of its precursor pyruvate.
[0491] The enzyme acetate kinase catalyzes the conversion of
acetylphosphate to the fermentative end product acetate whereas the
phosphate group is transferred from acetylphosphate to adenosine
diphosphate (ADP) so adenosine triphosphate (ATP) is formed. An
inactivation or a reduction of the enzymatic activity of this
enzyme can therefore result in a higher level of acetylphosphate
and maybe acetyl-CoA in the cell.
[0492] Reducing the enzymatic activity or knocking out of the gene
encoding phosphotransacetylase (PTA) can be important, since this
enzyme is at the branch point of acetate generation via
acetylphosphate. Acetylphosphate itself is an important
intermediate, because it is needed for ADP regeneration to ATP and
it stimulates the activity of polyhydroxybutyrate (PHB) synthase.
Knock out of the PTA therefore can avoid loss of acetyl-CoA into
the acetate branch and additionally can minimize PHB generation.
Thus acetyl-CoA can be channeled to the ethanol generating
branch.
[0493] The inventors found out that a reduction in the enzymatic
affinity or activity of the enzymes of the complete acetate
fermentation pathway, in particular phosphotransacetylase and
acetate kinase can lead to an increase in the ethanol production
rate without reducing the photosynthetic capacity of the
photoautotrophic host cells. For example a knock out of both genes
coding for phosphotransacetylase and acetate kinase can enhance the
ethanol production rate compared to a photoautotrophic host cell
harboring only at least one overexpressed enzyme for ethanol
formation as a second genetic modification but lacking the first
genetic modification, the knock out mutations of both enzymes.
[0494] On the other hand acetylphosphate is the natural precursor
of fermentative EtOH synthesis via acetaldehyde and therefore
overexpressing the phosphotransacetylase together with the
acetaldehyde forming enzyme and knocking-out or reducing the
enzymatic activity of the PHB synthase can also increase the level
of biosynthesis of acetaldehyde in the genetically modified host
cell.
[0495] In some bacterial cells both enzymes phosphotransacetylase
and acetate kinase can also catalyze the reverse reaction from
acetate to acetylphosphate and from acetylphosphate to acetyl-CoA.
In the case that the level of biosynthesis of acetyl-CoA should be
raised compared to the wild type cells the activity or affinity of
both enzymes can be enhanced for example via overexpression in
different first genetic modifications. Alternatively only acetate
kinase can be overexpressed in a first genetic modification in the
case that the second genetic modification comprises at least
acetaldehyde dehydrogenase converting the acetylphosphate to
acetaldehyde and further Adh, such as AdhI and/or AdhII converting
the acetaldehyde into ethanol.
[0496] Another possible target enzyme for down-regulation to
increase the level of biosynthesis of pyruvate is pyruvate
dehydrogenase, which catalyzes the thiamine pyrophosphate (TPP)
cofactor dependent decarboxylation of pyruvate resulting in
acetyl-CoA, NADH and CO2.
[0497] With regard to the enzymes forming reserve compounds for the
cell, the gene for glycogen synthase can be disrupted, for example
by inserting a heterologous nucleic acid sequence encoding for a
biocide resistance cassette into the gene. The inventors found out
that such a knockout of both glycogen synthase genes glgA1 and
glgA2 in the phototropic genetically modified host cell of the
genera Synechocystis results in an enhanced pyruvate level of up to
50-fold compared to the unmodified wild type host cell.
[0498] In particular, the enzymes forming one of the following
reserve compounds can be a prime target for a reduction of their
enzymatic activity of even for knockout: Glycogen,
polyhydroxyalkanoates like, for example poly-3-hydroxybutyrate or
poly-4-hydroxybutyrate, polyhydroxyvalerate, polyhydroxyhexanoate,
polyhydroxyoctanoate, amylopectin, starch, cyanophycin and their
copolymers, glucosyl glycerol and bacterial extracellular polymeric
substances such as extracellular polysaccharides. Enzymes which are
involved in the synthesis of these reserve compounds are for
example beta-ketothiolase, acetoacetyl-CoA reductase,
polyhydroxybutyrate synthase, glucosylglycerolphosphate
synthase.
[0499] Polyhydroxybutyrate is synthesized from acetyl-CoA via three
enzymatic reactions: 3-thiolase (EC 2.3.1.9) converts two
acetyl-CoA molecules to an acetoacetyl-CoA molecule,
NADPH-dependent acetoacetyl-CoA reductase (EC 1.1.136) converts
acetoacetyl-CoA to D-3-Hydroxybutyryl-CoA with NADPH oxidation, and
the last enzyme. PHB synthase, catalyzes the linkage of the
D-3-hydroxybutyryl moiety to an existing PHB molecule by an ester
bond.
[0500] The biosynthetic pathway of glucosyl glycerol begins with
ADP-glucose and glycerol-3-phosphate (G3P), which are used by the
GG-phosphate synthase (GGPS), and proceeds via the intermediate
GG-phosphate (GGP), which is dephosphorylated to GG by the
GGphosphate phosphatase (GGPP).
[0501] Hydrolyzed EPSs (bacterial extracellular polymeric
substances) showed the compositional involvement of four sugar
moieties viz. mannose, glucose, xylose and ribose in varying
combinations. Chemical analysis of EPS revealed a
heteropolysaccharidic nature, with xylose, glucose, galactose, and
mannose the main neutral sugars found.
[0502] In the case that a genetically modified host cell exhibits a
reduced enzymatic activity for the formation of any of the
above-mentioned reserve compounds, it is expected that the
precursors for these reserve compounds are fed into the glycolysis
pathway or the citric acid cycle, thereby resulting in an enhanced
level of, pyruvate, acetyl-CoA, acetaldehyde or their precursors.
This in turn can result in a higher ethanol production in the case
that pyruvate, acetyl-CoA or acetaldehyde are used by the at least
one overexpressed enzyme for ethanol formation in order to produce
ethanol.
[0503] In yet a further embodiment of the host cell of the
invention, the at least one overexpressed enzyme for the formation
of ethanol is an alcohol dehydrogenase.
[0504] An alcohol dehydrogenase catalyzes the reduction of a
substrate to ethanol. This reaction is normally dependent on the
cofactor NADH. Alternatively there are alcohol dehydrogenases which
are NADPH-dependent.
[0505] Furthermore, the alcohol dehydrogenase can be a thermophilic
alcohol dehydrogenase. Thermophilic alcohol dehydrogenase can, for
example, be obtained from a host cell which can normally grow well
at temperatures above 45.degree. C. Thermophilic alcohol
dehydrogenases can be more stable and probably more active than
alcohol dehydrogenases obtained from mesophilic host cells, which
normally grow at temperatures below 45.degree. C. One possible
example for such a thermophilic alcohol dehydrogenase is the
alcohol dehydrogenase AdhE obtained from the thermophilic
cyanobacterium Thermosynechococcus sp. or from E. coli.
[0506] One possible substrate for alcohol dehydrogenase can be
acetyl-CoA, which for example can be directly converted to ethanol
by the above-mentioned alcohol dehydrogenase AdhE from
Thermosynechococcus or E. coli. Overexpressing such an alcohol
dehydrogenase in a genetically modified host cell has the advantage
that only one enzyme has to be overexpressed in order to enhance
the level of ethanol production. In the case that the level of
biosynthesis of acetyl-CoA of the host cell is increased due to
overexpression of acetyl-coenzyme A forming enzymes and due to the
reduction of enzymatic activity of acetyl-CoA converting enzymes, a
high level of ethanol formation can result.
[0507] In addition the enzymatic activity or affinity of AdhE can
be increased by introducing mutations, in particular point
mutations into the protein via site directed or random mutagenesis.
The AdhE is an iron-dependent, bifunctional enzyme containing a
CoA-depending aldehyde dehydrogenase and an alcohol dehydrogenase
activity. One characteristic of iron-dependent alcohol
dehydrogenases (AdhII) is the sensitivity to oxygen. In the case of
the AdhE from E. coli a mutant was described that shows in contrast
to the wildtype also Adh activity under aerobic conditions. The
site of the mutation was determined in the coding region at the
codon position 568. The G to A nucleotide transition in this codon
results in an amino acid exchange from glutamate to lysine (E568K).
The E568K derivate of the E. coli AdhE is active both aerobically
and anaerobically. This mutation is therefore a solution for the
use of this Oxygen-sensitive enzyme in an oxygen-producing
photosynthetic host cell.
[0508] [Holland-Staley et al., Aerobic activity of Escherichia coli
alcohol dehydrogenase is determined by a single amino acid, J.
Bacteriol. 2000 November; 182(21):6049-54].
[0509] In a further embodiment of the invention, a genetically
modified host cell can be provided, which further comprises:
[0510] pyruvate decarboxylase converting pyruvate to acetaldehyde,
wherein the alcohol dehydrogenase converts the acetaldehyde to
ethanol.
[0511] In this case, the substrate for the alcohol dehydrogenase is
provided by a further overexpressed enzyme, for example pyruvate
decarboxylase, which is introduced into the host cell via a further
second genetic modification. Due to the fact that the level of
biosynthesis of pyruvate of the host cell is increased due to the
above-mentioned modifications of the pyruvate forming and
converting enzymatic activities by way of the first genetic
modification, more acetaldehyde is formed via the enzymatic
activity of pyruvate decarboxylase. Therefore there is an increased
synthesis of acetaldehyde, which is then further converted by
alcohol dehydrogenase to ethanol resulting in a higher
intracellular or extracellular ethanol level in the host cell. The
alcohol dehydrogenase, as well as the pyruvate decarboxylase can be
obtained from alcohol-fermenting organisms such as Zymomonas
mobilis, Zymobacter palmae or the yeast Saccharomyces
cerevisiae.
[0512] In another embodiment of the invention the genetically
modified host cell comprises two second genetic modifications, one
comprising alcohol dehydrogenases Adh converting acetaldehyde into
ethanol and another second genetic modification comprising a
CoA-dependent acetaldehyde dehydrogenase converting acetyl-CoA into
acetaldehyde. One example of such an acetylating CoA-dependent
acetaldehyde dehydrogenase is mhpF from E. coli.
[0513] In yet a further embodiment of the invention the genetically
modified host cell harbors a pyruvate decarboxylase enzyme as the
only second genetic modification. Such a single second genetic
modification is particularly advantageous in genetically modified
host cells, which already have an endogenous alcohol dehydrogenase
enzyme. The inventors surprisingly found that the activity of such
an endogenous alcohol dehydrogenase enzyme can be high enough in
order to convert all or almost all of the acetaldehyde formed by
the overexpressed pyruvate decarboxylase enzyme into ethanol.
[0514] For example all cyanobacterial host cells harbor at least
one endogenous alcohol dehydrogenase enzyme. A preferred example is
the cyanobacterium Synechocystis in particular Synechocystis
PCC6803 or nitrogen fixing cyanobacteria such as Nostoc/Anabaena
spec. PCC7120 and Anabaena variabilis ATCC 29413.
[0515] The alcohol dehydrogenase can be a zinc-dependent
dehydrogenase. In comparison to iron-dependent dehydrogenases, a
zinc-dependent dehydrogenase is less oxygen-sensitive and therefore
can exhibit a higher enzymatic activity in a photoautotrophic host
cell compared to an iron-dependent alcohol dehydrogenase. For
example, the alcohol dehydrogenase AdhI obtained from Zymomonas
mobilis is a zinc-dependent alcohol dehydrogenase, which can
convert acetaldehyde to ethanol by using NADH as a reductant.
Alternatively a zinc-dependent alcohol dehydrogenase can be
obtained from the cyanobacterium Synechocystis, which also depends
on the cofactor NADH.
[0516] Alternatively or additionally the alcohol dehydrogenase can
comprise AdhII for example from Zymomonas mobilis, which is a
Fe.sup.2+ dependent alcohol dehydrogenase converting acetaldehyde
into ethanol.
[0517] In one embodiment, the photoautotrophic ethanol producing
host cell comprises at least three second genetic modifications,
wherein the at least three overexpressed enzymes for ethanol
production have at least three different substrate
specificities.
[0518] In one embodiment thereof, the three substrate specificities
are for the substrates pyruvate, acetaldehyde and acetyl-CoA. For
example the three different overexpressed enzymes for ethanol
formation can be AdhE converting acetyl-CoA to ethanol, Pdc
converting pyruvate to acetaldehyde and AdhI or AdhII converting
the acetaldehyde to ethanol. In another embodiment the three
different overexpressed enzymes for ethanol formation can be a
CoA-dependent acetaldehyde dehydrogenase converting acetyl-CoA to
acetaldehyde and Pdc converting pyruvate to acetaldehyde and AdhI
or AdhII converting the acetaldehyde to ethanol.
[0519] In a further embodiment thereof, the three substrate
specificities are for the substrates pyruvate, acetaldehyde and
acetylphosphate. In this case the three different overexpressed
enzymes for ethanol formation can be acetaldehyde dehydrogenase
converting acetylphosphate to acetaldehyde, Pdc converting pyruvate
to acetaldehyde and AdhI or AdhII converting the acetaldehyde to
ethanol.
[0520] In another embodiment, the photoautotrophic ethanol
producing host cell comprises at least four second genetic
modifications, wherein the at least four overexpressed enzymes for
ethanol production have at least four different substrate
specificities. In one embodiment thereof, the four substrate
specificities are for the substrates pyruvate, acetaldehyde and
acetyl-CoA and acetylphosphate.
[0521] A further embodiment of the invention provides a genetically
modified host cell further comprising:
[0522] a host cell genome, wherein
[0523] a gene encoding the at least overexpressed enzyme for the
formation of ethanol is integrated into the host cell genome.
[0524] The host cell genome can be arranged in at least one
chromosome containing coding as well as non-coding sequences. The
coding sequences of the genome encode all the proteins and nucleic
acids present in a wild type host cell. The gene encoding the at
least one overexpressed enzyme for the formation of ethanol can be
integrated into the host cell genome, for example via homologous
recombination. Integration of the gene coding for the at least one
overexpressed enzyme for ethanol formation into the host cell
genome can be advantageous for host cells, which exhibit a natural
competence for homologous recombination, for example the
cyanobacterium Synechocystis sp.
[0525] Yet another embodiment of the invention provides a
genetically modified host cell further comprising:
[0526] at least one host gene encoding the enzyme converting
pyruvate or acetyl-CoA or acetaldehyde or forming reserve
compounds,
[0527] wherein a heterologous or endogenous gene encoding the at
least one overexpressed enzyme for the formation of ethanol is
integrated into that host gene thereby disrupting the host
gene.
[0528] Such a genetically modified host cell can be produced in
just one genetic engineering step, by simply inserting the
heterologous or endogenous gene, encoding the at least one
overexpressed enzyme for ethanol formation into the host genome
into a gene encoding an enzyme converting pyruvate or Acetyl-CoA or
forming reserve compounds. Such a procedure knocks out the gene for
the enzyme with the undesired activity and at the same time
provides a genetic modification Introducing an ethanol producing
enzyme into a host cell. These genetically modified host cells are
therefore easier to obtain than other genetically modified host
cells wherein the reduction of enzymatic activity of the enzymes
converting pyruvate, actyl-CoA or acetaldehyde and the introduction
of a gene encoding the overexpressed enzyme for ethanol formation
is done in two separate steps.
[0529] Furthermore, the gene encoding the heterologously or
endogenously expressed enzyme can be under the transcriptional
control of a promoter endogenous to the host cell. This has the
advantage that no exogenous promoter has to be introduced into the
host cell. In the case that an exogenous promoter is introduced
into a genetically modified host cell a further heterologous gene
encoding a transcription factor which recognizes the heterologous
promoter, can be introduced into the host cell as well, which
complicates the genetic engineering step. Therefore, the
introduction of an endogenous promoter, which is also present in an
genetically unmodified wild type host cell, has the advantage that
this promoter is easily recognized by the genetically modified host
cell without the need to introduce further genetic modifications.
For example, an inducible promoter such as isiA, which can be
induced under iron starvation and stationary growth phase
conditions for the host cells can be Introduced into Synechocystis
PCC 6803 as an endogenous promoter. Further non-limiting examples
for suitable promoters will be explained later on.
[0530] The gene encoding the heterologously or endogenously
expressed enzyme for ethanol formation can also be under the
transcriptional control of a heterologous promoter, which is not
present in a wild type host cell. For example, heat inducible
promoters such as the CI-PL promoter from the bacteriophage lambda
can be used to control the transcription of genes.
[0531] According to another embodiment of the invention the gene
encoding the heterologously or endogenously expressed enzyme for
ethanol formation is under the transcriptional control of an
inducible promoter.
[0532] Such a genetically modified host cell can accumulate large
amounts of acetyl-CoA, pyruvate, acetaldehyde or their precursors
in the uninduced state due to the above-mentioned modifications and
can then, after induction of the promoter, produce high amounts of
ethanol via the enzymatic action of the enzyme for ethanol
formation, which is now induced. Ethanol can be harmful to the
cell. Therefore, larger amounts of ethanol can be produced by first
accumulating the substrate necessary for ethanol formation without
producing ethanol (uninduced state of the host cell) and then after
induction directly converting these substrates into large amounts
of ethanol. Therefore inducible promoters can be a good genetic
tool in order to decouple the accumulation of acetyl-CoA, pyruvate,
acetaldehyde or their precursors in host cells from the ethanol
production.
[0533] Inducible promoters can be induced for example by nutrient
starvation of the host cell, by stationary phase growth of the host
cell culture or by subjecting the host cell to stressful
conditions.
[0534] These kind of promoters are useful, because a genetically
modified host cell culture can grow and reach a certain density,
thereby leading to a nutrient starvation of the host cell and also
increasing the stress for the host cell culture in the case that
the growth medium is not continuously supplemented with nutrients.
In this case a genetically modified cell culture can accumulate for
example acetyl-CoA, pyruvate or their precursors in the exponential
growth phase in the non-induced state without producing ethanol,
and upon having reached the stationary growth phase can convert
these metabolic products into ethanol due to induction of the
promoters. For example, the inducible promoters can be inducible by
nitrogen starvation or by trace element starvation, such as iron or
copper. Examples of such kinds of promoters are the ntcA promoter,
the nblA promoter as well as the sigB promoter from Synechocystis,
which are inducible by nitrogen starvation and the isiA promoter
which is inducible upon iron starvation. The petJ promoter is
inducible by copper starvation. In addition, the isiA or sigB
promoter can be also inducible by stationary growth phase of the
host cell culture. The sigB promoter can also be induced by
subjecting the host cell culture to darkness. Further stressful
conditions can be heat shock for induction (sigB hspA, htpG, hliB
or clpB1-promoter) and cold shock, which induces for example the
crhC promoter, Heat shock can be induced, for example by raising
the growth temperature of the host cell culture from 30.degree. C.
to 40.degree. C. In contrast to that, a cold shock can be induced
by reducing the growth temperature of the cell culture from
30.degree. C. to 20.degree. C. A further example of an inducible
promoter is the nirA promoter, which can be repressed by ammonia
and induced if nitrate is the sole nitrogen source.
[0535] Further relevant promoters are a promoter of a gene encoding
light repressed protein A homolog (1rtA promoter), which can be
induced by a transition from light to dark conditions. In addition
the promoter of gene of P700 apoprotein subunit Ia (psaA promoter),
which can be induced under low white light and orange light and
repressed in darkness. Furthermore the petE promoter (promoter of
the plastocyanin gene) is inducible by addition of traces of
copper.
[0536] Alternatively the gene encoding the heterologously or
endogenously expressed enzyme for ethanol formation can be under
the transcriptional control of a constitutive promoter, which
allows a certain level of transcription and therefore enzymatic
activity of the overexpressed enzyme for ethanol formation during
the whole period of cultivation even without induction. This can be
advantageous in the case that the metabolic intermediate converted
by the overexpressed enzyme for ethanol formation is harmful to the
cell, as for example acetaldehyde. In this case the acetaldehyde is
continuously converted to ethanol and is not present in the
genetically modified host cell in high amounts.
[0537] A further embodiment of the invention provides a genetically
modified photoautotrophic, ethanol producing host cell
comprising:
[0538] at least one first genetic modification changing the
enzymatic activity or affinity of an endogenous host enzyme of the
host cell,
[0539] the first genetic modification resulting in a level of
biosynthesis of a first metabolic intermediate for energy
production of the host cell, which is enhanced compared to the
level of biosynthesis in the respective wild type host cell,
[0540] at least one second genetic modification different from the
first genetic modification comprising an overexpressed first enzyme
for the formation of ethanol from the first metabolic
intermediate.
[0541] The first metabolic intermediate can be any metabolic
intermediate involved in the energy production of the host cell or
in the formation of reserve compounds in the claim, for example
starch, glycogen or polyhydroxybutyrate. This first metabolic
intermediate can, for example, be formed during the Calvin-cycle,
the light-independent part of photosynthesis, the glycolysis, the
fermentation pathway, the amino acid metabolism or the citric acid
cycle. Some non-limiting examples for the first metabolic
intermediate are pyruvate, acetyl-CoA or acetaldehyde.
[0542] Due to the fact that the level of biosynthesis of this first
metabolic intermediate is enhanced compared to the wild type host
cell and due to the fact that this first intermediate is used by
the first enzyme for ethanol formation in order to produce ethanol,
these genetically modified photoautotrophic host cells can produce
a high amount of ethanol.
[0543] For example, the first metabolic intermediate can comprise
acetyl-CoA and the at least one overexpressed first enzyme can
comprise the alcohol dehydrogenase AdhE directly converting
acetyl-CoA to ethanol. In this case only one overexpressed enzyme
is necessary in order to produce a increased amount of ethanol.
[0544] It is also possible that the genetically modified host cell
further comprises:
[0545] at least one overexpressed second enzyme, converting the
first metabolic intermediate into a second metabolic intermediate,
wherein
[0546] the at least one overexpressed first enzyme converts the
second metabolic intermediate into ethanol.
[0547] In this case, the first enzyme uses another metabolic
intermediate provided by a second overexpressed enzyme in order to
produce ethanol.
[0548] For example, the first metabolic intermediate can comprise
pyruvate and the second metabolic intermediate can comprise
acetaldehyde and the at least one overexpressed second enzyme can
comprise pyruvate decarboxylase converting pyruvate into
acetaldehyde and the at least one overexpressed first enzyme can
comprise alcohol dehydrogenase Adh, converting acetaldehyde into
ethanol.
[0549] Some host cells, for example, cyanobacteria, normally do not
have a pyruvate decarboxylase. Therefore, the transformation of
cyanobacteria with a pyruvate decarboxylase and in addition the
overexpression of an alcohol dehydrogenase which already can be
present in the wild type cyanobacterial cell can result in
increased amounts of ethanol.
[0550] Another embodiment of the invention provides a genetically
modified host cell, which further comprises:
[0551] at least one host enzyme for conversion of the first
metabolic intermediate, wherein
[0552] the activity of said host enzyme is reduced compared to the
respective wild type host cell by genetic engineering.
[0553] As mentioned above, the activity of host enzymes can be
reduced, for example by site directed mutagenesis or random
mutagenesis of the gene encoding the host enzyme, which results in
a protein with a lower activity.
[0554] Alternatively or additionally the promoter sequences
controlling the transcriptional activity of the genes encoding this
host enzyme also can be genetically modified in order to reduce the
transcriptional activity. Another example is to disrupt the gene
encoding the host enzyme for conversion of the first metabolic
intermediate with a heterologous nucleic acid sequence. The host
enzyme, for example, can be any enzyme of the Calvin-cycle, the
glycolysis pathway, the intermediate steps of metabolism, the amino
acid metabolism or the citric acid cycle converting the first
metabolic intermediate, which for example, can be pyruvate. In this
case the host enzymes whose activity is reduced can, for example,
be selected from a group consisting of pyruvate water dikinase,
pyruvate dehydrogenase, phosphotransacetylase, acetate kinase,
lactate dehydrogenase or alanine dehydrogenase.
[0555] In addition or alternatively the genetically modified host
cell can further comprise: [0556] at least one host enzyme for
forming the first metabolic intermediate, wherein
[0557] the activity of said host enzyme is enhanced compared to the
respective wild type host cell by genetic engineering.
[0558] In the case that the first metabolic intermediate is, for
example, pyruvate the at least one host enzyme can be selected from
the above-mentioned enzymes, which are: malate dehydrogenase, malic
enzyme, pyruvate kinase, enolase, and phosphoglycerate mutase.
[0559] In the case that the first metabolic intermediate is, for
example, acetyl-CoA the at least one host enzyme in addition to the
above latter mentioned enzymes also can be selected from pyruvate
dehydrogenase.
[0560] In yet another embodiment of the invention a genetically
modified photoautotrophic, ethanol producing host cell is provided,
comprising:
[0561] at least one first genetic modification changing the
enzymatic activity or affinity of an endogenous host cell
enzyme,
[0562] at least one second genetic modification different from the
first genetic modification comprising an overexpressed enzyme for
the formation of ethanol,
[0563] the first and second genetic modification resulting in an
increased rate of ethanol production compared to the respective
photoautotrophic, ethanol producing host cell harboring the second
genetic modification but lacking the first genetic
modification.
[0564] This genetically modified photoautotrophic, ethanol
producing host can comprise any of the above mentioned genetic
modifications.
[0565] There are several methods for genetic engineering, which are
useful in enhancing the enzymatic activity or affinity of an
enzyme, for example introducing point mutations (site directed
mutagenesis or random mutagenesis) into a gene encoding the host
enzyme for forming the first metabolic intermediate in order to
enhance the enzymatic activity of this enzyme. Furthermore,
additional gene copies encoding the host enzyme can be introduced
into the host cell therefore enhancing the amount of protein in the
host cell. Alternatively or in addition, the promoter region
controlling the transcriptional activity of the gene encoding the
enzyme can be mutated in order to enhance the transcriptional
activity of the gene. Overexpression can also be achieved by
introducing a heterologous enzyme into the host cell, which
exhibits the same enzymatic activity as the host cell enzyme, which
should be overexpressed. For example if PGA mutase should be
overexpressed in the cyanobacterium Synechocystis a plasmid
comprising a heterologous gene encoding PGA mutase from Zymomonas
mobilis can be introduced into the host cell. Another non-limiting
example is the overexpression of pyruvate kinase from E. coli in
Synechocystis, thereby raising the enzymatic activity of the
endogenous host cell enzyme pyruvate kinase in Synechocystis. In
addition homologous genes from other cyanobacterial sources such as
Synechocystis can be overexpressed in photoautotrophic host cells.
Non-limiting examples for overexpression are: PGA mutase genes
slr1124, slr1945, sll0395 and slr1748 and the enolase homolog
slr0752 from Synechocystis PCC 6803.
[0566] Yet another embodiment of the invention provides a construct
for the transformation of a photoautotrophic host cell by
disrupting a host gene sequence encoding a host enzyme in order to
increase the biosynthetic level of pyruvate, acetyl-CoA,
acetaldehyde or precursors thereof in the host cell comprising:
[0567] a heterologous nucleic acid sequence comprising a promoter
and a biocide resistance conferring gene under the transcriptional
control of the promoter, wherein
[0568] the heterologous nucleic sequence is flanked at its 5' and
3' end by nucleic acid sequences that bind to the host gene
sequence encoding a host enzyme.
[0569] Such a construct can, for example, be used in order to knock
out unwanted host enzymes which convert an important first
metabolic intermediate into another metabolic compound. Due to the
biocide resistance conferring gene, genetically modified host cells
resulting from the transformation with such a construct can be
selected by exposing the transformed host cells to a growth medium
containing the biocide. The 5' and 3' flanking nucleic acid
sequences are preferably homologous to the nucleic acid sequence of
the host gene encoding the host enzyme for conversion of the first
metabolic intermediate.
[0570] The term "binds to" is used herein to refer to the annealing
or hydrogen bonding of one nucleic acid (polynucleotide) to another
nucleic acid (polynucleotide) in a particularly preferred
embodiment, binding occurs in vive or within a cell between a
heterologous nucleic acid sequence and a genomic or chromosomal
nucleic acid sequence. This is particularly useful in promoting
homologous recombination. In other circumstances, the term may
refer to hybridization in a non-natural environment, particularly
under stringent conditions in the laboratory. "Hybridization
stringency" is a term well understood to those of ordinary skill in
the art. A particular, non-limiting example of stringent (e.g. high
stringency) hybridization conditions are hybridization in 6.times.
sodium chloride/sodium citrate (SSC) buffer at about 45 degrees
Celsius, followed by one or more washes in 0.2.times.SSC, 0.1% SDS
at 50-65 degrees Celsius. Hybridization stringency may also be
varied and used to identify and isolate nucleic acid sequences
having different percent identity with the probe sequence.
[0571] In various embodiments of the invention, 5' and 3' flanking
sequences of the invention are selected from a host cell enzyme
gene sequence described herein. Moreover, in the Examples section
provided herewith, the construction of various nucleic acid
constructs is provided. As one of ordinary skill in the art would
recognize, the invention is not limited to only those sequences
disclosed herein because these examples provide ample teaching to
select similar 5' and 3' sequences from host cell enzyme identified
in sequence databases.
[0572] These sequences can, for example, have an identity at least
80%, 85%, 90%, 95% and 100% to the corresponding nucleic acid
sequences of the host cell enzyme gene.
[0573] Another embodiment of the invention provides a construct for
the transformation of a photoautotrophic host cell by disrupting a
host cell gene sequence encoding a host cell enzyme in order to
increase the biosynthetic level of pyruvate, acetyl-CoA,
acetaldehyde or precursors thereof in the host cell,
comprising:
[0574] a heterologous nucleic acid sequence comprising a promoter
and a first gene encoding at least one overexpressed first enzyme
for the formation of ethanol from the first metabolic intermediate
under the transcriptional control of the promoter, wherein
[0575] the heterologous nucleic acid sequence is flanked at its 5'
and 3' end by nucleic acid sequences that bind to said host
gene.
[0576] Such a construct can, for example, be used in order to knock
out a gene encoding a host enzyme for conversion of a first
metabolic intermediate, which can be pyruvate, acetyl-CoA,
acetaldehyde or precursors thereof and at the same time, introduce
via genetic engineering a gene encoding a first enzyme for the
formation of ethanol. Such a construct can therefore be used in
order to enhance the level of a first metabolic intermediate in a
genetically modified host cell and at the same time use this first
metabolic intermediate as a substrate for ethanol production.
[0577] The 5' and 3' flanking nucleic acid sequences are preferably
highly identical, more preferably completely identical, to the
corresponding parts of the host cell gene encoding the host cell
enzyme. Such a construct is integrated into the host genome of a
host cell via homologous recombination.
[0578] Homologous recombination involves the alignment of similar
sequences, preferably homologous nucleic aid sequences located in
different nucleic acid strands, for example a recombinant
integrative plasmid and the chromosome of a host cell. After a
crossover between the aligned nucleic acid strands, the nucleic
acid strands are broken and repaired in order to produce an
exchange of nucleic acid material between the chromosome and the
recombinant integrative plasmid. The process of homologous
recombination naturally occurs in many host cells, for example
cyanobacteria such as Synechocystis and can be utilized as a
molecular biology technique for genetically engineering organisms
and introducing genetic changes into the organisms. The 5' and 3'
flanking nucleic acid sequences each can have a length of a few
hundred base pairs, preferably at least around 500 base pairs or
more, in order to enable homologous recombination. The length can
be up to 1.5 kilobases or even 2 kilobases.
[0579] In various embodiments of the invention, the heterologous
nucleic acid sequence further comprises a second gene encoding at
least one overexpressed second enzyme converting the first
metabolic intermediate into a second metabolic intermediate,
wherein the at least one overexpressed first enzyme converts the
second metabolic intermediate into ethanol.
[0580] In such a case the first metabolic intermediate can comprise
pyruvate and the second metabolic intermediate can comprise
acetaldehyde and the second gene can encode pyruvate decarboxylase
converting pyruvate into acetaldehyde, and the first gene can
encode alcohol dehydrogenase converting acetaldehyde into
ethanol.
[0581] Alternatively, the first metabolic intermediate can comprise
pyruvate and the second metabolic intermediate can, for example,
comprise acetyl-CoA. In this case the first gene can encode
pyruvate dehydrogenase, pyruvate formate lyase or
pyruvate-ferredoxin-oxidoreductase which can convert pyruvate to
acetyl-CoA. The second gene then can encode a coenzyme A dependent
aldehyde dehydrogenase which can convert acetyl-CoA to
acetaldehyde. In this case a third gene can be introduced into the
construct which encodes alcohol dehydrogenase which can convert
acetaldehyde to ethanol. Therefore, constructs according to certain
embodiments of the inventions can comprise more than two or even
more than three genes encoding more than two or three enzymes
involved in ethanol formation.
[0582] Alternatively the first metabolic intermediate can comprise
acetyl-CoA and the first gene can be alcohol dehydrogenase AdhE
directly converting acetyl-CoA into ethanol. In this case one
enzyme can be sufficient to trigger ethanol formation in a
genetically modified host cell.
[0583] Furthermore a co-expression of the enzymes AdhE, Adh and Pdc
in parallel is also able to convert acetyl-CoA into ethanol (e.g.
in combination with a blocked or reduced acetate and lactate
pathway) and to convert pyruvate into ethanol in parallel. This
could avoid that pathways are shifted to acetyl-CoA in case of Pdc
and Adh expression or to pyruvate in case of AdhE expression.
[0584] A further embodiment of the invention is directed to a
genetically modified photoautotrophic, ethanol producing host cell
comprising:
[0585] a first genetic modification comprising at least one genetic
modification of at least one host cell enzyme that is not pyruvate
decarboxylase or alcohol dehydrogenase, wherein the first genetic
modification results in an enhanced level of biosynthesis of
acetaldehyde, pyruvate, acetyl-CoA or precursors thereof compared
to the respective wild type host cell, and
[0586] a second genetic modification comprising at least one
overexpressed enzyme for the formation of ethanol.
[0587] The subject matter of a further embodiment of the invention
is a construct for the transformation of a host cell by disrupting
a host gene encoding a host enzyme for conversion of a first
metabolic intermediate for energy production of the host cell or
forming reserve compounds, comprising +
[0588] a heterologous nucleic acid sequence comprising an inducible
promoter and a gene encoding the host enzyme for conversion of the
first metabolic intermediate for energy production of the host cell
or forming the reserve compounds under the transcriptional control
of the inducible promoter, wherein
[0589] the heterologous nucleic acid sequence is flanked at its 5'
and 3' end by nucleic acid sequences which are able to bind to at
least parts of said host gene.
[0590] As mentioned above, the 5' and 3' flanking nucleic acid
sequences are necessary in order to ensure the insertion of this
construct into the host cell genome, for example via homologous
recombination. Such a construct can be useful in the case that the
host enzyme for conversion of the first metabolic intermediate or
for forming reserve compounds is a very crucial enzyme for the
metabolism of the host cell so that it might not be possible to
completely knock out this enzyme without killing the host cells
during this process. Such a construct can be used in order to
replace the uncontrollable wild type host gene by a copy of the
gene which is under the control of an inducible promoter. Such a
construct enables the controlling of the enzymatic activity of an
important metabolic enzyme of the host cell without completely
knocking out the enzymatic activity of this enzyme.
[0591] The host gene, for example, can encode glycogen synthase.
Due to the fact that two copies are sometimes present in the genome
of a host cell, two different constructs have to be designed in
order to knock out both glycogen synthase coding genes.
[0592] The above-mentioned constructs can be part of a recombinant
plasmid which further can comprise other genes, which for example
encode biocide resistance conferring genes.
[0593] Subject matter of a further embodiment of the invention is a
method for producing genetically modified host cells comprising the
method steps:
A) Providing a wild type host cell showing a wild type level of
biosynthesis of a first metabolic intermediate for energy
production of the host cell, B) enhancing the level of biosynthesis
of the first metabolic intermediate in comparison to the wild type
level by genetic engineering, C) introducing a first heterologous
or endogenous gene into the host cell, the first gene encoding at
least one overexpressed first enzyme for the formation of ethanol
from the first metabolic intermediate.
[0594] Such a method enhances in method step B) the level of
biosynthesis of a useful first metabolic intermediate and then
introduces in method step C) a gene into the host cell encoding a
protein which can use the first metabolic intermediate for ethanol
synthesis.
[0595] Alternatively first method step C) then method step B) can
be carried out. Such a method, can be healthier for the cell due to
the fact that the metabolic intermediate, which can be harmful
would not accumulate in the cells, e.g. in case of
acetaldehyde.
[0596] According to a further embodiment of the method of the
invention in step C) a second heterologous or endogenous gene can
be introduced into the host cell, the second heterologous or
endogenous gene encoding at least one overexpressed second enzyme
converting the first metabolic intermediate into a second metabolic
intermediate, wherein the at least overexpressed first enzyme
converts the second metabolic intermediate into ethanol.
[0597] As mentioned above, the first metabolic intermediate can
comprise pyruvate and the second metabolic intermediate can
comprise acetaldehyde so that the second gene can encode pyruvate
decarboxylase converting pyruvate into acetaldehyde and the first
gene can encode alcohol dehydrogenase converting acetaldehyde into
ethanol.
[0598] Alternatively the first metabolic intermediate can comprise
acetyl-CoA and the first gene can encode the alcohol dehydrogenase
AdhE, which directly converts acetyl-CoA into ethanol.
[0599] In a further modification of the method of the invention in
step A) a wild type host cell can be provided which further
comprises a first host gene encoding at least one first host enzyme
for conversion of the first metabolic intermediate or for forming
reserve compounds, the first host gene is under the transcriptional
control of a first host promoter. Then in step B) the activity of
the at least one first host enzyme can be reduced by genetic
engineering.
[0600] In particular, in step B) the activity of the at least one
host enzyme can be reduced by mutating either the first host
promoter or the first host gene or by disrupting the first host
gene by introducing a heterologous nucleic acid sequence into the
first host gene.
[0601] According to a further embodiment of the method of the
invention, in step A) a wild type host cell can be provided which
further comprises a second host gene encoding at least one second
host enzyme for forming the first metabolic intermediates or
precursors thereof, the second host gene is under the
transcriptional control of a second host promoter, and then in step
B) the activity of the at least one second host enzyme is enhanced
by genetic engineering. The activity of the at least one second
host gene can be enhanced by mutating either the second host
promoter or the second host gene or by overexpressing the second
host enzyme.
[0602] Another embodiment of the invention furthermore provides a
genetically modified photoautotrophic, ethanol producing host cell
comprising:
[0603] an overexpressed pyruvate decarboxylase converting pyruvate
to acetaldehyde, and
[0604] an overexpressed zinc-dependent alcohol dehydrogenase,
converting acetaldehyde to ethanol.
[0605] As already mentioned above, the pyruvate decarboxylase as
well as the alcohol dehydrogenase can be heterologously or
endogenously overexpressed which means that they can already be
present in an unmodified wild type host cell or be introduced as a
heterologous enzyme which naturally only occurs in a different host
cell into the genetically modified host cell of this embodiment of
the invention. Zinc-dependent alcohol dehydrogenases are much more
oxygen-insensitive than iron-dependent alcohol dehydrogenases which
can result in a higher activity of Zinc-dependent alcohol
dehydrogenases.
[0606] Furthermore experimental data show that the Adh enzyme from
Synechocystis is a member of the Zn.sup.2+-binding GroES-like
domain alcohol dehydrogenase phylogenetic family and does not
catalyze the disadvantageous back-reaction, the oxidation of the
formed ethanol back into acetaldehyde or only catalyzes this
reaction to a very small extent. This results in a higher ethanol
production rate and in addition in a higher growth rate of the
genetically modified cells compared to genetically modified cells
containing an Adh enzyme, which also catalyzes the oxidation of
ethanol back to acetaldehyde, such as AdhI or Adh II from Zymomonas
mobilis. These enzymes are also not cyanobacterial enzymes.
[0607] In a further embodiment of this invention the Zn.sup.2+
dependent alcohol dehydrogenase enzyme is therefore selected from a
group consisting of the sub-clades A, sub-clades B and sub-clades C
of the Zinc-binding GroES-like domain alcohol dehydrogenases as
determined by the phylogenetic analysis mentioned below. In
particular the Adh enzyme from Synechocystis is a member of the
sub-clade B of the GroES-like domain alcohol dehydrogenases clade
(see FIG. 47A). The Zn.sup.2+ dependent alcohol dehydrogenase
enzyme can furthermore be selected from a cyanobacterial Zn.sup.2+
dependent alcohol dehydrogenase enzyme. In yet another embodiment
of the invention the Zn.sup.2+ dependent alcohol dehydrogenase
enzyme has at least 60% preferred at least 70% or 80% or most
preferred 90% sequence identity to the amino acid sequence of
Synechocystis Adh.
[0608] Genetically modified photoautotrophic, ethanol producing
host cells comprising an overexpressed pyruvate decarboxylase
converting pyruvate to acetaldehyde, and an overexpressed
zinc-dependent alcohol dehydrogenase, converting acetaldehyde to
ethanol can reach the following high ethanol production rates under
continuous exposure to light for 24 hours a day (rates in % EtOH
(v/v)):
[0609] Over a period of 10 days a daily production of 0.005 can be
reached, more preferred 0.01% per day and most preferred 0.02% per
day. One example is a photoautotrophic cyanobacterial host cell
such as Synechocystis, which is transformed with the integrative
construct pSK10-PisiA-PDC-ADHII. If normalized to OD.sub.750 nm, a
rate of 0.0032% EtOH (v/v) per OD1 and day can be reached.
[0610] Over a period of 25 days a daily production of 0.005 can be
reached, more preferred 0.01% per day and most preferred 0.015% per
day by using a photoautotrophic cyanobacterial host cell such as
Synechocystis transformed with the self-replicating construct
pVZ-PnirA-PDC-SynAdh. If normalized to OD.sub.750 nm1, a rate of
0.0018% EtOH (v/v) per OD1 and day can be achieved.
[0611] Over a period of 40 day a daily production of 0.004 can be
reached, more preferred 0.008% per day and most preferred 0.012%
per day for a photoautotrophic cyanobacterial host cell transformed
with the self-replicating construct pVZ-PpetJ-PDC-SynAdh). If
normalized to OD.sub.750 nm 1, a rate of 0.0013% EtOH (v/v) per OD1
and day can be reached.
[0612] The following ethanol production rates can be reached for
photoautotrophic cyanobacterial host cells under 12 hours light/12
hours dark cycle (day/night cycle) in % EtOH (v/v):
[0613] Over a period of few hours (3-4 hours) a daily production of
0.008 is reached, more preferred 0.016% per day and most preferred
0.024% per day. These ethanol production rates can be achieved by
using for example a cyanobacterium such as Synechocystis
transformed with the integrative construct pSK10-PisiA-PDC-ADHII.
If normalized to OD.sub.750 nm 1, a rate of 0.0048% EtOH (v/v) per
OD1 and day can be measured.
[0614] Over a period of 10 days a daily production of 0.004 is
reached, more preferred 0.009% per day and most preferred 0.014%
per day by using the integrative construct pSK10-PisiA-PDC-ADHII in
a cyanobacterial host cell such as Synechocystis. If normalized to
OD.sub.750 nm 1, a rate of 0.0035% EtOH (v/v) per OD1 and day can
be reached.
[0615] Over a period of 20 days a daily production of 0.004, more
preferred 0.008% per day and most preferred 0.01% per day is
reached by using the self-replicating construct
pVZ-PnirA-PDC-SynAdh or using the se-replicating construct
pVZ-PhspA-Pdc-SynAdh in a for example a cyanobacterial host cell.
If normalized to OD.sub.750 nm 1, a rate of 0.0017% EtOH (v/v) per
OD1 and day can be achieved.
[0616] Over a period of 50 days a daily production of 0.003 is
reached, more preferred 0.005% per day and most preferred 0.008%
per day by using the self-replicating construct
pVZ-PnirA-PDC-SynAdh or the self-replicating construct
pVZ-PhspA-PDC-SynADH. If normalized to OD.sub.750 nm 1, a rate of
0.0010% EtOH (v/v) per OD1 and day can be reached.
[0617] All maximal given rates were obtained and measured only in
the culture. Losses of ethanol by evaporation are not considered. A
person of ordinary skill in the art can calculate a loss of 1% of
present ethanol in the culture per day, resulting in a loss of 14%
after 30 days and 22% after 50 days.
[0618] In general, short term experiments as well as continuous
illumination result in higher rates. Different Adh enzyme types
differ not significantly in their maximal rates but in the duration
of ethanol synthesis and SynAdh experiments result in a longer
production caused by a better longevity of the cells because of the
missing back reaction from ethanol to acetaldehyde.
[0619] In one further embodiment, the invention provides a
genetically modified photoautotrophic, ethanol producing host cell
comprising:
[0620] (a) an overexpressed pyruvate decarboxylase enzyme
converting pyruvate to acetaldehyde,
[0621] (b) an overexpressed Zn.sup.2+ dependent alcohol
dehydrogenase enzyme, converting acetaldehyde to ethanol; and
[0622] (c) at least one overexpressed ethanol producing enzyme
having a different substrate specificity than (a) or (b).
[0623] In a further embodiment thereof, (c) comprises an
overexpressed ethanol producing enzyme with a substrate specificity
for acetyl-CoA or acetylphosphate. In a further embodiment thereof,
(c) comprises AdhE converting acetyl-CoA into ethanol, or
acetaldehyde dehydrogenase converting acetylphosphate into
acetaldehyde, or a CoA-dependent acetaldehyde dehydrogenase
converting acetyl-CoA into acetaldehyde.
[0624] Another embodiment of this invention also provides a
construct for the transformation of a photoautotrophic host cell,
the photoautotrophic host cell comprising a host genome, the
construct comprising:
[0625] a coding nucleic acid sequence comprising a first gene
encoding a Zinc-dependent alcohol dehydrogenase, wherein
[0626] the coding nucleic acid sequence is flanked at its 5' and 3'
end by nucleic acid sequences which are able to bind at least parts
of that host genome for integration of the coding nucleic acid
sequence into the host genome.
[0627] Such a construct can be used, for example, in an integrative
plasmid in order to introduce a gene encoding a Zinc-dependent
alcohol dehydrogenase into the genome of a host cell, for example
the cyanobacterium Synechocystis via homologous recombination.
[0628] The construct furthermore can comprise a heterologous or
endogenous promoter controlling the transcription of the first
gene. This embodiment of the invention also provides a construct
for the transformation of a photoautotrophic host cell,
comprising:
[0629] a coding nucleic acid sequence comprising a promoter and a
first gene encoding a Zink-dependent alcohol dehydrogenase wherein
the first gene is under the transcriptional control of the
promoter.
[0630] The above-mentioned constructs can be part of a recombinant
circular plasmid.
[0631] Another embodiment of the invention provides a genetically
modified photoautotrophic ethanol producing host cell
comprising:
[0632] an overexpressed alcohol dehydrogenase directly converting
acetyl-CoA to ethanol.
[0633] Such a genetically modified photoautotrophic host cell only
requires one overexpressed alcohol dehydrogenase enzyme, for
example AdhE which can be a thermophilic alcohol dehydrogenase, for
example obtained from the cyanobacterium Thermosynechococcus in
order to produce ethanol from the metabolic products naturally
occurring in this host cell or which can be from E. coli.
[0634] In addition the enzymatic activity or affinity of AdhE can
be increased by introducing mutations, in particular point
mutations into the protein via site directed or random mutagenesis.
The AdhE is an iron-dependent, bifunctional enzyme containing a
CoA-depending aldehyde dehydrogenase and an alcohol dehydrogenase
activity. One characteristic of iron-dependent alcohol
dehydrogenases (AdhII) is the sensitivity to oxygen. In the case of
the AdhE from E. coli a mutant was described that shows in contrast
to the wildtype also Adh activity under aerobic conditions. The
site of the mutation was determined in the coding region at the
codon position 568. The G to A nucleotide transition in this codon
results in an amino acid exchange from glutamate to lysine (E568K).
The E568K derivate of the E. coli AdhE is active both aerobically
and anaerobically. This mutation is therefore a solution for the
use of this oxygen-sensitive enzyme in an oxygen-producing
photosynthetic host cell. [Holland-Staley et al., Aerobic activity
of Escherichia coli alcohol dehydrogenase is determined by a single
amino acid, J. Bacteriol. 2000 November; 182(21):6049-54].
[0635] In one embodiment, the invention provides a genetically
modified photoautotrophic, ethanol producing host cell
comprising:
[0636] (a) an overexpressed alcohol dehydrogenase enzyme, directly
converting acetyl-CoA to ethanol;
[0637] (b) at least one overexpressed ethanol producing enzyme
having a different substrate specificity than (a).
[0638] In one embodiment thereof, the at least one an overexpressed
ethanol producing enzyme of (b) has a substrate specificity for
acetaldehyde or acetylphosphate. In a further embodiment thereof,
(b) comprises Adh or acetaldehyde dehydrogenase.
[0639] Another embodiment of the invention provides a construct for
the transformation of a photoautotrophic host cell, the
photoautotrophic host cell comprising a host genome, the construct
comprising:
[0640] a coding nucleic acid sequence comprising a gene encoding an
alcohol dehydrogenase, directly converting acetyl-CoA to ethanol,
wherein
[0641] the coding nucleic acid sequence is flanked at its 5' and 3'
end by nucleic acid sequences which are able to bind to at least
parts of said host genome for integration of the coding nucleic
acid sequence into the host genome.
[0642] Such a construct is be useful in order to introduce a
nucleic acid sequence encoding for an alcohol dehydrogenase such as
AdhE directly converting Acetyl-CoA to ethanol into a host genome,
for example via homologous recombination.
[0643] Such a construct furthermore can comprise a heterologous or
endogenous promoter controlling the transcription of the gene.
[0644] In one embodiment, the invention provides a genetically
modified photoautotrophic, ethanol producing host cell comprising
at least two overexpressed enzymes for ethanol production
comprising at least two substrate specificities. In a further
embodiment thereof, the at least two substrate specificities are
selected from a group consisting of acetyl-CoA, acetaldehyde and
acetylphosphate. In yet a further embodiment thereof, the at least
two overexpressed enzymes for ethanol production are selected from
a group consisting of Adh, AdhE, a CoA-dependent acetaldehyde
dehydrogenase and an acetaldehyde dehydroagenase converting
acetylphosphate into acetaldehyde.
[0645] Another embodiment of the invention provides a genetically
modified photoautotrophic, ethanol producing host cell comprising
an overexpressed NAD.sup.+/NADH cofactor specific alcohol
dehydrogenase, wherein the host cell comprises an enhanced level of
NAD.sup.+/NADH biosynthesis compared to the respective wild type
host cell.
[0646] Such a host cell exhibits an enhanced level of ethanol
formation due to the fact that the alcohol dehydrogenase is
overexpressed and its activity is enhanced due to the enhanced
intracellular level of NAD.sup.+/NADH biosynthesis.
[0647] For example, such a genetically modified host cell can
comprise a host NADH dehydrogenase converting NADH to NAD.sup.+
wherein the activity of the NADH dehydrogenase is reduced compared
to the wild type host cell by genetic engineering.
[0648] Such genetic engineering can, for example, be done by
introducing point mutations into the NADH dehydrogenase reducing
its enzymatic activity, by mutating the promoter region of the gene
encoding the NADH dehydrogenase which can result in a reduced
transcriptional activity or by disrupting the gene encoding the
NADH dehydrogenase.
[0649] Alternatively or in addition the genetically modified host
cell furthermore can comprise a NAD (P).sup.+ transhydrogenase
converting NADPH to NADH (electrons are transferred from NADPH to
NAD.sup.+, so NADP.sup.+ and NADH are generated) wherein the
activity of this NAD (P).sup.+ transhydrogenase is enhanced
compared to the activity of the enzyme in a wild type host cell.
This can, for example, be done by overexpressing the NAD (P).sup.+
transhydrogenase.
[0650] A further embodiment of the invention provides a genetically
modified photoautotrophic ethanol-producing host cell
comprising:
[0651] a heterologous or endogenous nucleic acid sequence
comprising a promoter and a gene encoding at least one
overexpressed enzyme for the formation of ethanol under the
transcriptional control of the promoter, wherein
[0652] the promoter can be induced by nutrient starvation,
oxidative stress, light, darkness, heat shock, cold shock, salt
stress, by a change of the nutrient source, by an increase in the
concentration of one nutrient or stationary growth of the host
cell.
[0653] The nutrient can be a metal such as a trace metal for
example iron or cooper. Furthermore the nutrient can comprise
non-metals such as nitrogen or phosphorus. One example for a
nitrogen source is ammonium NH.sub.4.sup.+ or nitrate
NO.sub.3.sup.-. The nirA promoter for example can be induced by a
switch from NH.sub.4.sup.++ to NO.sub.3.sup.- as a nitrogen
source.
[0654] Such a genetically modified photoautotrophic host cell can
be produced, for example, by introducing a heterologous gene
encoding the at least one overexpressed enzyme into the host cell
or by introducing an endogenous gene encoding an enzyme, which is
already present in the wild type host cell, into the host cell, in
order to ensure that a higher level of this enzyme is produced in
the genetically modified host cell compared to the wild type host
cell.
[0655] In the case that the promoter can be induced by nutrient
starvation, by a change of the nutrient source or stationary growth
of the host cell, these host cells can simply grow into a condition
of nutrient starvation or stationary growth in the case that no new
growth medium or nutrients is/are added while culturing these host
cells. In the case of for example nirA, the preferred nitrogen
source ammonium is first used by the cells and then the
transcription is induced or increased by changing to the less
preferred nitrogen source nitrate. This can, for example, be done
by batch culturing these host cells. In this case the host cells
are automatically induced when reaching the condition of nutrient
starvation or stationary growth and the additional method step of
providing an exogenous stimulus for induction of the host cells can
be omitted, thereby simplifying the culturing of these host
cells.
[0656] The inducible promoters can furthermore be selected from a
group of promoters consisting of: ntcA, nblA, isiA, petJ, petE,
sigB, IrtA, htpG, ggpS, psaA, psbA2, nirA, hspA, clpB1, hliB and
crhC.
[0657] The promoters hspA, clpB1, and hliB can be induced by heat
shock (raising the growth temperature of the host cell culture from
30.degree. C. to 40.degree. C.), cold shock (reducing the growth
temperature of the cell culture from 30.degree. C. to 20.degree.
C.), oxidative stress (for example by adding oxidants such as
hydrogen peroxide to the culture), or osmotic stress (for example
by increasing the salinity). The promoter sigB can be induced by
stationary growth, heat shock, and osmotic stress.
[0658] The promoters ntcA and nblA can be induced by decreasing the
concentration of nitrogen in the growth medium and the promoters
psaA and psbA2 can be induced by low light or high light
conditions. The promoter htpG can be induced by osmotic stress and
heat shock. The promoter crhC can be induced by cold shock. An
increase in copper concentration can be used in order to induce the
promoter petE, whereas the promoter petJ is induced by decreasing
the copper concentration. A further embodiment of the invention
provides a method for producing ethanol, comprising the method
steps of:
[0659] A) providing and culturing any of the genetically modified
host cells as described above in a growth medium under the exposure
of light and CO.sub.2 the host cells accumulating ethanol while
being cultured, [0660] B) isolating the ethanol from the host cells
and/or the growth medium.
[0661] The method step A) of this method can comprise the step of
providing host cells, which comprise a genetically modified gene
encoding at least one enzyme for the formation of ethanol under the
transcriptional control of an inducible promoter, which can be
induced by exposure to an exogenous stimulus. In this case the
method step A) can further comprise the sub-steps:
[0662] A1) culturing the host cells under the absence of the
exogenous stimulus or under a low presence of the exogenous
stimulus, and thereafter
[0663] A2) providing or enhancing the exogenous stimulus, thereby
inducing or enhancing ethanol production
[0664] In particular inducible promoters can be used, which show a
small level of basal transcription even in the absence of the
exogenous stimulus or which are active at least to a certain degree
even if the nutrient is present in the growth medium. Such examples
are the nirA or the petJ promoter. Furthermore someone can let the
photoautotrophic cells grow into a condition of nutrient starvation
by not supplying a desired nutrient to the cells. The cells then
consume the nutrient and gradually grow into a condition of
nutrient starvation if these promoters are used the ethanol
production rate can gradually increase during cultivation rather
than be turned on immediately after induction.
[0665] In the above described methods for producing ethanol, the
exogenous stimulus can be provided by changing the environmental
conditions of the host cells. This can be done for example by
changing the growth medium via centrifugation of the cells and
re-suspending the cells in a growth medium, which lacks the
nutrient (for promoters, which are induced by nutrient starvation)
or which contains a different source for the nutrient. Furthermore
the cells might simply grow into the condition of nutrient
starvation.
[0666] A further variant of the method for producing ethanol is
described, wherein
[0667] the exogenous stimulus comprises nutrient starvation,
and
[0668] method step A2) comprises letting the host cell culture grow
into a condition of nutrient starvation by consuming the nutrient
while growing, and
[0669] after method step A2) the nutrient is added in order to
reduce or abolish the exogenous stimulus, thereby leading to method
step A1).
[0670] The nutrient starvation can also lead to a reduction in the
photosynthetic activity of the photoautotrophic cells, if for
example iron and nitrogen starvation are used for induction. In
these cases it is possible to supply the nutrient to the
photoautotrophic cells again after a certain period of time of
induction and ethanol production in order to allow the cells to
recover their photosynthetic activity in an uninduced state.
Furthermore by periodically supplying the nutrient to the
photoautotrophic cells a "biphasic" long term culture can be
maintained, wherein an uninduced state (method step A1) alternates
with an induced state (method step A2). The promoters, which are
inducible by nutrient starvation or by a change of the nutrient
source are preferably selected from a group consisting of: isiA,
nblA, ntcA, nirA, and petJ.
[0671] Furthermore the impact of nitrogen starvation on the
photosynthetic activity also can be reduced if the host cell
culture comprises nitrogen (N.sub.2) fixing so called diazotrophic
host strains. Upon reaching the condition of nitrogen starvation,
these strains can switch to nitrogen fixation. The reduction of
N.sub.2 to ammonia is catalyzed by the nitrogenase, an enzyme
complex, which is Irreversibly inactivated by O.sub.2-Therefore
photosynthetic N.sub.2-fixing organisms have two conflicting
metabolic systems in one cell: oxygen evolving photosynthesis and
oxygen-sensitive nitrogen fixation. Among cyanobacteria many
unicellular and filamentous strains are able to fix nitrogen and
have evolved various strategies to protect the nitrogenase from
oxygen. For example, certain strains of Gloeothece and
Synechococcus evolved a temporal separation of oxygenic
photosynthesis and nitrogen fixation, while other (filamentous)
strains developed specialized cells, called heterocysts, for
N.sub.2-fixation. These heterocysts are able to supply the fixed
nitrogen to the so-called vegetative cells of the filament, which
cannot fix nitrogen, but maintain photosynthesis instead. Examples
for nitrogen fixing cyanobacteria are filamentous cyanobacteria
from the genus Anabaena such as Nostoc/Anabaena spec. PCC7120 and
Anabaena variabilis ATCC 29413.
[0672] Nitrogen fixing photoautotrophic host cells such as
cyanobacteria can also be transformed with constructs containing
genes encoding ethanologenic enzymes under the control of inducible
promoters, which can be induced by other conditions than nitrogen
starvation, for example iron starvation (isiA promoter) or even by
an increase in the copper concentration (petE promoter).
[0673] The construct used for transforming and manufacturing these
genetically modified host cells can, for example, be a construct
for the transformation of photoautotrophic host cell,
comprising:
[0674] a heterologous or endogenous nucleic acid sequence
comprising a promoter, which can be induced by nutrient starvation
of the host cell, and
[0675] a gene encoding at least overexpressed enzyme for the
formation of ethanol under the transcriptional control of the
promoter.
[0676] The constructs can be introduced into the host cell, for
example via electroporation, conjugation, by using natural
competence for DNA uptake or any other method for genetic
transformation known in the art.
[0677] In a further embodiment of this Invention, the construct for
the transformation of the photoautotrophic host cell furthermore
comprises flanking 5' and 3' nucleic acid sequences for the
heterologous or endogenous nucleic acid sequences, which are able
to bind to at least parts of the host genome of the host cell to be
transformed for integration of the heterologous nucleic acid
sequence into the host genome. The integration can, for example, be
done by homologous recombination.
Embodiment of Screening Strains
[0678] A further embodiment of the invention provides a method for
testing a photoautotrophic strain for a desired growth property
selected from a group of properties consisting of ethanol
tolerance, salt tolerance, above neutral pH tolerance, mechanical
stress tolerance, temperature tolerance and light tolerance,
comprising the method steps of:
a) providing a photoautotrophic strain to be tested, b) cultivating
the photoautotrophic strain to be tested in a liquid growth medium
and subjecting the photoautotrophic strain to a condition selected
from a group of conditions of:
[0679] adding ethanol to the growth medium,
[0680] adding salt to the growth medium,
[0681] increasing the pH of the growth medium.
[0682] agitating the growing culture,
[0683] increasing the temperature of the growing culture,
[0684] subjecting the photoautotrophic strain to light,
c) determining the viability of the cells of the photoautotrophic
strain cultivated in the step b).
[0685] Such a method can be used in order to identify
photoautotrophic strains tolerant to certain growth conditions to
which they are subjected during the cultivation and production of,
for example, ethanol.
[0686] Ethanol can be harmful to cells. Therefore, searching for
and identifying ethanol-tolerant strains Improves the ethanol
production, because these cells can produce a high amount of
ethanol without being affected too much by the produced
ethanol.
[0687] In addition, salt-tolerant strains can also be cultured in
brackish or even sea water, which is easier to obtain and cheaper
than fresh water. The inventors made the observation that fresh
water photoautotrophic cells, in particular fresh water
cyanobacterial cells often have a higher photosynthesis rate than
comparable marine species. Due to that it can be advantageous
trying to culture fresh water species, having a high tolerance to
salt water in brackish water or even in sea water. In this case the
higher photosynthesis rate of the fresh water species can also
result in a higher ethanol production rate of a genetically
modified fresh water species, because the carbohydrates and other
metabolic products produced by the photosynthesis can serve as
substrates for the overexpressed enzymes for ethanol formation.
[0688] A tolerance to above neutral pH conditions in the growth
medium can also have a positive effect on the rate of
photo-synthesis. This can be due to the fact that at pH values
above neutral (in the range pH 8) hydrogencarbonate
(HCO.sub.3.sup.-, bicarbonate) has a higher mole fraction in a
liquid aqueous growth medium than at a lower pH. Therefore, more
hydrogencarbonate can be used by the genetically modified cells
having a high pH tolerance for carbon fixation. Although
cyanobacteria fix carbon as CO.sub.2 by the enzyme RubisCO most of
the carbon that is taken up by the cell can be HCO.sub.3.sup.-,
which is than converted into CO.sub.2 by the enzyme carbonic
anhydrase before its fixation as CO.sub.2 by the RubisCO. In
cyanobacterial cells the enzymes RubisCO (ribulose-1,5-bisphosphate
carboxylase/oxygenase) and carbonic anhydrase are both arranged in
the so called carboxysomes, an intracellular CO.sub.2-concentrating
structure (microcompartment). Carboxysomes are mainly found in all
cyanobacterial species and some other bacteria, for example
nitrifying bacteria. Due to the complexation of RubisCO with the
enzyme carbonic anhydrase, which catalyzes the following
reaction:
HCO.sub.2.sup.-+H.sup.+.fwdarw.H.sub.2CO.sub.2.fwdarw.CO.sub.2+H.sub.2O
[0689] The hydrogencarbonate taken into the cell from the liquid
growth medium around the cell can be converted back to carbon
dioxide, which then in turn can be used by RubisCO in order to
transfer carbon dioxide to ribulose-1,5-bisphosphate thereby
producing two molecules of 3-phosphoglycerate. As a result high
alkaline pH of the growth medium, which favors formation of
hydrogencarbonate greatly enhances the carbon fixation of the cells
cultured in this growth medium. Therefore, if more carbon is fixed
by the cell as a precursor of pyruvate, acetyl-CoA, or
acetaldehyde, then there may be more substrate for the
overexpressed enzyme for production of ethanol and ethanol
production of these genetically modified cells is increased. In
particular, some photoautotrophic strains, for example some
cyanobacterial strains can be adapted to grow in alkaline growth
media having a pH of >8, preferably 9. some preferred 11 to
12.
[0690] Screening for and identifying photoautotrophic strains
having a high mechanical stress tolerance can improve the culturing
of these cells. During culturing the growth medium containing the
cells can be stirred or the growth medium containing the cells can
be pumped from one location to another location thereby subjecting
the cells to a high mechanical stress.
[0691] Furthermore, the photoautotrophic cells can be cultivated in
regions having a high daytime temperature and high solar radiation.
As a result, identifying photoautotrophic strains, which are
tolerant to a high temperature in the growth medium and to a high
amount of solar light can improve the cultivation of these cells
in, for example, sunny and dry or even desert landscapes.
[0692] Determining the viability of the screened photoautotrophic
strains can comprise determining at least one parameter selected
from a group of parameters consisting of:
[0693] growth rate of the photoautotrophic strain,
[0694] ratio of living to dead cells,
[0695] ability to be recultivable in a liquid growth medium in the
absence of the stressful conditions,
[0696] microscopic analysis of the photoautotrophic strain.
[0697] Any of these methods and parameters can be suitable to
determine whether a strain subjected to any of the above-mentioned
stressful growth conditions is still viable or not. The growth rate
of the photoautotrophic strains can, for example, be determined by
measuring the optical density of the cells, for example at a
wavelength of 750 nm in a photometer. The optical density can be
measured before subjecting the photoautotrophic strain to a
stressful condition and can also be taken at certain points of time
while cultivating the photoautotrophic strain under the stressful
condition. For example the optical density of a 0.1-1 ml aliquot
taken from the cell culture can always easily be determined in a
photometer at a wavelength of 750 nm for cyanobacterial cells,
especially in case of non-filamentous cyanobacterial strains.
Further possible methods are cell counting or determination of the
biovolume of the cells.
[0698] The ratio of living to dead cells in a cell culture can, for
example, be determined by detecting the presence of a photopigment
in the photoautotrophic cells. The photopigment can be a chemical
entity that undergoes a physical or chemical change when subjected
to light, for example chlorophyll, carotenoids and phycobilins.
These photopigments are normally excited to a high energy state
upon absorbing a photon. The photopigments can relax from this
excited high energy state by converting the energy into chemical
energy and, for example, induce light-driven pumping of ions across
biological membranes, as is the case, for example for
bacteriorodopsin or via excitation and transfer of electrons
released by photolysis. This reaction can lead to a light-driven
electron transfer chain pumping protons across the membranes of the
cells.
[0699] The presence of the photopigment can, for example, be
detected by measuring the fluorescence of the photopigment, for
example the red auto fluorescence of chlorophyll. The maximum of
the fluorescence emission spectrum is at approx. 700 nm.
[0700] The ability to be recultivable in a liquid growth medium
without the stressful condition to which the photoautotrophic
strain was subjected beforehand can also be an indication of the
viability of the strain. For example a recultivation is considered
to be successful in the case that within, for example 72 hours
after starting with the recultivation the recultivated culture is
growing again and e.g. an increase in the optical density of the
cell culture can be observed.
[0701] Microscopic analysis can be an easy to handle tool to assess
the amount of cell debris and of bleached photoautotrophic cells,
which do not contain photopigments any more. For example an initial
ethanol tolerance can be conducted, subjecting the strain to a
stepwise increased ethanol concentration (for example 5 vol % to 20
vol % of ethanol increased in 5 vol % steps) and a photoautotrophic
strain can be considered as an ethanol tolerant strain in the case
that at least 50% of the cells an survive a concentration of around
10 vol % of ethanol in the growth medium. Living cells can be
distinguished from dead and often lysed cells for example under a
microscope.
[0702] A further embodiment of this method of the invention
comprises the modification that steps b) and c) are repeated
alternatively and in a subsequent step b2) after a first step b1)
the conditions are changed in comparison to the foregoing step b1)
by at least one of:
[0703] increasing the amount of ethanol in the growth medium,
[0704] increasing the amount of salt in the growth medium,
[0705] increasing the pH in the growth medium,
[0706] increasing the rate of agitation during cultivation, and
[0707] increasing the temperature during cultivation.
[0708] In this embodiment of the method, a first step b1) is
performed in which a stressful condition is introduced for the host
cells, for example adding ethanol, adding salt, increasing the pH
of the growth medium or increasing the rate of agitation in the
growth medium or raising the temperature of the growth medium for
the first time. In a subsequent step c1) the viability of the cells
subjected to the stressful conditions in step b1) is analyzed and
then, in a further subsequent step b2) the already present
stressful conditions are increased by increasing, for example, the
amount of ethanol, the amount of salt, the pH of the growth medium
or the rate of agitation and the temperature of the growth medium
during cultivation in comparison to the condition present in step
b1).
[0709] Such a method is a good evaluation tool in order to find out
how a photoautotrophic strain can react to an increasing stress
condition and up to which levels a certain stress condition is
tolerated by a photoautotrophic strain.
[0710] In particular, the amount of ethanol in the growth medium
can be increased stepwise between successive steps b1), b2) and
even further steps b3) and so on. For example, the amount of
ethanol in the growth medium of the photoautotrophic strain can be
increased starting with 5 volume percent of ethanol and increasing
the amount of ethanol in the growth medium in 5 volume percent
steps up to a concentration of 20 volume percent ethanal in the
growth medium. Such an administration scheme of ethanol can be
suitable for a so called "initial ethanol tolerance test" in order
to evaluate whether a photoautotrophic strain has a certain degree
of tolerance to ethanol or not.
[0711] For example, a 5 volume percent concentration of ethanol in
the growth medium can be introduced into the growth medium for at
least 10 minutes and then an aliquot of the photoautotrophic strain
culture, for example a 1 ml aliquot can be taken in order to
determine the optical density (OD) of the cell culture, for example
at a wavelength of 750 nm.
[0712] This cell density can then be compared to an OD measurement
taken before the addition of ethanol to the culture. Such a 10
minute 5 volume percent concentration test of ethanol can be
sufficient in order to determine whether a photoautotrophic strain
has some degree of tolerance to ethanol or not. In the case that a
microscopical observation of the culture shows that there is no
change, that means there is still about the same amount of living
cells in the culture, the ethanol concentration can then be
increased in 5 volume percent steps. For example, it is possible to
subject the strain to a concentration of 10 volume percent of
ethanol for 24 hours and after that determine the optical density
again and also perform the microscopic check of the culture. After
that the concentration of ethanol can be raised again to 15 volume
percent for at least 24 hours and then maybe, after having taken
another aliquot for OD measurement, the culture can be subjected to
a concentration of 20 volume percent of ethanol for two hours.
[0713] The recultivation of the culture subjected to high ethanol
concentrations can be done, for example by centrifuging the
photoautotrophic cell cultures in a centrifuge, for example for 10
minutes at 3,000 rounds per minute (about 3,000 to 4,000 g) in the
case of cyanobacterial cells. The pellet can then be resuspended in
fresh media without ethanol and then again this culture can be
cultivated for at least, for example 72 hours, and the optical
density can, for example, be measured again after 24, 48 and 72
hours and also at the starting point of the culture in order to
determine the growth rate of the recultivated culture.
[0714] Beside varying the end concentrations or values of ethanol,
salt, pH, temperature, rate of agitation and so on, also the
increments of increasing steps of stresses (e.g. 2.5% or 7.5% steps
of ethanol increase) as well as the time period of stress treatment
(e.g. 12 hours or 48 hours at 10% ethanol) can be varied. The
general principle of screening allows for an adaptation of the
detailed screening parameter subject to the results and the
experiences collected.
[0715] Alternatively the amount of ethanol can be continuously
increased during step b). For example it is possible that during
step b) the ethanol is added to the growth medium with a certain
flow rate, for example by using a pump such as an liquid
chromatography (LC) pump and the flow rate is increased between
successive steps b) until a maximum flow rate is reached and then
the flow rate is reduced between the further successive steps b)
again. Such an adding scheme of ethanol results in a sigmoid curve
of the concentration of ethanol in the growth medium. Such a
sigmoidal curve, for example, can mimic the ethanol production by a
genetically altered photoautotrophic ethanol producing cell (see
publication "Ethanol production by genetic engineering in
Cyanobacteria" from Deng and Coleman, 1999; Applied and
Environmental Microbiology, February 1999, pages 523-528). This was
also observed by the inventors. Therefore such an administration
scheme can be particularly valuable in order to determine whether a
certain photoautotrophic strain is tolerant to a rising ethanol
concentration produced by overexpressed enzymes in the cell. Such a
test which is also called a "exact ethanol tolerance test" can also
be used in order to assess the exact ethanol concentration
tolerated by a certain photoautotrophic strain.
[0716] It is also possible to conduct a so-called "long term
ethanol tolerance test" by testing for a long time, for example for
weeks or months, whether a certain photoautotrophic strain can
tolerate relatively low ethanol concentrations of, example given,
0.2, 1 or 5 volume percent in the growth medium. Depending on the
growth rate of the strain this test can also be carried out for a
period of time corresponding to a certain number of cell divisions
for example up to 30 or 40 cell divisions.
[0717] Ethanol tolerant strains, which can tolerate a high amount
of ethanol for a short time, can be tolerant to between 13 to 17 or
more volume percent of ethanol for a short term of around 24 to 26
hours, whereas photoautotrophic strains tolerant to a small amount
of ethanol for a long period of time can tolerate 0.2 to 5 volume
percent of ethanol for weeks or around 30 to 40 cell divisions.
[0718] Another method of the invention furthermore can comprise
that
[0719] method step b) comprises the sub steps b1) and b2) and
method step c) comprises the sub steps c1) and c2)
[0720] a plurality of different photoautotrophic strains to be
tested are first subjected to a first condition including adding a
first amount of ethanol to the growth medium in the method step b1)
and
[0721] cultivating the different photoautotrophic strains for a
first period of time during method step b1) and identifying the
photoautotrophic strains found to be tolerant to the first
condition in method step c1) and thereafter
[0722] subjecting the photoautotrophic strains identified in method
step c1) to a second amount of ethanol for a second period of time
in a subsequent step b2), and
[0723] identifying the photoautotrophic strains tolerant to the
second condition in a method step c2),
[0724] the first amount of ethanol being higher than the second
amount of ethanol, and
[0725] the first period of time being smaller than the second
period of time.
[0726] Such an embodiment of the method of the invention is able to
identify photoautotrophic strains which can tolerate a high amount
of ethanol for a small time in the method steps b1) and c1) and
also can tolerate small amounts of ethanol for a relative long time
in the method steps b2) and c2).
[0727] The tests whether the photoautotrophic strain can tolerate a
high amount of ethanol for a small time can be used to mimic the
ethanol production when using genetically modified photoautotrophic
host cells with enzymes for ethanol production under the
transcriptional control of inducible promoters. In such a case the
cells can be first grown to a very high cell density in an
uninduced state without the production of ethanol and then, after
induction, a high amount of ethanol can be produced in a short
period of time.
[0728] In contrast, the test for ethanol tolerance of relatively
small amounts of ethanol for a long time can mimic the ethanol
production using long cultivation times with constitutive
promoters, which are active without induction. In such a case the
cells always produce relatively small amounts of ethanol during the
whole period of cultivation.
[0729] Furthermore during method step b) salt can be added to the
growth medium by adding for example brackish water, salt water or
so-called "artificial sea water".
[0730] In a further modification of the method of the invention,
during method step b) the growth medium is stirred during the
cultivation in order to mimic mechanical stress conditions. This
can be done, for example by using a magnetic stirrer at a velocity
of 5,000 rounds per minute and the optical density of such a cell
culture can be checked before applying the mechanical stress and
after 48 hours and 96 hours of the cultivation with the mechanical
stress. A photoautotrophic strain found to be tolerant to such a
mechanical stress situation preferably should still grow so that an
increase in the optical density can be observed.
[0731] In particular a stress tolerant photoautotrophic strain can
be distinguishable from a photoautotrophic strain, which is not
stress tolerant by its ability to grow in moved and pumped water in
comparison to a non-mechanical stress tolerant strain, which can
only grow in relatively still water.
[0732] In order to assess the tolerance for high temperature
conditions during method step b), the photoautotrophic strain is
cultivated in a growth medium at elevated temperatures of at least
42.degree. C. or even at 45.degree. C. for more than 48 or 96
hours. Again the optical density can be measured before increasing
the temperature of the growth medium and also after 48 and 96 hours
in order to determine the growth rate of the photoautotrophic
strain under high temperature conditions. A photoautotrophic strain
which is high temperature tolerant still has to grow under these
conditions whereas a photoautotrophic strain which is not tolerant
to these conditions cannot grow anymore.
[0733] If someone wants to determine, what growth speed and which
maximal optical density can be reached by certain strains and
whether a certain photoautotrophic strain can tolerate relatively
low ethanol concentrations of, e.g., 0.2, 1 or 5 volume percent in
the growth medium during method step b), the photoautotrophic
strain can be subjected to a first light intensity and a first
CO.sub.2 concentration in the lag phase and in the exponential
growth phase and after having reached a stationary phase the light
intensity and the carbon dioxide concentration can be increased to
a second light intensity and to a second carbon dioxide
concentration.
[0734] In addition, samples of the strain can be taken at different
growth phases (lag, log, stationary phase and stationary phase
after addition of ethanol) for later analysis of the intracellular
metabolites.
[0735] Such a test can determine the growth behavior of the
photoautotrophic strain to be cultured under high light conditions
and high carbon dioxide concentrations. The light intensity and the
carbon dioxide concentrations can be increased when the
photoautotrophic strain has reached stationary growth in order to
test whether the light intensity and the carbon dioxide
concentrations are limiting factors for the growth of the cells. If
this is the case the cells can start growing again after having
reached stationary phase when exposed to higher light intensity and
higher carbon dioxide concentrations. For example, the first light
intensity can be 40 .mu.E/m.sup.2s per day and then it can be
increased to 120 .mu.E/m.sup.2s and further to 220 .mu.E/m.sup.2s
once the stationary phase is reached. In general the light
intensity can vary between 40 .mu.E/m.sup.2s and 100
.mu.E/m.sup.2s. The carbon dioxide concentration can be increased
from 0.5 vol % to 5 vol % or can vary between 2 vol % and 5 vol
%.
[0736] For the determination of light tolerance strains with
defined cell densities at the beginning of the experiment should be
cultivated under certain light intensities (e.g. 100, 250, or 500
.mu.E/m.sup.2s) for at least 5 days and growth rates should be
measured as it was done in the other tests.
[0737] In addition to testing the ability of a photoautotrophic
strain to be tolerant to certain stressful growth conditions, it
also can be useful to test the presence and the amount of toxins
produced by the photoautotrophic strain. This, for example, can be
done by high performance liquid chromatography (HPLC) and/or mass
spectrometry (MS). Using analytical standards both methods can
identify and quantify a toxin. In case of the HPLC the
quantification is usually more exact by using toxin-specific
absorption maxima for the quantification whereas in case of MS the
identification is more exact by detecting the molecular mass of a
toxin. Toxins produced by the photoautotrophic strains can also be
released into the environment during cultivation and can pose harm
to any people involved in the cultivation of these strains or to
the environment. Therefore these strains have to be filtered out
from the above screening procedures and normally cannot be used for
ethanol production. Or the genes responsible for the toxin
producing enzymes have to be knocked out by genetic
engineering.
[0738] Furthermore, the photoautotrophic strains identified to be
tolerant to certain stressful cultivation conditions also should be
genetically transformable. This is due to the fact, that enzymes
for ethanol production might have to be introduced into these
photoautotrophic cells in order to obtain a sufficient ethanol
production rate. Due to that an abovementioned screening method
also can comprise the method step of:
[0739] subjecting the photoautotrophic strain to a transforming
factor, conferring a marker property,
[0740] detecting the presence of a marker property in the
strain.
[0741] The marker property can be any easily detectable marker
property, for example an antibiotic resistance or for example
fluorescence. The transforming factor can be a plasmid, which can
be introduced into the photoautotrophic cell for genetic
modification. The plasmid can be an extra chromosomal,
self-replicative plasmid, which is introduced into the cell without
being integrated into the genome of the host cell. Additionally or
alternatively an integrative plasmid can be used, which can be
integrated into the genome of the host cell, for example via
homologous recombination. Tests for genetic transformability can,
for example, include a test for conjugation or a test for the
natural competence of a strain to take up DNA. In addition,
electroporation tests can also be performed. In order to identify
transformable photoautotrophic strains the strains can be
cultivated on agar plates or liquid cultures including the
corresponding antibiotic after transformation. False positive
strains due to naturally occurring resistances of cells can be
eliminated by performing a polymerase chain reaction (PCR) in order
to detect the plasmid in the transformed cells.
[0742] Another reporter for a transformation event could be the
green fluorescence protein (GFP) allowing for the detection of an
transformed plasmid under the microscope. The expression of gene
encoding for the green fluorescence protein (GFP) on a plasmid
leads to autofluorescence after a successful transformation after
UV excitation of the cell.
[0743] In addition the method for screening the photoautotrophic
strains also can comprise the further step of determining the
photosynthetic activity of the photoautotrophic strain to be
tested. The rationale behind this additional testing step can be
that, on the one hand someone can screen for photoautotrophic
strains having a high tolerance for stressful cultivation
conditions, but on the other hand someone also wants to identify a
photoautotrophic strain having a high photosynthetic activity. Such
a method step can be useful in order to further distinguish high
stress tolerant photoautotrophic strains with a low photosynthesis
rate from other high stress tolerant photoautotrophic strains with
a high photosynthesis rate. The photosynthesis rate, for example,
can be measured by the oxygen generation of the photoautotrophic
strain in different growth phases using an oxygen electrode.
[0744] A minimum rate that should be observed in the test should be
150 .mu.Mol O.sub.2/hmg chlorophyll (180 .mu.Mol O.sub.2/hmg
chorophyll e.g. corresponds to the Model organism Synechocystis
PCC6803).
[0745] In particular a photoautotrophic strain with a high oxygen
production is desirable because high oxygen production correlates
with a high CO.sub.2 fixation, which result in high levels of
ethanol formation. Also a strain can be subjected to certain growth
conditions (e.g. marine media, higher pH, higher temperature,
higher bicarbonate content etc.) and then be checked for the
photosynthesis rate under these conditions.
[0746] A so-called "Initial growth test" can be carried out in
order to get a good hint of the photosynthetic activity of the
photoautotrophic strains by a more or less simple comparison of
growth speed of strains allowing for an easier test and a higher
throughput of strains. Beside the optical density or biovolume also
the dry weight production should be determined as a growth
parameter, that also corresponds to the carbon fixation in the same
way as the generation of oxygen.
[0747] A further embodiment of the above-mentioned screening method
can include a method for identifying a photoautotrophic strain with
a tolerance for at least a first and a second growth condition
selected from the above-mentioned growth conditions from a
plurality of different photoautotrophic strains, comprising:
[0748] culturing the plurality of different photoautotrophic
strains under a first growth condition in method step b1),
[0749] identifying the photoautotrophic strains tolerant to the
first growth condition in method step c1) and thereafter
[0750] culturing the photoautotrophic strains identified in method
step c1) under a second growth condition in a further step b2), the
second growth condition being different from the first growth
condition,
[0751] identifying the photoautotrophic strains tolerant to the
second growth condition in method step c2).
[0752] During such a screening method photoautotrophic strains
found to be tolerant to a first stressful condition are then
selected for screening for a second, different stressful growth
condition. Such a screening method is useful in order to identify
photoautotrophic strains having multiple tolerances for different
stressful growth conditions.
[0753] For example the stressful growth conditions can be high
light intensity as well as high concentrations of ethanol, or other
stressful growth conditions, such as above neutral pH growth media
and high salinity growth media. Such a method can also be used in
order to identify photoautotrophic strains having a tolerance to
more than two stressful growth conditions. This can simply be done
by extending the above-described method by further method steps for
example b3) and c3) using the photoautotrophic strains found to be
tolerant to the second stressful growth condition for further
screening.
[0754] In a further embodiment of the method of the invention, the
method can be used to identify a photoautotrophic strain with a
tolerance for at least the first an the second stressful condition
and additionally at least one desired property selected from a
group consisting of:
[0755] high photosynthetic activity, lack of ability to produce
toxins and ability to be genetically transformable
from the plurality of different photoautotrophic strains,
comprising at least one further method step d) selected from a
group of method steps consisting of:
[0756] determining the photosynthetic activity of the
photoautotrophic strain,
[0757] subjecting the photoautotrophic strain to a transforming
factor, conferring a marker property, and detecting the presence of
the marker property in the strain, and
[0758] determining the presence and amount of toxins produced by
the photoautotrophic strain, and
[0759] identifying the photoautotrophic strain having any of the
above abilities in a further method step d),
[0760] wherein the method steps d) and e) can be performed before
or after the method steps b1) and c1) or b2) and c2).
[0761] Such a method can be used in order to additionally screen
for photoautotrophic strains which have a high photosynthetic
activity, a lack of ability to produce toxins and the ability to be
genetically transformable. These further tests can be done before
or after the screening tests for stressful growth conditions.
[0762] In a further variant of the method of the invention the
method steps d) and e) are performed before method steps b1) and
c1) or b2) and c2).
[0763] In particular in one embodiment of the method of the
invention the first method step d) of the screening method
comprises determining the photosynthetic activity of the
photoautotrophic strain. This can for example be done by carrying
out the "test for photosynthetic activity" as later described
therein. Photoautotrophic strains are identified as being positive
in this test if they show a minimum photosynthetic activity of at
least 150 .mu.Mol O.sub.2/hmg chlorophyll, more preferred 200
.mu.Mol, most preferred at least 250 .mu.Mol O.sub.2/hmg
chlorophyll.
[0764] An evaluation of around 180 photoautotrophic strains tested
in the screening method of the invention shows that only roughly
30% of the tested strains exhibit photosynthetic rates, which
satisfy these above values. In contrast to that, most of the tested
photoautotrophic strains would pass the test for short term and
long term ethanal tolerance (roughly 75% and 65%, respectively)
Further roughly 75% of the tested photoautotrophic strains passed
the salt tolerance test, the test for the ability to grow in
brackish or salt water such as marine media. Only about 25% of the
tested photoautotrophic strains passed the test for
thermo-tolerance. However this test is strongly dependent on the
ambient temperature of each intended production site and therefore
has to be adapted on a case by case basis.
[0765] These data therefore show that by first conducting the test
for the photosynthetic activity or capacity most of the tested
photoautotrophic strains can be discarded in the first test step,
which makes it easier to further process the few remaining
photoautotrophic strains, which have passed this test, through the
other tests.
[0766] After having carried out the first test for photosynthetic
activity method step b1) can for example be conducted, comprising
the step (i) of adding ethanol to the growth medium.
[0767] In particular the "short term ethanol tolerance test", which
can be carried out quickly can be performed wherein during step b1)
the photoautotrophic strains are subjected to at least between 13
to 17 vol % of ethanol for around 24 to 26 hours, preferably to at
least 10 vol %, more preferred to at least 15 vol % most preferred
to at least 20 vol % of ethanol for at least 52 hours as a first
growth condition.
[0768] The photoautotrophic strains tolerant to these ethanol
conditions can then be identified in method step c1) via
microscopic analysis and/or their ability to be recultivable.
[0769] The strains, which fail this "short term ethanol tolerance
test" are discarded and the other photoautotrophic strains which
passed this test are in a further method step b2) subjected to 0.2
to 5 vol % of ethanol for around 30 to 40 cell divisions,
preferably to around 1 vol % of ethanol for around 5 weeks, more
preferred for around 10 weeks most preferred for at least 15 weeks
as a second growth condition. This test is the so-called "long term
ethanol tolerance test" intended to test the long term ability of
the photoautotrophic strains to withstand relatively small amounts
of ethanol for a longer period of time.
[0770] After this method step b2), the photoautotrophic strains
tolerant to the growth conditions of method step b2) are identified
via microscopic analysis and/or their ability to be recultivable in
a further method step c2).
[0771] After that, an additional method step b3) can be carried out
by culturing the photoautotrophic strains, which passed the second
growth test under a third growth condition, such as a salt
tolerance test and further conducting the method step c3) of
identifying the photoautotrophic strains tolerant to the third
growth condition.
[0772] Subsequently further testing steps b4), b5), b6) and their
respective analysis steps c4), c5, c6) can be carried out to test
for even more growth conditions and/or desired properties of the
photoautotrophic strains.
[0773] For example the fourth growth condition to be tested in the
method steps b4) and c4) respectively can be the ability of the
photoautotrophic strains to tolerate an increase in the temperature
to 45.degree. C., the so-called "thermo tolerance test". Afterwards
the photoautotrophic strains having passed these four growth
conditions can be subjected to further growth conditions and
additionally can be tested for at least one desired property,
selected from a group consisting of:
[0774] lack of ability to produce toxins, ability to be genetically
transformable, increasing the pH of the growth medium, agitating
the growing culture, the maximal optical density or dry weight per
volume, pool size of intracellular metabolites in different growth
phases in the absence or presence of EtOH, and light tolerance of a
strain.
[0775] The further tests for these growth conditions and the at
least one desired property can be tested sequentially or in
parallel. These tests can be used in order to further characterize
the photoautotrophic strains without discarding strains, which do
not perform well in one of these tests.
[0776] Summing it up, one preferred embodiment of the screening
method of the invention comprises the following method steps:
[0777] a) providing various photoautotrophic strains to be tested
for example by obtaining photoautotrophic strains from public
depositories or by picking photoautotrophic strains from natural
habitats.
[0778] d) Test for photosynthetic activity or capacity,
[0779] e) Identifying the photoautotrophic strains having a desired
value of photosynthetic activity,
[0780] b1) short term ethanol tolerance test,
[0781] c1) identifying the photoautotrophic strains being tolerant
to the short term ethanol tolerance test,
[0782] b2) long term ethanol tolerance test,
[0783] c2) identifying the photoautotrophic strains being tolerant
to the long term ethanol tolerance test,
[0784] b3) salt tolerance test,
[0785] c3) identifying the photoautotrophic strains being tolerant
to the salt tolerance test,
[0786] b4) thermo-tolerance test,
[0787] c4) identifying the photoautotrophic strains being tolerant
to the thermo-tolerance test,
[0788] Then further test being carried out sequentially or in
parallel consisting of a group of:
[0789] lack of ability to produce toxins, ability to be genetically
transformable, increasing the pH of the growth medium, agitating
the growing culture, the maximal optical density or dry weight per
volume, pool size of intracellular metabolites in different growth
phases in the absence or presence of EtOH, and light tolerance of a
strain
[0790] In an alternative embodiment of the screening method of the
Invention, the method steps d) and e) are performed after the
method steps b1) and c1). In this case the method step b1) can for
example comprise the step (i) of adding ethanol to the growth
medium. In particular during the method steps b1) and c1) can
comprise the short term ethanol tolerance test and identifying the
strains being tolerant to this short term ethanol tolerance
test.
[0791] Such as screening method has the advantage that the short
term ethanol tolerance test is not so laborious as the
determination of the photosynthetic activity so that a large number
of photoautotrophic strains can be tested in a relative small
period of time.
[0792] The further method steps b2) and c2) can then comprise the
long term ethanol tolerance test and identifying the strains being
tolerant to this long term ethanol tolerance test. Afterwards
further tests b3) and c3) related to photosynthetic activity and
method steps b4) c4) directed to the evaluation of salt stress
tolerance can be conducted.
[0793] In another concrete example of the screening method of the
invention, it is possible to first conduct the above-mentioned
"initial ethanol tolerance test", followed by a test of the
photosynthesis rate of the strain and an "initial growth test".
Then the so-called "exact ethanol tolerance test" can be performed
in order to find out the exact amounts of ethanol to which a
certain strain is tolerant. In a subsequent test the
photoautotrophic strain can be subjected to a so-called "long term
ethanol tolerance test" in order to find out whether this strain
can tolerate small amounts of ethanol for a long period of time.
After that the so-called "thermo-tolerance" and "mechanical stress
tolerance test" can be conducted with the strains, which were found
to be highly tolerant to ethanol and were found to have a high
photosynthetic activity. Afterwards an HPLC and/or MS analysis of
the content of the natural products of the photoautotrophic
strains, which tolerate any of the above stressful conditions can
be performed in order to find out whether any of the highly ethanol
tolerant strains produces natural toxins. Subsequently, an alkaline
media test can be conducted in order to test for the above neutral
pH tolerance of the strains identified so far. One of the last
tests can be a test for the ability to grow the selected fresh
water strains in marine media or brackish media. There is the
possibility to transform a strain in order to increase the salt
resistance, if necessary. The last test then can be the so-called
"exact growth test" in order to determine the growth behavior under
high light conditions.
[0794] By carrying out the above mentioned different embodiments of
the screening method of the invention photoautotrophic strains can
be identified, which passed all high priority tests, namely the
test for photosynthetic activity, the long and short term ethanol
tolerance test and the salt and thermo tolerance test as shown in
FIG. 50-15. For example Synechocystis PCC 6803 passed all four of
these tests whereas Nostoc sp. PCC 7120 did not pass at least the
thermo tolerance test.
[0795] The photoautotrophic strains to be tested can, for example,
be selected or picked from a collection of different
photoautotrophic strains, for example obtained from publicly
available strain databases, for example the PCC-Pasteur Culture
Collection found on the world wide web at
pasteur.fr/recherche/banques/PCC or the SAG, the so-called
"Algensammlung aus Gottingen" algal collection of the university of
Gottingen found on the world wide web at
epsag.uni-goettingen.de.
[0796] In particular it is advantageous to pre-select the strains
found in publicly available strain databases for strains known to
be fast-growing strains, dominant strains with high photosynthetic
activity and strains known to be able to produce mass populations
in nature. For example it is useful to select Synechocystis,
Synechococcus, Spirulina, Arthrospira, Nostoc, Anabaena,
Trichodesmium, Leptolyngbya, Plectonema, Myxosarcina, Pleurocapsa,
Oscillatoria, Phormidium, Anabena, Pseudanabena or comparable
genera, because these strains are for example known to produce
algal blooms, which are a sign of cyanobacterial mass populations
in nature (e.g. Trichodesmium) or are known from industrial large
scale-processes (e.g. Spirulina).
[0797] The photoautotrophic strains, in particular the algal and
cyanobacterial strains selected in any of the above screening
methods can then be used for genetic transformation in order to
produce any of the photoautotrophic genetically modified ethanol
producing host cells already mentioned above in this patent
application. In particular enzymes for the formation of ethanol can
be introduced into these selected photoautotrophic strains. These
enzymes for the formation of ethanol can be selected from a group
consisting of: Adh, Pdc, CoA-dependent acetaldehyde dehydrogenase,
AdhE, and an acetaldehyde dehydrogenase converting acetylphosphate
into acetaldehyde.
[0798] Further at least one genetic modification can be introduced
into these selected photoautotrophic strains,
[0799] this genetic modification changing the enzymatic activity or
affinity of an endogenous host cell enzyme of the photoautotrophic
strains,
the genetic modification resulting in an enhanced level of
biosynthesis of acetaldehyde, pyruvate, acetyl-CoA or precursors
thereof compared to the respective wild type host cell.
Embodiments of Algae and Bacteria
[0800] In a further embodiment of the invention the genetically
modified photoautotrophic ethanol producing host cell is an aquatic
organism. This aquatic organism can, for example, be a fresh water
species living in lakes, rivers, streams or wetlands. Alternatively
the aquatic organism can be a marine organism, which lives in salty
water, for example oceans. The aquatic organism also can be a fresh
water species, which shows a high tolerance for brackish water or
even salt water. The inventors also found fresh water strains that
can grow in marine media with the same growth rate as in fresh
water media, which were selected from a large variety of different
cyanobacterial strains by using the method for testing a
photoautotrophic strain for a desired growth property disclosed in
this patent application.
[0801] In a further embodiment the genetically modified host cell
is selected from a group consisting of: algae and bacteria.
[0802] Algae are a diverse group of simple plant-like organisms
which include unicellular or multicellular forms. Algae are
photosynthetically active organisms, in particular photoautotrophs,
which produce organic compounds from Inorganic molecules such as
CO.sub.2 and water using light as an external source of energy.
[0803] Algae are considered to be eukaryotic organisms in
particular protists. Protists are relatively simple eukaryotic
organisms which are unicellular or multicellular without highly
specialized tissues.
[0804] In particular, protist algae can include Chlorophytes, which
are green algae, such as Ulva chlatrata, Rhodophytes, red algae or
heterokontophytes, which are brown algae. A preferred green algal
species is Chlorella. One example of a green algae is
Chlamydomonas, which are unicellular flagellates. A particular well
known example of Chlamydomonas is Chlamydomonas reinhardtii, which
is a motile single-celled green algae found in, for example, fresh
water. Chlamydomonas reinhardtii is also known to produce minor
amounts of ethanol via fermentation under dark conditions (Gfeller
and Gibbs, Fermentative Metabolism of Chlamydomonas reinhardtii,
Plant Psychology (1984) 75, pages 212 to 218).
[0805] Various methods for transformation of eukaryotic algae are
known. For example the Chlamydomonas reinhardtii chloroplast genome
was transformed by using microprojectile particle bombardment. This
method involves introducing gold or tungsten particles into the
cell, which are coated with DNA for transformation. These particles
are accelerated into the target cells by helium-driven particle
guns. This technique can be used in order to transform
undifferentiated plant cells, which for example grow on a gel
medium in a Petri dish and which are subjected to a nanoparticle
beam of the DNA-coated gold or tungsten particles. This technique
has been successfully used in order to transform Chlamydomonas
reinhardtii. References describing the particle gun method are for
example, Boynton, J. E., Gillham, N. W., Harris, E. H., Hosler, J.
P., et al (1988) "Chloroplast transformation in Chlamydomonas with
high velocity microprojectiles". Science 240: 1534-1538; Debuchy,
R., Purton, S, and Rochaix, J. D. (1989) "The argininosuccinate
lyase gene of Chlamydomonas reinhardtii: an important tool for
nuclear transformation and for correlating the genetic and
molecular maps of the ARG7 locus". EMBO J. 8: 2803-2809; Kindle, K.
L., Schnell, R. A., Fernandez, E. and Lefebvre, P (1989) "Stable
nuclear transformation of Chlamydomonas using the Chlamydomonas
gene for nitrate reductase". J Cell Biol 109: 2589-2601; Dunahay,
T. G., Jarvis, E. E., Davis, S. S, and Roessler, P. G. (1995)
"Genetic transformation of the diatoms Cyclotella cryptica and
Navicula saprophila". J Phycol 31: 1004-1012; Apt, K. E.,
Kroth-Pancic, P. and Grossman, A. R. (1996) "Stable nuclear
transformation of the diatom Phaeodactylum tricomutum". Mol Gen
Gent 252: 572-579; Falciatore, A. et al. (1999) "Transformation of
nonselectable reporter genes in marine diatoms". Mar Biotechnol 1:
239-251; Zaslayskaia, L. A., Lippmeier, J. C., Kroth, P., Grossman,
A. and Apt, K. E. (2000) "Transformation of the diatom
Phaeodactylum tricomutum (Bacillariophyceae) with a variety of
selectable marker and reporter gene". J. Phycol. 36: 379-386.
[0806] Another method of transforming eukaryotic or prokaryotic
algae is, for example, the introduction of genes into host cells by
electroporation. This method involves applying an external electric
field to a probe containing the eukaryotic or prokaryotic cells to
be transformed. This electrical field leads to a significant
increase of the electrical conductivity and permeability of the
cell plasma membranes of the cells to be transformed. Therefore,
DNA can be taken up by cells subjected to such an external
electrical field.
[0807] A further method of genetic transformation of eukaryotic
algae is the glass bead agitation. This method involves vortexing
the algal cells to be transformed with glass beads in the presence
of DNA and, for example, polyethylene glycol. This method can be
used in order to transform cell wall deficient mutants of
microalgae species, for example Chlamydomonas. An overview of
different methods of genetic transformation of microalgae is
presented in the review article of Banares at al. (Banares et al.:
Transgenic Microalgae as Green Cell Factories, Trends in
Biotechnology, vol. 22, no. 1 (2004), pages 45 to 52).
[0808] In a further embodiment of the invention the genetically
modified host cell comprises a cyanobacterium. Cyanobacteria are
also known as Cyanophyta or blue green algae and are prokaryotic
bacteria, which are photosynthetically active. Cyanobacteria
include unicellular or multicellular species. Cyanobacteria include
fresh water species or marine species. In addition, cyanobacterial
species also can be found in brackish water. In contrast to
eukaryotic algae cyanobacteria lack a nucleus, mitochondria or
chloroplasts. Examples of cyanobacterial species include
Synechococcus. Synechocystis and Phormidium.
[0809] A genetically modified cyanobacterial cell according to the
invention can be derived from cyanobacteria, which perform ethanol
fermentation even in the genetically unmodified wild-type state.
Examples of ethanol fermenting wild type cyanobacterial species
are, for example, Oscillatoria limosa (Stal L; Heyer H.; Bekker S.;
Villbrandt M.; Krumbein W. E. 1989. Aerobic-anaerobic metabolism in
the cyanobacterium Oscillatoria limosa. In: Cohen, Y., and
Rosenberg, E. (ed.), Microbial mat: Physiological ecology of
benthic microbial communities. American Society for Microbiology
Washington, D.C.). Another example of an ethanol-fermenting
cyanobacterial species is the cyanobacterium Microcystis PCC7806
(Moezelraar et al., A Comparison of Fermentation in the Cyano
Bacterium Microcystis PCC7806 grown under a Light/Dark Cycle and
continuous Light, European Journal of Phycology (1997), 32, pages
373 to 378). Further examples of ethanol fermenting cyanobacteria
are Cyanothece PCC 7822, Microcystis aeruginosa PCC 7806,
Oscillatoria sp., and Spirulina platensis ("The ecology of
cyanobacteria, Their Diversity in Time and Space, Edited by Brian
A. Whitton and Malcolm Potts, Kluwer Academic Publishers, Chapter 4
by L. J. Stal Cyanobacterial Mats and Stromatolites").
[0810] In another aspect the invention provides a method for the
production of ethanol comprising the method steps of:
[0811] A. Providing and culturing any of the above-mentioned
genetically modified host cells in a growth medium under the
exposure of light and carbon dioxide, the host cells accumulating
ethanol while being cultured, and [0812] B. Isolating the ethanol
from the host cells and/or the growth medium.
[0813] As discussed above, the genetically modified host cells can
comprise cyanobacteria, algal cells or other phototropic organisms.
The photoautotrophic genetically modified host cell can produce the
ethanol intercelluarily from sunlight, carbon dioxide and water and
then excrete the ethanol into the growth medium. The growth medium
can, for example, be sea water in the case of marine strains, or
fresh water in the case of freshwater strains or brackish water,
which can be supplemented with trace elements for example a
fertilizer liquid. The ethanol can then be separated from the
liquid growth medium, for example by distillation.
[0814] During method step A host cells can be provided which
comprise a genetically modified gene encoding at least one enzyme
for the formation of ethanol under the transcriptional control of
an inducible promoter, which can be induced by exposure to an
exogenous stimulus, wherein the method step A further
comprises:
A1. Culturing the host cells under the absence of the exogenic
stimulus, and thereafter A2. Providing the exogenic stimulus,
thereby inducing ethanol production.
[0815] During such a variant of the method of the invention the
host cells can grow without producing ethanol in the un-induced
state. Due to the fact that ethanol can be harmful to the host
cells, the host cells can reach a higher cell density when cultured
in an un-induced state compared to a situation where the host cells
continuously produce ethanol. This variant of the method of the
invention can, for example, be used in the case that the substrate
for the at least one enzyme for the formation of ethanol is not
harmful to the host cells and is, for example, pyruvate or
acetyl-CoA. These compounds can easily be further metabolized even
by an uninduced host cell.
[0816] The exogenic stimulus can, for example, be provided by
changing the environmental conditions of the host cells depending
on the inducible promoter. For example, the stimulus can be
provided by subjecting the cell culture to darkness, for example in
the case that the inducible promoter is the sigB promoter or the
IrtA promoter. The exogenic stimulus can also be provided by a
nutrient starvation in the case that the inducible promoter is, for
example, the ntcA promoter, the nblA promoter, the isiA promoter,
the petJ promoter, or the sigB promoter. One way of subjecting a
growing cell culture to nutrient starvation can be that the cell
culture consumes the nutrients in the growth medium while growing
and therefore automatically reaches a state of nutrient starvation
in the case that no new nutrients are supplemented into the growth
medium. The nutrients required for the growth of the host cells
can, for example, be trace elements such as phosphorous, nitrogen
or iron.
[0817] The exogenic stimulus can furthermore be provided by
subjecting the growing cell culture to oxidative stress, for
example by adding oxidants such as hydrogen peroxide to the
culture. Inducible promoters which can be induced by oxidative
stress are, for example the isiA promoter. Further examples of
exogenic stimuli are, for example, heat shock or cold shock which
can be induced by raising the temperature of a growing culture from
30.degree. C. to, for example, 40.degree. C. or by reducing the
temperature of a growing culture from 30.degree. C. to 20. .degree.
C. An example of a heat shock inducible promoter are the htpG,
hspA, clpB1, hliB and sigB promoter and an example of a cold shock
inducible promoter is the crhC promoter. A further example of an
exogenic stimulus can be stationary growth which automatically is
reached by a growing culture in the case that the culture is not
diluted and no new nutrients are added. Examples of stationary
growth inducible promoters are the isiA promoter or the sigB
promoter. Furthermore the exogenic stimulus can be provided by
addition of a nutrient, for example by adding copper in the case of
copper inducible petE promoter.
[0818] Alternatively the at least one enzyme for ethanol production
can be under the transcriptional control of a constitutive
promoter, for example the rbcLS promoter. Such a culture produces
ethanol during all phases of the cell growth, in particular during
the lag phase, during the exponential growth phase and even after
having reached the stationary phase. Such a method of producing
ethanol can be particularly valuable in the case that the
genetically modified host cells comprise a first genetic
modification which results in an increased affinity or activity of
a host metabolic enzyme, which produces metabolic intermediates
harmful to the cell, for example acetaldehyde. In such a case the
genetically modified host cells normally also comprise a second
genetic modification resulting in an overexpression of an enzyme
for ethanol formation from the harmful metabolic intermediate. Due
to the fact that the second enzyme for the formation of ethanol is
under the transcriptional control of a constitutive promoter and
therefore is expressed during all stages of the growth, the harmful
metabolic intermediate can quickly be further converted by this
enzyme for ethanol formation into ethanol. Therefore, the metabolic
intermediate which is harmful for the cell normally cannot
accumulate in high amounts in the cell or in the growth medium of
the cells.
[0819] According to another aspect of the invention the method step
A further comprises the method step:
A3. Adding a substrate to the growth medium, the substrate used by
the at least one overexpressed enzyme for ethanol formation to
produce ethanol.
[0820] The substrate, for example, can be acetaldehyde. The
inventors experienced that often the availability of a substrate is
limiting for ethanol production in the case that genetically
modified host cells with at least one overexpressed enzyme for
ethanol formation are used to produce ethanol. In such a case the
further addition of the substrate of the overexpressed enzyme for
ethanol formation can greatly enhance the ethanol production.
[0821] In a further embodiment the method comprises determining an
optimum concentration range for the substrate used by the at least
one overexpressed enzyme for ethanol formation in the growth
medium. The substrate can then be added in an amount within this
optimum concentration range. The inventors determined that an
optimum concentration range for a substrate like acetaldehyde is
between 150 .mu.M and 200 .mu.M.
[0822] According to a further aspect of the method of the
invention, the method can comprise the additional method step C of
using the host cells after having isolated the ethanol in method
step B as a substrate for a heterotrophic fermentative organism.
The heterotrophic organism can ferment the biomass provided by the
host cells for ethanol production in order to produce different
fermentative products, depending on the fermentation mechanism.
[0823] For example the heterotrophic organism can comprise
ethanol-fermenting organisms, such as yeast, which can produce
ethanol from fermentation of the host cell biomass. In another
embodiment of the method of the invention methane can be produced
by methanogenic microorganisms while fermenting the host cell
biomass. These methanogens, for example can produce methane from
acetic acid, which is produced by other fermentative microorganisms
from the biomass provided by the host cells.
[0824] According to another embodiment of the method of the
invention during method step A, the genetically modified host cells
produce a first metabolic intermediate and at least partially
secret the first metabolic intermediate into the growth medium, and
during method step A a microorganism is added to the growth medium,
the microorganism converting the first metabolic intermediate into
ethanol.
[0825] Especially in the case that the genetically modified host
cells comprise a first genetic modification changing the affinity
or activity of a host cell enzyme leading to a higher production of
a first metabolic intermediate, the first metabolic intermediate is
often excreted from the host cells into the growth medium. In such
a case ethanol formation can be enhanced by adding a microorganism,
for example a fungus which can metabolize the excreted metabolic
intermediate into ethanol.
[0826] More detailed description of the embodiments with reference
to the figures
[0827] In the following the inventions will be explained in more
detail with reference to figures and certain embodiments.
[0828] FIGS. 1A to C depict general schemes of metabolic pathways
in Cyanobacteria with marked enzymes for overexpression and
down-regulation or knock-out for the increase of biosynthesis of
different metabolic intermediates.
[0829] FIG. 2 shows a flow chart including some ethanologenic
enzymes for ethanol production.
[0830] FIG. 1A shows some general metabolic pathways in
cyano-bacteria as a non-limiting example, in particular the Calvin
cycle as the light independent part of the photosynthesis is shown
starting with the carbon dioxide fixation reaction catalyzed by the
enzyme RubisCO. Further the glycolysis pathway, the pentose
phosphate pathway and the citric acid cycle are shown. The general
metabolic pathways depict boxed and circled enzymes, whose activity
or affinity can be changed as part of at least one first genetic
modification of an endogenous host enzyme of the cyanobacterial
host cell. Boxed enzymes either have been overexpressed compared to
the respective wild type cyanobacterial cells or are prime
candidates for overexpression. Circled enzymes either have been
knocked out or down regulated or are prime targets for knock-out or
down-regulation. The main reason for the knock-out or
overexpression is to enhance the level of pyruvate biosynthesis in
the genetically modified cell by knocking-out or reducing the
activity or affinity of enzymes consuming pyruvate or its
metabolites and to enhance the enzymatic activity of enzymes
producing pyruvate or its precursors such as phosphoenolpyruvate
(PEP). The cyanobacterial host cell can comprise more than one
first genetic modification. For example enzymes enhancing the level
of pyruvate biosynthesis such as enolase or malic enzyme can be
overexpressed and the activity or affinity of enzymes consuming
pyruvate, such as lactate dehydrogenase or alanine dehydrogenase
can be reduced or abolished by knock-out of the respective genes in
one cyanobacterial host cell.
[0831] In addition two second genetic modifications resulting in an
overexpression of enzymes for ethanol formation have been
introduced into the metabolic cyanobacterial pathways shown in FIG.
1A. These enzymes are indicated by the thickly framed boxes denoted
with the reference sign "A". In particular these enzymes are
alcohol dehydrogenase (abbreviated as Adh) and pyruvate
decarboxylase (abbreviated as Pdc), which also have to be
introduced into most cyanobacteria via genetic engineering.
[0832] FIG. 1B shows the same general metabolic pathways in
cyano-bacteria as already presented in FIG. 1A for the case that
the level of biosynthesis of acetyl-CoA is raised compared to a
wildtype cyanobacterial cell. The enzymes, which are part of the
first and second genetic modification are marked in the same way as
in FIG. 1A. In addition the direct conversion of acetyl-CoA to
ethanol catalyzed by the enzyme aldehyde-alcohol dehydrogenase
AdhE, which has to be introduced into most cyanobacteria via a
second genetic modification is denoted. AdhE is for example an
endogenous enzyme in the cyanobacterium Thermosynechococcus or an
heterologous enzyme from E. coli. In this case the expression of
AdhE can be enhanced in a second genetic modification in
Thermosynechococcus, for example by introducing additional gene
copies into the cell or by mutating the promoter of the wildtype
gene encoding AdhE in order to enhance transcription and
translation. In the case of overexpression of AdhE the enzyme
pyruvate dehydrogenase can be overexpressed (shown as a boxed
enzyme). In addition to overexpression of AdhE It is still possible
to overexpress Pdc and Adh simultaneously. Alternatively only AdhE
can be overexpressed.
[0833] FIG. 1C gives an overview of metabolic enzymes in
cyanobacteria, which can be overexpressed (boxed enzymes) or
knocked out or downregulated (circled enzymes) in the case that the
level of biosynthesis of acetaldehyde is to be increased in the
cell. In this case the enzymes phosphotransacetylase and
acetaldehyde dehydrogenase are overexpressed in comparison to the
situation shown in FIG. 1B. The enzyme acetaldehyde dehydrogenese
converting acetylphosphate to acetaldehyde is for example disclosed
in the publication Stal (Stal, Moezelaar, "Fermentation in
cyanobacteria", FEMS Microbiology Reviews 21, (1997), pages
179-211). The enzymes, which are part of the first and second
genetic modification are marked in the same way as in FIGS. 1A and
1B.
[0834] FIG. 1D depicts the exemplary metabolic pathway of other
bacteria. In contrast to the metabolic pathways shown in the FIGS.
1A to 1C, the enzyme acetate kinase in addition also catalyzes the
reaction in the other direction from acetate to acetylphosphate. In
the case that the enzyme acetaldehyde dehydrogenase is
overexpressed or its affinity or activity is enhanced in other ways
described in this patent application, Overexpression of acetate
kinase enzyme can enhance the level of biosynthesis of
acetylphosphate, thereby enhancing ethanol formation by Adh. In
addition the other ethanol forming enzyme AdhE can also be
overexpressed.
[0835] FIG. 1E shows the same metabolic pathway as depicted in FIG.
1D with the exception that in addition to the acetate kinase enzyme
the phosphotransacetylase enzyme also catalyzes the reverse
reaction from acetylphosphate to acetyl-CoA. In this case
phosphotransacetylase can be overexpressed in addition to acetate
kinase enzyme in order to enhance the level of biosynthesis of
acetyl-CoA in a first genetic modification. The second genetic
modification comprises overexpression of AdhE, which converts the
acetyl-CoA into ethanol. In addition the second genetic
modification also can comprise overexpression of Adh and Pdc.
[0836] FIG. 1F shows some relevant metabolic pathways of
cyanobacteria with different overexpressed enzymes for ethanol
formation, which can be introduced into a photoautotrophic
cyanobacterial host cell by second genetic modifications. In one
aspect of the invention a CoA-dependent acetaldehyde dehydrogenase
can be overexpressed in the host cell, which converts acetyl-CoA
into acetaldehyde. The acetaldehyde can then further be converted
to ethanol by a further enzyme for ethanol formation Adh, which can
be AdhI enzyme or AdhII enzyme or a combination of both
enzymes.
[0837] In addition or alternatively Pdc enzyme can be present in
the host cell as a further overexpressed enzyme for ethanol
formation introduced via a second genetic modification, which can
convert pyruvate into acetaldehyde.
[0838] FIG. 2 shows in a more detailed way the last steps of
ethanol synthesis in genetically modified cyanobacteria.
[0839] FIG. 3 depicts a further non-limiting representation of
metabolic pathways of a cyanobacterium. In contrast to the FIGS. 1A
to 1F a NAD dependent acetaldehyde dehydrogenase is shown, which
can convert acetate into acetaldehyde, which then can be converted
into ethanol by Adh enzyme.
Working Example of Genetic Knockout
[0840] In the following one embodiment of the invention, in
particular a genetically modified host cell comprising a host
enzyme forming reserve compounds, wherein the gene encoding this
enzyme is disrupted by genetic engineering, is explained in more
detail with reference to a working example, The host enzyme is
glycogen synthase, which is encoded by two genes in the host cell
Synechocystis sp. PCC 6803. In order to knock-out both genes a
double knock-out mutant has to be generated
Laboratory Protocols
[0841] Protocols for the Generation of a Glycogen Synthase Double
Mutant of Synechocystis sp. PCC 6803
[0842] In the genome database of Synechocystis sp. PCC 6803 two
genes encoding glycogen synthases are annotated (available on the
world wide web at bacteria.kazusa.or.jp/cyano).
[0843] One glycogen synthase of Synechocystis sp. PCC 6803 is
encoded by the gene sll0945 (glgA1) annotated as glycogen synthase
1 (GlgA1). The Accession number of the protein is P74521 (EC
2.4.1.21), its amino acid sequence is presented in FIG. 4A.
[0844] A second glycogen synthase of Synechocystis sp. PCC 6803 is
encoded by the gene sll1393 (glgA2), annotated as glycogen (starch)
synthase 2 (GlgA2). The Accession number of the protein is P72623
(EC 2.4.1.21), its amino acid sequence is presented in FIG. 4B.
[0845] Construction of DNA-vectors (knock-out-constructs) for the
two glycogen synthase encoding genes (glgA1 and glgA2) of
Synechocystis sp. PCC 6803
In general:
[0846] DNA sequences encoding genes of interest are amplified by
polymerase chain reaction (PCR) using specific primers. When the
genomic sequence does not contain appropriate restriction sites for
cloning, primers are designed containing restriction sites. Genomic
DNA from Synechocystis sp. PCC 6803 are used as template. The
amplified PCR fragments are digested with the appropriate
restriction enzymes and ligated into a cloning vector.
[0847] An antibiotic resistance cassette is then inserted into
selected sites of the cloned genes. Upstream and downstream on each
site of the antibiotic resistance cassette at least 500 bps remain
for homologous recombination.
[0848] Genetic engineering of constructs as well as PCRs, ligations
into cloning vectors, insertions of antibiotic resistance cassettes
and transformations into E. coli are done using standard procedures
(state of the art) or according to the manufacturers
instructions.
[0849] To generate a glycogen deficient mutant in Synechocystis sp.
PCC 6803, constructs were created for inactivation both glycogen
synthase genes. The resulting glycogen deficient mutant described
below is named mutant M8.
[0850] For creating a knock-out construct to Inactivate glgA1, a
1341 bp fragment containing the major part of the coding sequence
from glycogen synthase 1 (sll0945) was amplified by PCR using the
following primers:
(SEQ ID NO: 140) #glgA-1fw: 5'-CGACGGTATGAAGCTTTTATTTG-3', primer
contains a HindIII restriction site for cloning (marked in bold
letters). (SEQ ID NO: 141) #glgA-1rv: 5'-CCGGCGGAACGGTACCAAC-3',
primer contains a Kpnl restriction site for cloning (marked in bold
letters)
[0851] The PCR fragment was digested with HindIII and Kpnl and
cloned into plasmid pUC19 (Ac. No M77789). A single BstXI site
present in the middle of glgA1 gene was used to insert a
chloramphenicol resistance cassette (named Cm). The chloramphenicol
resistance cassette, encoding a chloramphenicolacetyltransferase
(cat) gene, was cut out of plasmid pACYC184 (Ac. No X06403) using
BsaAI and BsaBI. The orientation of the antibiotic cassette was
analyzed by digestion with HindIII and EcoRI; a restriction map is
presented in FIG. 4C.
[0852] A knock-out-construct, named pUC-glgA1-Cm, has the structure
presented in FIG. 4D, and the nucleotide sequence of the construct
pUC-glgA1-Cm is presented in FIG. 4E.
[0853] For creating a knock-out construct to inactivate glgA2, a
1673 bp fragment containing the entire coding sequence from
glycogen synthase 2 (sll1393) was amplified by PCR using the
following primers:
(SEQ ID NO: 142) #glgA-2fw: 5'-GGCCAGGGGAATTCTCCTCCAG-3', primer
contains an EcoRI restriction site for cloning (marked in bold
letters). (SEQ ID NO: 143) #glgA-2rv:
5'-GCGGATAATACTGAACGAAGCTTTG-3', primer contains a HindIII
restriction site for cloning (marked in bold letters).
[0854] The PCR fragment was digested with EcoRI and HindIII and
cloned into plasmid pUC19. A single HincII site present in the
middle of glgA2 gene was used to insert a kanamycin resistance
cassette (named Kan). The kanamycin resistance cassette, encoding
an aminoglycoside 3''-phosphotransferase (aph) gene, was cut out of
plasmid pUC4K (Ac. No X06404) using HincII. The orientation of
antibiotic cassette was analyzed with the restriction enzyme
HindIII. A restriction map of this done is presented schematically
in FIG. 4G.
[0855] The knock-out-construct used, named pUC-glgA2-Kan, has the
structure presented in FIG. 4G and the nucleotide sequence
presented in FIG. 4H.
[0856] Mutagenesis by transformation of the DNA-vectors
(knock-out-constructs) using the natural competence of
Synechocystis sp. PCC 6803 for DNA uptake and its system for
homologous recombination.
[0857] The transformation was done in two steps. The first
transformation knocks out gene sll0945 (glgA1) in the wild type of
Synechocystis, and the corresponding mutant .DELTA.glgA1 was
selected. In a second step, gene sll1393 (glgA2) was knocked out in
the .DELTA.glgA1 mutant and the double mutant
.DELTA.glgA1/.DELTA.glgA2 was selected.
General Transformation Protocol:
[0858] Spin down 10 ml of exponentially growing culture of
Synechocystis sp. at room temperature (RT) and remove the
supernatant [0859] Resuspend the pellet in 0.5-1.0 ml of BG11
medium (media recipe: [0860] NaNO.sub.3: 1.5 g [0861]
K.sub.2HPO.sub.4: 0.04 g [0862] MgSO.sub.4.7H.sub.2O: 0.075 g
[0863] CaCl.sub.2.2H.sub.2O: 0.036 g [0864] Citric acid: 0.006 g
[0865] Ferric ammonium citrate: 0.006 g [0866] EDTA (disodium
salt): 0.001 g [0867] NaCO.sub.3: 0.02 g [0868] Trace metal mix
A5.sub.--1.0 ml [0869] Agar (if needed): 10.0 g [0870] Distilled
water: 1.0 L [0871] The pH should be 7.1 after sterilization [0872]
Trace metal mix A5: [0873] H.sub.3BO.sub.3: 2.86 g [0874]
MnCl.sub.2.4H.sub.2O: 1.81 g [0875] ZnSo.sub.4.7H.sub.2O: 0222 g
[0876] NaMoO.sub.4.2H.sub.2O: 0.39 g [0877] CuSO.sub.4.5H.sub.2O:
0.079 g [0878] Co(NO.sub.3).sub.2.6H.sub.2O: 49.4 mg [0879]
Distilled water: 1.0 L)
[0880] Add 1-10 .mu.g plasmid DNA (knock-out-construct carrying
gene of interest and an antibiotic cassette for screening for
homologous recombination) [0881] Incubate on a table top shaker for
5-6 hours in the light at RT
[0882] Plate 500 .mu.l of a 1/100 dilution of the transformation
mixture on a BG11 agar plate. Plate the remainder of the cell
suspension on another plate. Include control plate (transformation
mixture with water instead of plasmid DNA).
[0883] Incubate 48 h in the light at room temperature (RT) when
chloramphenicol is used for selection or over night when kanamycin
is used for selection.
[0884] Pipet 500 .mu.l of the corresponding antibiotic in a
suitable concentration under the agar for the selection of mutant
clones (initial concentration for chloramphenicol: 1 .mu.g/ml BG11
agar; initial concentration for kanamycin: 5 .mu.g/ml) [0885]
Incubate for approx. 2 weeks in the light at RT [0886] Transfer
individual colonies to plates containing the corresponding
antibiotic
[0887] Thereafter, the concentrations of antibiotics were increased
stepwise when the cells were transferred onto another agar plate or
into liquid culture (for kanamycin from initially 5 to 150 .mu.g/ml
BG11, for chloramphenicol from initially 1 to 15 .mu.g/ml BG11
medium) in order to get fully segregated (homozygous) mutants.
Transfers were done every 2 weeks. In case of kanamycin, the
concentration in the range from 50 to 150 .mu.g/ml agar was
increased gradually over the course of 4 weeks.
Cultivation of Cyanobacterial Wild Type and Mutant Strains
[0888] Wild type and mutant strains of Synechocystis PCC 6803 were
grown as batch cultures in BG11 medium at 29.degree. C. under
continuous illumination with white light (intensity: 40
.mu.m.sup.-2 s.sup.-1) and aeration with air. For cultivation of
mutants, the appropriate antibiotics were added to the medium
(kanamycin 75 mg/l; chloramphenicol 15 ms/l).
[0889] Samples were analyzed briefly before the nitrogen step down
("+N"), directly after resuspension of the cells in BG11 medium
lacking a nitrogen source ("-N", 0 h) and after 3, 6 and 24
hours.
Generation of Knock-Out Mutants of Synechocystis sp. PCC 6803 and
Other Cyanobacteria Affecting the Following Genes: [0890] a)
alanine dehydrogenese (ald) [0891] b) ADP-glucose pyrophosphorylase
(glgC) [0892] c) pyruvate water dikinase (ppsA) [0893] d) lactate
dehydrogenase (Idh) [0894] e) acetate kinase (ack) [0895] f)
phosphoacetyltransacetylase (pta) [0896] g) PHB knockout mutant
(AphaC) h) knockout mutant of ADP-glucose-pyrophosphorylase, agp,
glgC in the filamentous, diazotrophic cyanobacteria Nostoc/Anabaena
spec. PCC7120 and Anabaena variabilis ATCC 29413 Protocols for
Generation of Knock-Down Mutants of Synechocystis sp. PCC 6803 and
Other Cyanobacteria Affecting the Following Gene: [0897] a)
pyruvate dehydrogenase (pdhB)
[0898] Protocols for the generation of knock-out mutants of
Synechocystis sp. PCC 6803 and other cyanobacteria
Construction of DNA-Vectors for Generation of Knock-Out Mutants
[0899] In general:
[0900] DNA sequences encoding genes of interest were amplified by
polymerase chain reaction (PCR) using specific primers. When the
genomic sequence did not contain appropriate restriction sites for
cloning, primers were designed containing restriction sites.
Genomic DNA from Synechocystis sp. PCC 6803 was used as template.
The amplified PCR fragments were digested with the appropriate
restriction enzymes and ligated into a cloning vector.
[0901] An antibiotic resistance cassette was then inserted into
selected sites of the cloned genes. Upstream and downstream on each
site of the antibiotic resistance cassette at least 500 bps
remained for homologous recombination. The following antibiotic
resistance cartridges were used: kanamycin resistance cassette
(named Kan) from pUC4K vector (Ac. No X06404) from the NCBI
database available on the world wide web at
ncbi.nlm.nih.gov/sites/nucleotide encoding aminoglycoside
3'-phosphotransferase (aph) gene or chloramphenicol resistance
cartridge (named Cm) from pACYC184 vector (Biolabs, Ac No. X06403)
encoding chloramphenicolacyltransferase (cat) gene. Genetic
engineering of constructs as well as PCRs, ligations into cloning
vectors, insertions of antibiotic resistance cassettes and
transformations into E. coli were done using standard procedures
(state of the art) or according to the manufacturer
instructions.
[0902] Sequences and structures of the used cloning and expression
plasmids are described below (see 3.). Knock-outs were generated
via homologous recombination of the wild type gene with the mutant
genes. The method of transformation of the DNA-vectors
(knock-out-constructs) using the natural competence of
Synechocystis sp. PCC 6803 for DNA uptake was already described in
detail for the generation of the glycogen deficient mutant.
a) Construction of a DNA-Vector for Generation of an Alanine
Dehydrogenase Knock-Out Mutant (.DELTA.ald)
[0903] The open reading frame (ORF) sll1682 encodes alanine
dehydrogenase (EC 1.4.1.1), Ac. No BAA16790. The amino acid
sequence of this protein is presented in FIG. 5A.
[0904] Two constructs were generated for knock-out of alanine
dehydrogenase differing in orientation of the inserted kanamycin
resistance cartridge (in sense and in antisense orientation to the
aid ORF) using the following primers:
TABLE-US-00001 (SEQ ID NO: 144) #Ald50.fw:
5'-GGCTGACCCCCAGTAGTGTA-3 (SEQ ID NO: 1454) #Ald104.2.rv:
5'-ATTTTCCGGCTTGAACATTG-3'
[0905] A 993 bp aid PCR fragment was amplified by a BIOTAQ.TM. DNA
Polymerase (BIOLINE), cloned into the pGEM-T vector (Promega) and
restricted with Smal (blunt ends; Fermentas). The kanamycin
cartridge was remained by a restriction of the pUC4K vector with
EcoRI (5' overhangs; Fermentas) and a following "fill in reaction"
via the T4 DNA Polymerase (Promega). Plasmids were analyzed by
restriction digest in order to select constructs with both
orientations of the inserted kanamycin cartridge.
[0906] A construct designated as pGEM-T/.DELTA.ald-antisense has
the structure presented schematically in FIG. 5B.
[0907] The sequence of the insert for this construct
(pGEM-T/.DELTA.ald-antisense) is presented in FIG. 5C.
[0908] In the other construct, designated as
pGEM-T/.DELTA.ald-sense the kanamycin resistance cartridge is
inserted in the other direction.
b) Construction of DNA-Vector for Generation of an ADP-Glucose
Pyrophosphorylase Knock-Out Mutant (.DELTA.glgC)
[0909] The open reading frame (ORF) slr1176 encodes ADP-glucose
pyrophosphorylase (EC 2.7.7.27), Ac. No BAA18822. The amino acid
sequence of this protein is presented in FIG. 6A.
[0910] Four constructs were generated for knock out of ADP-glucose
pyrophosphorylase differing in the locus of insertion (EcoRI,
BsaBI) and in orientation of the resistance (kanamycin-Km,
chloramphenicol-Cm) cartridge (in sense and in antisense
orientation to the glgC gene). Both insertion sites were tested
because of a putative small non-coding RNA at the 5'-terminus of
the glgC gene (in antisense orientation). Therefore, the insertion
of the chloramphenicol cartridge at the BsaBI-site might affect the
expression of the putative small non-coding RNA.
[0911] The following primers were used for PCR
TABLE-US-00002 EcoRI: G.dwnarw.AATTC #GlgC5.fw:
5'-GTTGTTGGCAATCGAGAGGT-3' (SEQ ID NO: 146) #GlgCIR.rv:
5'-GTCTGCCGGTTTGAAACAAT-3' (SEQ ID NO: 147) BsaBI:
GATNN.dwnarw.NNATC (SEQ ID NO: 148) #GlgCIR.fw:
5'-ACCCCATCATCATACGAAGC-3' (SEQ ID NO: 149) #GlgC1233.rv:
5'-AGCCTCCTGGACATTTTCCT-3' (SEQ ID NO: 150)
[0912] The first 1579 bp glgC PCR fragment was amplified by a
BIOTAQ.TM. DNA Polymerase (BIOLINE), cloned into the pGEM-T vector
(Promega) and restricted with EcoRI (5' overhangs; Fermentas). The
kanamycin cartridge was remained by a restriction of the pUC4K
vector with EcoRI (5' overhangs; Fermentas).
[0913] Plasmids were analyzed by restriction digest in order to
select constructs with both orientations of the inserted kanamycin
cartridge, respectively.
[0914] The construct pGEM-T/.DELTA.glgC-KMantisense has the
structure shown in FIG. 6B, and its insert the nucleotide sequence
presented in FIG. 5C.
[0915] In the other construct, designated as
pGEM-T/.DELTA.glgC-KMsense the kanamycin resistance cartridge is
inserted in the other direction.
[0916] The second 1453 bp glgC PCR fragment was amplified by a
BIOTAQ.TM. DNA Polymerase (BIOLINE), cloned into the pDrive vector
(Qiagen) and restricted with BsaBI (blunt ends; Biolabs). The
chloramphenicol cartridge was remained by restriction of the
pACYC184 vector (Biolabs, Ac No. X06403) with BsaAI (blunt ends;
Biolabs).
[0917] Plasmids were analyzed by restriction digest in order to
select constructs with both orientations of the inserted
chloramphenicol resistance (Cm) cartridge, respectively.
[0918] A construct designated as pDrive/.DELTA.glgC-CMantisense was
selected; its structure is presented schematically in FIG. 6D and
the nucleotide sequence of the insert is presented in FIG. 6E.
[0919] In the other construct, designated as
pDrive/.DELTA.glgC-CMsense the chloramphenicol resistance cartridge
is inserted in the other direction.
c) Construction of DNA-Vector for Generation of a Pyruvate Water
Dikinase Knock-Out Mutant (.DELTA.ppsA)
[0920] The open reading frame (ORF) slr0301 encodes pyruvate water
dikinase/PEP synthase (EC 2.7.9.2), Ac. No BAA10558. This protein
has the amino acid sequence that is presented in FIG. 7A.
[0921] Two constructs were generated for knock-out of pyruvate
water dikinase differing in orientation of the inserted kanamycin
resistance cartridge (in sense and in antisense orientation to the
ppsA ORF) using the following primers:
TABLE-US-00003 (SEQ ID NO: 151) #PpsA547.fw:
5'-TTCACTGACCGGGCTATTTC-3' (SEQ ID NO: 152) #PpsA2329.rv:
5'-CTTGGCCACAGATACCGATT-3'
[0922] A 1783 bp ppsA PCR fragment was amplified by a BIOTAQ.TM.
DNA Polymerase (BIOLINE), cloned into the pGEM-T vector (Promega)
and restricted with Smal (blunt ends; Fermentas). The kanamycin
cartridge was remained by a restriction of the pUC4K vector with
EcoRI (5' overhangs; Fermentas) and a following "fill in reaction"
via the T4 DNA Polymerase (Promega). Plasmids were analyzed by
restriction digest in order to select constructs with both
orientations of the inserted kanamycin cartridge.
[0923] The construct used, designated as
pGEM-T/.DELTA.ppsA-antisense, has the structure presented in FIG.
7B. The nucleotide sequence of it insert is presented in FIG.
7C.
[0924] In the other construct, designated as
pGEM-T/.DELTA.ppsA-sense the kanamycin resistance cartridge is
inserted in the other direction.
d) Construction of a DNA-Vector for Generation of a Lactate
Dehydrogenase Knock-Out Mutant (.DELTA.Idh)
[0925] The open reading frame (ORF) slr 1556 encodes a putative
lactate dehydrogenase (EC 1.1.1.28), annotated as
2-hydroxyaciddehydrogenase homolog (P74586). This amino acid
sequence for this protein is presented in FIG. 8A.
[0926] A 1931 bp fragment containing the entire coding sequence
from lactate dehydrogenase (slr1556) was amplified by PCR using the
following primer:
TABLE-US-00004 (SEQ ID NO: 153) #ldh-1fw:
5'-GCGAACTACCCAACGCTGACCGG-3' (SEQ ID NO: 154) #ldg-2rv:
5'-GCATCAAGTGTTGGGGGATATCCCTG-3', primer contains a EcoRV
restriction site (GATATC) for cloning (marked in bold letters).
[0927] The PCR fragment was digested with NheI/EcoRV (NheI site is
present in the genomic sequence) and cloned into pBluescript SK+
vector using XbaI/EcoRV. The kanamycin resistance cassette was used
from the DNA vector pUC4K and ligated into the BglII/BclI
restriction sites of slr1556. A restriction map of this is
presented in FIG. 8B.
[0928] The knock-out-construct used, named pBlue Idh-Kan-a, has the
structure presented in FIG. 5C, and the nucleotide sequence for its
insert is presented in FIG. 80.
e) Construction of a DNA-Vector for Generation of an Acetate Kinase
Knock-Out Mutant (Rack)
[0929] The open reading frame (ORF) sll 1299 encodes a putative
acetate kinase (EC 2.7.2.1), Ac No. P73162. The amino acid sequence
for this protein is presented in FIG. 9A.
[0930] A 2316 bp fragment containing the entire coding sequence
from acetate kinase (sll1299) was amplified by PCR using the
following primer:
TABLE-US-00005 (SEQ ID NO: 155) #ack-1 fw:
5'-CCGGGACGTGACAGAACGGGTGG-3' (SEQ ID NO: 156) #ack-2 RV:
5'-GCGTTGGCGATCGCCGTCACTAG-3'
[0931] The PCR fragment was digested with SpeI (both sites are
located in the genomic sequence) and cloned into pBluescript SK+
vector. The kanamycin resistance cassette was used from the DNA
vector pUC41K and ligated into the HpaI restriction sites of
slr1299. A restriction enzyme map of this region is presented in
FIG. 9B.
[0932] The orientation of the kanamycin resistance cassette was
either in the same direction as sll1299 (designed "a") or in the
opposite direction (designed "b").
[0933] The knock-out-construct used, named pBlue ack-Kan-b, has the
structure presented in FIG. 9C, and the nucleotide sequence of its
insert is presented in FIG. 90.
f) Construction of DNA-Vector for Generation of a
Phosphoacetyltransacetylase (Phosphoacyltransferase) Knock-Out
Mutant (.DELTA.pta)
[0934] The open reading frame (ORF) slr2132 encodes a
phosphoacetyltransacetylase (EC 2.3.1.8), Ac No. P73662. The amino
acid sequence for this protein is presented in FIG. 10A.
[0935] A 2869 bp fragment containing the entire coding sequence
from phosphoacetyl-transacetylase (slr2132) was amplified by PCR
using the following primer:
TABLE-US-00006 (SEQ ID NO: 157) #pta-1fw:
5'-GCCATTGTGGGGGTGGGTCAG-3' (SEQ ID NO: 158) #pta-2rv:
5'-CAGTTTATGCCCCGCTACCGGG-3',
[0936] The PCR fragment was digested with MfeI/HindIII (both sites
present in the genomic sequence) and cloned into pUC19
(EcoRI/HindIII) vector. The chloramphenicol resistance cassette was
used from plasmid pACYC184 and ligated into the ClaI/PstI
restriction sites of slr2132. A restriction map of this region is
presented in FIG. 10B.
[0937] The knock-out-construct selected is named pUC pta-Cm. It's
structure is presented schematically in FIG. 10C, and the
nucleotide sequence of the insert for this clone is presented in
FIG. 10D.
g) Construction of DNA-Vector for Generation of PHB Knockout Mutant
(.DELTA.phaC)
[0938] The open reading frame (ORF) slr1830 encodes
poly(3-hydroxyalkanoate) synthase [EC:2.3.1], Ac. No BAA17430. The
amino acid sequence for this protein is presented in FIG. 11A.
[0939] One construct was generated for knock out of
poly(3-hydroxyalkanoate) synthase by deletion/insertion (resistance
cartridge: kanamycin) mutagenesis.
TABLE-US-00007 (SEQ ID NO: 159) # phaC-25_XbaI.fw:
5'-CCGATGtcTAGaTAATTCACCATC-3' (SEQ ID NO: 160) # phaC404_BamHI.rv:
5'-TCTAGGGggAtCCAACGATCG-3' (SEQ ID NO: 161) # phaC711_BamHI.fw:
5'-CCAGGGGATccTCTTAACCTAG-3' (SEQ ID NO: 162) # phaC1133_ClaI.rv:
5'-TGTCGTatCGATAGCCAATGG-3'
[0940] Two PCR products (pos. 24 to pos. 404; pos. 711 to pos.
1133) of the phaC fragment were amplified by a BIOTAQ.TM. DNA
Polymerase (BIOLINE), ligated via BamHI sites and cloned into the
pIC20H vector. The kanamycin cartridge was remained by a
restriction of the pUC vector (available on the world wide web at
seq.yeastgenome.or/vectordb/vector_descrip/COMPLETE/PUC4K.SEQ.html)
with BamHI (Fermentas). Plasmids were analyzed by restriction
digest Knockouts were generated via homologous recombination of the
wild type gene with the mutant genes.
[0941] The construct selected is pIC20H/AphaC-KM and has the
structure presented schematically in FIG. 11B. The nucleotide
sequence for the insert of this clone is presented in FIG. 11C.
h) Construction of DNA-Vectors for Generation of Knockout Mutants
of ADP-Glucose-Pyrophosphorylase, agp (glgC) in the Filamentous,
Diazotrophic Cyanobacteria Nostoc/Anabaena spec. PCC7120 and
Anabaena Variabilis ATCC 29413
[0942] In order to generate ethanol producing Anabaena strains,
different constructs were created for conjugation into Anabaena
PCC7120 and Anabaena variabilis ATCC29413. Constructs for genome
integration of ethanologenic genes were created for both Anabaena
strains. As integration site into the genome the
glucose-1-phosphate adenylyltransferase gene
(ADP-glucose-pyrophosphorylase, agp, glgC) was chosen. Thus, by
integration of the ethanologenic genes simultaneously an agp
knock-out mutant was created.
[0943] Glucose-1-phosphate adenylyltransferase
(ADP-glucose-pyrophosphorylase, agp, glgC), EC 2.7.7.27, of
Anabaena spec. PCC7120 is encoded by ORF a114645, Ac. No. P30521.
The amino acid sequence of ORF a114645 is shown in FIG. 11D.
[0944] Constructs for conjugation into Anabaena PCC7120 were cloned
as followed:
[0945] Two fragments representing the 5' and 3' part of the
ADP-glucose-pyrophosphorylase (agp) gene, ORF all4645, were
amplified by PCR using the following primers:
TABLE-US-00008 (SEQ ID NO: 163) #agp1.1
5'-CATCCATCATGAGCTCTGTTAAC-3' (SacI site inserted) (SEQ ID NO: 164)
#agp2.1 5'-GTATCTCGAGCGATGCCTACAGG-3' (XhoI site inserted) (SEQ ID
NO: 165) #agp3.1 5'-CGCATTGGTTTCTAGATGGCGC-3' (XbaI site inserted)
(SEQ ID NO: 166) #agp4.1 5'-CGATAACTCTAGACGAGTCATTG-3' (XbaI site
inserted)
[0946] Inserted restriction sites in primer sequences are marked in
bold letters
[0947] As indicated in FIG. 11E, in between these agp fragments a
C.K3 cassette (coding for kanamycin/neomycin resistance) was
ligated into the XbaI site. [C.K3 cassette is described in Elhai,
J. & Wolk, C. P. (1988) Gene, 68119-138.]
[0948] The entire "agp knock-out" fragment was cloned into suicide
vector pRL271 (Ac. No. L05081). The pdc/adh genes, or only pdc,
were cloned downstream of the inducible promoter PpetE and
Integrated into the "agp-C.K3" construct.
[0949] The following constructs have been generated:
pRL271 agp (a114645)::C.K3 pRL271 agp
(a114645)::C.K3-PpetE-pdc-AdhI pRL271 agp
(a114645)::C.K3-PpetE-pdc
[0950] The structures of the constructs are depicted in FIG.
11-2.
[0951] The sequence of the insert of pRL271 agp
(all4645)::C.K3-PpetE-pdc-AdhII is shown in FIG. 11F.
[0952] The same strategy was used to create constructs for
expression in Anabaena variabilis ATCC29413. The nucleotide
sequences of the agp genes from both strains are 97%, their protein
sequences are 99.3% identical.
[0953] Glucose-1-phosphate adenylyltransferase
(ADP-glucose-pyrophosphorylase, agp, glgC). EC 2.7.7.27, of
Anabaena variabilis ATCC29314 is encoded by ORF Ava.sub.--2020, Ac.
No. 43 MBJ4, and has the amino acid sequence as shown in FIG.
11G.
[0954] For PCR amplification of the genomic fragments of Anabaena
variabilis the following primers were used:
TABLE-US-00009 (SEQ ID NO: 167) #agp1.2
5'-GAGGCAATGAGCTCCACTGGACG-3' (SacI site inserted) (SEQ ID NO: 168)
#agp2.2 5'-CTGGCGTTCCACTCGAGCTTGG-3' (XhhoI site inserted) (SEQ ID
NO: 169) #agp3.1 5'-CGCATTGGTTTCTAGATGGCGC-3' (XhaI site inserted)
(SEQ ID NO: 170) #agp4.2 5'-CGATAACTCTAGACGAGTCATCG-3' (XhaI site
inserted)
[0955] Inserted restriction sites in primer sequences are marked in
bold letters.
[0956] Generation of the constructs was exactly as described for
the constructs of Anabaena PCC7120.
[0957] The following constructs have been generated:
pRL271 agp::C.K3 pRL271 agp::C.K3-PpetE-pdc-AdhII pRL271
agp::C.K3-PpetE-pdc
[0958] All described plasmids were conjugated into Anabaena strains
according the following method:
Conjugation of Nostoc spec. PCC7120/Anabaena variabilis
Cargoplasmids
[0959] Cargoplasmids (pRL593, pRL1049 or pRL271) were transformed
into competent E. coli HB101 (pRL528.sub.helperplasmid)
In Preparation for Conjugation
[0960] E. coli Cultures:
[0961] inoculation of overnight cultures in LB with the appropriate
antibiotics from [0962] Cargoplasmid in E. coli HB101
(pRL528.sub.helperplasmid) [0963] Helperstrain E. coli J53
(RP4)
[0964] preparation of well growing culture (for each
conjugation/plate 10 ml of HB101 (pRL528+cargo plasmid) and 10 ml
of J53 (RP4) is needed): inoculate 0.25 ml overnight culture in 10
ml LB+antibiotic, grow for 2.5 h/37.degree. C.
[0965] spin down the well grown E. coli cultures in "Falcons" 10
min 4800 rpm. [0966] (for J53 culture: take 2 Falcons).
[0967] "wash"/resuspend cells in equal volume of LB without
antibiotics.
[0968] for each conjugation spin 10 ml of resuspended HB101
(culture carrying pRL528+cargo plasmid) in 15 ml Falcon tube,
remover supernatant
[0969] add on the cell pellets 10 ml resuspended J53 (RP4) culture,
spin down, remove supernatant and resuspend combined cells in 1 ml
LB, transfer cells in Eppi tubes, resuspend again in 100 .mu.l LB
and incubate for 2 h at 30.degree. C.
Cyanos
[0970] determine the chlorophyll concentration of well grown
Anabaena cultures
[0971] for each conjugation, culture corresponding to about 10
.mu.g Chlorophyll is needed.
[0972] spin down the equivalent volume of Anabaena culture and
resuspend to a volume corresponding to 10 .mu.g Chlorphyll/100
.mu.l BG11 medium.
Conjugation
[0973] for each conjugation place one HATF filter on a plate
(BG11)
[0974] mix 100 .mu.l E. coli suspension=100 .mu.l Anabaena culture
and plate on filter
[0975] incubate plates at 30.degree. C. overnight wrapped in
paper
[0976] next day remove paper
[0977] after one day transfer filter on plates containing
antibiotics.
Construction of DNA-Vectors for Generation of Knock-Down
Mutants
[0978] a) Construction of a DNA-Vector for Generation of a Pyruvate
Dehydrogenase (pdhB) Knock-Down Mutant
[0979] The open reading frame (ORF) sll1721 encodes the
.beta.-subunit of the E1 component of the pyruvate dehydrogenase,
(EC 1.2.4.1), Ac. No BAA17445. This protein has the amino acid
sequence presented in FIG. 12A.
[0980] Two strategies were considered for knock-down of the
pyruvate dehydrogenese. A knock-down could be achieved by
regulation of the expression of the adequate antisense RNA (i) or
by insertion of a controllable wild type gene copy accompanied by a
knock-out of the original wild type gene (ii). Therefore, four
constructs were generated to knock-down the pyruvate
dehydrogenase.
[0981] The PCR fragments for the expression of the adequate
antisense RNA as well as for the controllable wild type gene copy
were amplified by a High-Fidelity DNA Polymerase (Phusion.TM.;
Finnzymes), adenylated (BIOTAQ.TM. DNA Polymerase; BIOLINE), cloned
into the pDrive vector (Qiagen) and restricted with ClaI/BglII (i)
or NdeI/BglII.sup.1 (ii) (Fermentas). These fragments were cloned
into the pSK9 vector, digested with ClaI/BglII (i) or NdeI/BglII
(i). The non-public pSK9 vector was generated in the lab of V. V.
Zinchenko (Moscow, Russia). The gene is incorporated into a
non-coding genome region via the integrated platform. The
expression of the enzyme and the antisense RNA is under the control
of the copper inducible promoter petJ. The termination of
transcription is achieved either by the gene-specific terminator
loop (ii) or by the loop-terminator of the lambda phage (i) (Topp
is part of the reverse-Primer), both amplified by PCR
reaction..sup.1 .sup.1BglII was used instead of ClaI because this
inserted ClaI cleavage side was affected by Dam-methylation. The
BglII cleavage side is part of the 3' end of the amplified PCR
product ad do not affect the translation termination loop.
#PdhBantiClaI.fw 5'-ATCGATATAATTTCCGGGTCGTAGCC-3' (SEQ ID NO: 171),
this primer contains a ClaI restriction site for cloning (marked in
bold letters) #PdhBantioopBglII.rv:
5'GATCTGGAATAAAAAACGCCCGGCGGCAACCGAGCGGCAGCC ATTCGGGATAATAA-3' (SEQ
ID NO: 172), this primer contains a BglII restriction site for
cloning (marked in bold letters) and the oop terminator region of
the lambda phage (underlined) #PdhBNdel.fw:
5'-CATATGGCTGAGACCCTACTGTTT-3' (SEQ ID NO: 173), this primer
contains a NdeI restriction site for cloning (marked in bold
letters) #PdhB1061ClaI.rv: 5'-ATCGATCTTACAAGCTCCCGGACAAA-3' (SEQ ID
NO: 174), this primer contains a ClaI restriction site for cloning
(marked in bold letters)
[0982] The 1142 bp pdhB PCR fragment for the knock-out of the
original wild type gene was amplified by a BIOTAQ.TM. DNA
Polymerase (BIOLINE), cloned into the pGEM-T vector (Promega) and
restricted with Eco147I (blunt ends; Fermentas). The kanamycin
cartridge was remained by a restriction of the pUC4 vector with
EcoRI (5' overhangs; Fermentas) and a following "fill in reaction"
via the T4 DNA Polymerase (Promega) and ligated into the Eco1471
site. Resulting plasmids were analyzed by restriction digest in
order to select constructs with both orientations of the inserted
kanamycin cartridge. Knock-outs were generated via homologous
recombination of the wild type gene with the mutant genes. The
following primers were used for PCR:
TABLE-US-00010 (SEQ ID NO: 175) # PdhB.fw:
5'-AATCGACATCCACCCTTGTC-3' (SEQ ID NO: 176) # PdhB.rv:
5'-GCCTTAACTGCGTCCACAAT-3'
[0983] (i) Knock-Down by Regulation of the Expression of the
Adequate Antisense RNA
[0984] The construct used, designated as psK9/pdhBanti, has the
structure presented in FIG. 12B, and the nucleotide sequence of its
Insert is presented in FIG. 12C.
(ii) Knock-Down by Insertion of a Controllable Wild Type Gene Copy
Accompanied by a Knock-Out of the Original Wild Type Gene
[0985] The construct used, designated as pSK9/pdhB, has the
structure presented in FIG. 12D, and the nucleotide sequence of the
insert for this clone is presented in FIG. 12E.
[0986] The knock-out construct used, designated as
pGEM-T/.DELTA.pdhB-KMantisense, has the structure presented in FIG.
12F. The sequence for the insert in this done is presented in FIG.
12G.
[0987] In the other construct, designated as
pGEM-T/.DELTA.pdhB-KMsense the kanamycin resistance cartridge is
inserted in the other direction.
[0988] In the following the cloning vectors, which were used are
described.
a) Cloning Vector pGEM.RTM.-T Structure and Sequence
[0989] PCR cloning vector pGEM.RTM.-T was from Promega corp.,
Madison Wis., USA. The structure of the plasmid is presented in
FIG. 13A, and it nucleotide sequence is presented in FIG. 13B.
b) Cloning Vector pDrive Structure and Sequence
[0990] Cloning vector pDrive was from Qiagen, Hilden, Germany. The
structure of this plasmid is presented in FIG. 14A and its
nucleotide sequence in FIG. 148.
c) Cloning Vector pBLueSK+ Structure and Sequence
[0991] Cloning vector pBluescript II.RTM. SK+ (Ac. No X52328) was
from Stratagene, La Jolla, Calif., USA.
[0992] The structure of this plasmid is presented in FIG. 15A and,
its nucleotide sequence is presented in FIG. 158.
d) Cloning Vector pUC1 Structure and Sequence
[0993] Cloning vector pUC19 (Ac. No M7779) is presented
schematically in FIG. 16A, and its nucleotide sequence is presented
in FIG. 168.
e) Plasmid pSK9 Structure and Sequence
[0994] The non-public pSK9 vector was generated in the lab of V. V.
Zinchenko (Moscow, Russia). A schematic of pSK9 structure is
presented in FIG. 17A, and its nucleotide sequence is presented in
FIG. 17B.
Protocols for Generation of Synechocystis sp. PCC 6803 Mutants
Overexpressing the Following Genes: a) malic enzyme b) malate
dehydrogenese c) malic enzyme and malate dehydrogenase d) pyruvate
kinase 1 e) pyruvate kinase 2 f) pyruvate kinase, enolase and
phosphoglycerate mutase g) enolase h) phosphoglycerate mutase i)
pyruvate kinase (1 or 2)/enolase/phosphoglycerate mutase j)
phosphoketolase k) phosphoacetyltransacetylase l)
phosphoketolase/phosphoacetyltransacetylase m) acetaldehyde
dehydrogenase n) PEP carboxylase o) ribulose-1,5-bisphosphate
carboxylase/oxygenase (RubisCO)
Construction of DNA-Vectors for Overexpression
[0995] In general:
[0996] DNA sequences encoding genes of interest were amplified by
polymerase chain reaction (PCR) using specific primers. When the
genomic sequence did not contain appropriate restriction sites for
cloning, primers were designed containing restriction sites.
Genomic DNA from Synechocystis sp. PCC 6803 was used as template.
The amplified PCR fragments were digested with the appropriate
restriction enzymes and cloned into either a self replicating
plasmid (pVZ series) or an integrative plasmid (pSK series). As
promoters either the genomic 5' region of the specific gene itself
was used or alternative an inducible promoter like PpetJ. (PpetJ,
pVZ, pSK, for description see below mentioned adh/pdc constructs).
An antibiotic resistance cassette for selection of positive clones
is present on the appropriate plasmid. The structures and sequences
of all used DNA-vectors are described below (see 2.).
[0997] Genetic engineering of constructs as well as PCRs, ligations
into cloning vectors, insertions of antibiotic resistance cassettes
and transformations into E. coli were done using standard
procedures (state of the art) or according to the manufacturer
instructions.
[0998] All pVZ plasmids were transferred to Synechocystis sp. PCC
6803 by conjugation. This method is described for the below
mentioned adh/pdc constructs. The pSK constructs were transferred
to Synechocystis sp. PCC 6803 by transformation. The method of
transformation using the natural competence of Synechocystis sp.
PCC 6803 for DNA uptake was already described in detail for the
generation of the glycogen synthase mutant
a) Construction of DNA-Vectors for Overexpression of Malic
Enzyme
[0999] The open reading frame (ORF) slr0721 encodes malic enzyme 1
(EC 1.1.1.38), Ac. No P72661. The amino acid sequence for this
protein is presented in FIG. 18A.
[1000] For overexpression of malic enzyme, the encoding me gene
together with its gene-specific terminator region was PCR-amplified
using the following primer:
Mae-NdeI.fw: 5'-CATATGGTTAGCCTCACCCCCAAT-3' (SEQ ID NO: 177),
primer contains a NdeI restriction site for cloning (marked in bold
letters) MeLongClaI.rv: 5'-ATCGATCGGGATGGCCTATTTATGG-3' (SEQ ID NO:
178), primer contains a ClaI restriction site for cloning (marked
in bold letters)
[1001] The PCR fragment was amplified by a High-Fidelity DNA
Polymerase (Phusion.TM.; Finnzymes), adenylated (BIOTAQ.TM.
DNA-Polymerase; BIOLINE), cloned into the pDrive vector (Qiagen)
and restricted with NdeI/ClaI (Fermentas). This fragment was cloned
into the pSK9 vector, digested with NdeI/ClaI. The gene is
incorporated into a non-coding genome region of Synechocystis sp.
PCC 6803 via the integrated platform. The expression of the enzyme
is under control of the copper dependent promoter PpetJ.
[1002] The construct used, designated as pSK9/me-long, has the
structure presented in FIG. 18B. The insert for this clone has the
nucleotide sequence presented in FIG. 18C
b) Construction of DNA-Vector for Overexpression of Malate
Dehydrogenese
[1003] An open reading frame (ORF) sll0891 encodes malate
dehydrogenase (EC 1.1.1.37), Ac. No Q55383. The amino acid sequence
for this protein is presented in FIG. 19A.
[1004] For overexpression of malate dehydrogenase a construct was
generated including start-codon and the gene specific termination
loop of the mdh gene using the following primers:
Mdh-NdeI.fw: 5'-CATATGAATATTTTGGAGTATGCTCC-3' (SEQ ID NO: 179),
primer contains a NdeI restriction site for cloning (marked in bold
letters) Mdh-ClaI.rv 5'-ATCGATAAGCCCTAACCTCGGTG-3' (SEQ ID NO:
180), primer contains a ClaI restriction site for cloning (marked
in bold letters)
[1005] The PCR fragment was amplified by a High-Fidelity DNA
Polymerase (Phusion.TM.; Finnzymes), adenylated (BIOTAQ.TM.
DNA-Polymerase; BIOLINE), cloned into the pDrive vector (Qiagen)
and restricted with NdeI/ClaI (Fermentas). This fragment was cloned
into the pSK9 vector, digested with NdeI/ClaI. The expression of
the enzyme is under the control of the copper dependent promoter
PpetJ.
[1006] The construct used, designated as pSK9/mdh, has the
structure presented in FIG. 19B; the nucleotide sequence for the
insert of this clone is presented in 19C
c) Construction of DNA-Vector for Co-Overexpression of Mac Enzyme
and Malate Dehydrogenese
[1007] This construct was generated for co-overexpression of malic
enzyme and malate dehydrogenase. These genes were amplified by PCR
using primers including the start and stop-codon of the me gene
(PCR fragment I) and including the ribosome binding site (RBS) and
termination loop of the mdh gene (PCR fragment II). The
co-expression of the enzymes is under the control of the copper
dependent promoter PpetJ.
[1008] The following primers were used for amplification
[1009] PCR fragment I:
Mae-NdeI.fw: 5'-CATATGGTTAGCCTCACCCCCAAT-3' (SEQ ID NO: 181),
primer contains a `NdeI restriction site for cloning (marked in
bold letters) MeShortClaI.rv 5'-ATCGATACAATTCCCGATTAACTATTGACC-3'
(SEQ ID NO: 182), primer contains a ClaI restriction site for
cloning (marked in bold letters)
[1010] PCR fragment I:
MdhRBSClaI.fw: 5'-ATCGATTTTTCTCCACCATCAACACC-3' (SEQ ID NO: 183),
primer contains a ClaI restriction site for cloning (marked in bold
letters) MdhBglII.rv: 5'-AGATCTAAGCCCTAACCTCGGTG-3' (SEQ ID NO:
184), primer contains a BglII restriction site for cloning (marked
in bold letters)
[1011] The PCR fragments were amplified by a High-Fidelity DNA
Polymerase (Phusion.TM.; Finnzymes), adenylated (BIOTAQ.TM.
DNA-Polymerase, BIOLINE), cloned into the pDrive vector (Qiagen)
and restricted with NdeI/ClaI and ClaI/BglII (Fermentas),
respectively. These fragments were cloned into the pSK9 vector,
first digested with NdeI/ClaI for integration of malic enzyme and
secondly with ClaI/BglII for integration of malate
dehydrogenase.
[1012] The construct used, designated as pSK9/me-mdh, has the
structure presented in FIG. 19D, and the nucleotide sequence of its
insert is presented in FIG. 19E
d) Construction of DNA-Vectors for Overexpression of Pyruvate
Kinase 1
[1013] The open reading frame (ORF) sll0587 encodes a pyruvate
kinase 1 (EC 2.7.1.40 (PK1)), Ac. No Q55863. The amino acid
sequence of this protein is presented in FIG. 20A. Two constructs
were generated in order to overexpress pyruvate kinase 1. One,
harboring the own pyruvate kinase promoter region, and another
construct on which pyruvate kinase 1 is under control of the
inducible promoter PpetJ.
[1014] For the construct with the genomic 5'-region of the pyruvate
kinase gene itself serving as promoter, a 2376 bp fragment
containing the entire coding sequence from pyruvate kinase 1 (sll
0587) plus 770 bp upstream of the gene (promoter region) and 320 bp
downstream of the gene (terminator region) was amplified by PCR
using the following primer
#pykA-5fw: 5'-CCTGTTATTGGCCACGGGCAGTA-3' (SEQ ID NO: 185)
#pykA-2rv: 5''-GGTTTACCCTGGGCTCGAGAATTAGG-3' (SEQ ID NO: 186),
primer contains a XhoI restriction site (CTCGAG) for cloning
(marked in bold letters).
[1015] The PCR fragment was digested with MfeI/XhoI (MfeI site was
present in the genomic sequence; MfeI shares compatible cohesive
ends with EcoRI), subcloned into pIC20H (using EcoRI/XhoI), cut out
of this plasmid with SalI/XhoI and ligated into the E.
coli-Synechocystis shuttle vector pVZ321 (self replicating
plasmid).
[1016] The construct used, named pVZ321-pyk1, has the structure
presented in FIG. 20B, and its insert nucleotide sequence is
presented in FIG. 20C.
[1017] For the construct on which pyruvate kinase 1 is under
control of the inducible promoter PpetJ, a 1763 bp fragment
containing the entire coding sequence from pyruvate kinase 1 (sll
0587) plus 320 bp downstream of the gene (terminator region) was
amplified by PCR using the following primer:
#pykA-3fw: 5'-CCCGGTGAAGCATATGAGACCCCT-3' (SEQ ID NO: 187), primer
contains a NdeI restriction site (CATATG) for cloning (marked in
bold letters). ATG in the restriction site represents the start
codon of the gene. .pi.pykA-2rv: 5''-GGTTTACCCTGGGCTCGAGAATTTAGG-3'
(SEQ ID NO: 188), primer contains a XhoI restriction site (CTCGAG)
for cloning (marked in bold letters).
[1018] The PCR fragment was digested with NdeI/XhoI, ligated to
PpetJ (SalI/NdeI) and cloned into the E. coli-Synechocystis shuttle
vector pVZ321 (self replicating plasmid).
[1019] The construct used, named pVZ321-PpetJ-pyk1, has the
structure presented in FIG. 20D, and the nucleotide sequence of its
insert is presented in FIG. 20E.
e) Construction of DNA-Vectors for Overexpression of Pyruvate
Kinase 2
[1020] The open reading frame (ORF) sll1275 encodes pyruvate kinase
2 (EC 2.7.1.40 (PK2)), Ac. No P73534. The amino acid sequence for
this protein is presented in FIG. 21A. Two constructs were
generated in order to overexpress pyruvate kinase 2. One, harboring
the own pyruvate kinase promoter region, and another construct on
which pyruvate kinase 2 is under control of the inducible promoter
PpetJ.
[1021] For the construct with the genomic 5' region of the pyk2
gene itself serving as promoter, a 2647 bp fragment containing the
entire coding sequence from pyk 2 (sll 1275) plus 600 bp upstream
of the gene (promoter region) and 280 bp downstream of the gene
(terminator region) was amplified by PCR using the following
primer:
#pykB-1fw: 5'-CCTAAATTCAGGTCGACCGGCAAAC-3' (SEQ ID NO: 189), primer
contains a SalI restriction site (GTCGAC) for cloning (marked in
bold letters). #pykB-2rv: 5'-CACCAACCAGGCTCGAGTGGG-3' (SEQ ID NO:
190), primer contains a XhoI restriction site (CTCGAG) for cloning
(marked in bold letters).
[1022] The PCR fragment was digested with SalI/XhoI and ligated
into the E. coli-Synechocystis shuttle vector pVZ321 (self
replicating plasmid).
[1023] The construct used, named pVZ321-pyk2, has the structure
presented in FIG. 21B, and the nucleotide sequence of its insert is
presented in FIG. 21C.
[1024] For the construct on which pyruvate kinase 2 is under
control of the inducible promoter PpetJ, a 2057 bp fragment
containing the entire coding sequence from pyruvate kinase 2 (sll
1275) plus 280 bp downstream of the gene (terminator region) was
amplified by PCR using the following primer:
#pykB-3fw: 5'-CCTAATTTCAGCCCCATATGCAAACG-3' (SEQ ID NO: 191),
primer contains a NdeI restriction site (CATATG) for cloning
(marked in bold letters). ATG in the restriction site represents
the start codon of the gene. #pykB-2rv: 5'-CACCAACCAGGCTCGAGTGGG-3'
(SEQ ID NO: 192), primer contains a XhoI restriction site (CTCGAG)
for cloning (marked in bold letters).
[1025] The PCR fragment was digested with NdeI/XhoI, ligated to
PpetJ (SalI/NdeI) and cloned into the E. coli-Synechocystis shuttle
vector pVZ321 (self replicating plasmid).
[1026] The resulting construct, pVZ321-PpetJ-pyk2, has the
structure presented in FIG. 21D, and the nucleotide sequence of its
insert is presented in FIG. 21E.
f) Construction of DNA-Vector for Overexpression of Pyruvate
Kinase, Enolase and Phosphoglycerate Mutase
[1027] A DNA-vector was constructed in order to express additional
genes coding for pyruvate kinase, phosphoglycerate mutase and
enolase A DNA fragment encoding these genes was cut out of plasmid
#67. This plasmid was constructed by Dr. John Coleman, University
of Toronto, Toronto, Canada.
[1028] The insert of plasmid #67 has the structure presented in
FIG. 22A.
[1029] The Insert of plasmid #67 contains a 357 bases long
cyanobacteria ribulose-1,5-bisphosphate carboxylase/oxygenase
(RubisCO) promoter (Prbc) from Synechococcus PCC 7942. Downstream
of this promoter there are three inserted open reading frames, the
first is pyruvate kinase I from E. coli, the second enolase and the
third phosphoglycerate mutase both from Zymomonas mobilis. The
pyruvate kinase region differs from E. coli K-12 pyruvate kinase 1
(Ac. No AAC74746) by 3 nucleotides and one amino acid. (G to D
mutation, underlined in the sequence below). The enolase gene from
Zymomonas mobilis (Ac. No YP.sub.--163343) is a 100% amino acid
match. The nucleotide sequence differs by two synonymous
substitutions in the enolase region. The phosphoglycerate mutase
gene is one amino acid different from Zymomonas (Ac. No
YP.sub.--162975), from G to D at 118th amino acid (underlined in
the sequence below). A HindIII site links the E. coli pyruvate
kinase and the Zymomonas enolase genes.
[1030] The amino aid sequences of the enzymes encoded by the
described insert are presented in FIG. 22B for pyruvate kinase I
(E. coli K.sub.12); in FIG. 22C for enolase (Zymomonas mobilis);
and in FIG. 22D for phosphoglycerate mutase (Zymomonas
mobilis).
[1031] The nucleotide sequence of the described insert of plasmid
#67 s presented in FIG. 22E.
[1032] The insert of plasmid #67 was cut out the vector using
restriction enzymes XmaI and SpeI and cloned into the E.
coli-Synechocystis shuttle vector pVZ321 and pVZ322 (self
replicating plasmids)(XmaI/XbaI); XbaI and SpeI share compatible
cohesive ends.
[1033] Plasmid pVZ321-p67 has the structure presented in FIG. 22F,
and plasmid pVZ322-p67 has the structure presented in FIG. 22G.
g) Construction of DNA-Vectors for Overexpression of Enolase
[1034] The open reading frame (ORF) sir 752 encodes the enclase
(eno, 2-phosphoglycerate dehydratase) (EC 4.2.1.11), Ac. No
BAA18749. The amino acid sequence for this protein is presented in
FIG. 23A.
[1035] A construct was generated for overexpression of enolase
under control of the inducible promoter PpetJ.
[1036] The construct includes the petJ promoter, the 1299 bp coding
sequence for enolase (slr0752) and 214 bp downstream of the gene
(terminator region). The enolase gene was amplified by PCR using
the following primer:
TABLE-US-00011 #Eno-SacI-ATG (SEQ ID NO: 193)
5'-TAGAGCTCTTAAGTAAAGTCCCCGCCAC CAT-3', #Eno-XhoI-rev (SEQ ID NO:
194) 5'-TACTCGAGGTCATTGCTTCCTTGGCTTA GAAC-3',
[1037] Primers contain a SacI or XhoI restriction site,
respectively, for coning (marked in bold letters).
[1038] The PCR fragment was digested with SacI/XhoI and ligated
downstream of the PpetJ promoter into pJet-PpetJ. The entire
PpetJ-enolase fragment was cut out of this plasmid with SalI/XhoI
and ligated into the E. coli-Synechocystis shuttle vector pVZ21
(self replicating plasmid).
[1039] The construct used, named pVZ321-PpetJ-eno, has the
structure presented in 23B, and the nucleotide sequence of its
insert is presented in 23C.
h) Construction of DNA-Vectors for Overexpression of
Phosphoglycerate Mutase
[1040] The open reading frame (ORF) slr1124 encodes the
phosphoglycerate mutase (EC 5.4.2.1), Ac. No BAA16651. The amino
acid sequence for this protein is presented in FIG. 24A.
[1041] A construct was generated for overexpression of
phosphoglycerate mutase under control of the Inducible promoter
PpetJ.
[1042] The construct includes the petJ promoter, the 1047 bp coding
sequence for phosphoglycerate mutase (slr1124) and 143 bp
downstream of the gene (terminator region). The phosphoglycerate
mutase gene was amplified by PCR using the following primer:
TABLE-US-00012 #Pgm-SacI-ATG (SEQ ID NO: 195)
5'-TAGAGCTCACCAAAGACGATGTGGCCC ACCAA-3' #Pgm-XhoI-rev (SEQ ID NO:
196) 5'-TACTCGAGTATGACCCCGCTGTTGCAG TTC-3'
[1043] Primers contain a SacI or XhoI restriction site,
respectively, for cloning (marked in bold letters).
[1044] The PCR fragment was digested with SacI/XhoI and ligated
downstream of the PpetJ promoter into pJet-PpetJ. The entire
phosphoglycerate mutase fragment was cut out of this plasmid with
SalI/XhoI and ligated into the E. coli-Synechocystis shuttle vector
pVZ321 (self replicating plasmid).
[1045] The construct used, named pVZ321-PpetJ-pgm, has the
structure presented in FIG. 24B, the nucleotide sequence of its
insert is presented in FIG. 24C.
i) Construction of DNA-Vectors for Co-Overexpression of Pyruvate
Kinase 1 or 2, Enolase and Phosphoglycerate Mutase
[1046] Further plasmids were generated in order to overexpress the
three glycolytic enzymes pyruvate kinase 1 or 2, enolase and
phosphoglycerate mutase from one transcript.
[1047] One construct was generated for overexpression of pyruvate
kinase 1 (ORF sll0587), enolase (ORF slr0752) and phosphoglycerate
mutase (ORF slr1124); the second construct encodes pyruvate kinase
2 (ORF sll1275), enolase (ORF slr0752) and phosphoglycerate mutase
(ORF slr1124). The protein sequences, EC and Accession numbers of
the enzymes are already described herein.
[1048] In both constructs the overexpression of the three genes is
under control of the inducible promoter PpetJ.
[1049] The glycolytic genes were amplified by PCR using the
following primers:
TABLE-US-00013 pyruvate kinase 1 (pyk1): (SEQ ID NO: 197) #pykA-3fw
5'-CCCGGTGAAGCATATGAGACCCCT-3' (NdeI-site: inserted) (SEQ ID NO:
198) #Pyk1-SacI-rev 5'-TAGAGCTCTTAAGAAATACGGTGAATCTTG- 3' pyruvate
kinase 2 (pyk2): (SEQ ID NO: 199) #pykB-3fw:
5'-CCTAATTTCAGCCCCATATGCAAACG-3' (NdeI-site inserted) (SEQ ID NO:
200) #Pyk2-SacI-rev 5'-TAGAGCTCCCTATCCTTTGGACACC-3' enolase (eno):
(SEQ ID NO: 201) #Eno-SacI-fw
5'-TAGAGCTCGTGTTTGGAGCATTACACACCGATG-3' (SEQ ID NO: 202)
#Eno-BglII-rev 5'-TAAGATCTTTTTAAGAATGTTTGGGACCCAG- 3'
phospgoglycerate mutase (pgm): (SEQ ID NO: 203) #Pgm-BglII-fw
5'-TCAGATCTGCCCCTCTGGGAAAAAATGACCA- 3' (SEQ ID NO: 204)
#Pgm-XhoI-rev 5'-TACTCGAGTATGACCCCGCTGTTGCAGTTC-3'
[1050] All primers contain restriction sites for cloning (marked in
bold letters).
[1051] PCR fragments were subcloned into PCR cloning plasmid
pJet1.2 blunt. The genes were cut out of these plasmids with the
appropriate restriction enzymes and ligated downstream of the PpetJ
promoter into plC-PpetJ as followed:
TABLE-US-00014 5'-XhoI-pIC-PpetI-NdeI-3' 5'-NdeI-pyk1-SacI-3'
5'-SacI-eno-BglII-3' 5'-BglII-pgm-XhoI-3'
[1052] The same construct was generated using fragment
5'-NdeI-pyk2-SacI-3' instead of 5'-NdeI-pyk1-SacI-3'.
[1053] The entire PpetJ-pyk1-eno-pgm or PpetJ-pyk2-eno-pgm
fragments were cut out of the cloning plasmid with PstI/XhoI and
ligated into the E. coli-Synechocystis shuttle vector pVZ322 (self
replicating plasmid).
[1054] The construct named pVZ322-PpetJ-pyk1-eno-pgm has the
structure presented in FIG. 24D and the construct
pVZ322-PpetJ-pyk2-eno-pgm has the structure presented in FIG. 24E.
The sequence of the insert of pVZ322-PpetJ-pyk1-eno-pgm is
presented in the FIG. 24F and the sequence of the insert of
pVZ322-PpetJ-pyk2-eno-pgm is presented in FIG. 24.
j) Construction of DNA-Vector for Overexpression of
Phosphoketolase
[1055] The open reading frame (ORF) slr0453 encodes the probable
phosphoketolase (phk), (EC 4.1.2-), Ac. No P74690. The amino acid
sequence of the protein is presented in FIG. 25A.
[1056] A construct was generated for overexpression of
phosphoketolase under control of the inducible promoter PpetJ.
[1057] The construct includes the petJ promoter, the 2418 bp coding
sequence for phosphoketolase (slr0453) and 307 bp downstream of the
gene (terminator region). The phosphoketolase gene was amplified by
PCR using the following primer:
TABLE-US-00015 (SEQ ID NO: 205) #phk1-NdeI
5'-GTGTCTCATATGGTTACATCCCCCTTTTCCCTT-3' (SEQ ID NO: 206) #phk2-XhoI
5'-CGAGCCCTGCTCGAGCAGGC-3'
[1058] Primers contain a NdeI or XhoI restriction site,
respectively, for cloning (marked in bold letters).
[1059] The PCR fragment was digested with NdeI/XhoI and ligated
downstream of the PpetJ promoter into plC-PpetJ. The entire
PpetJ-phosphoketolase fragment was cut out of this plasmid with
PstI/XhoI and ligated into the E. coli-Synechocystis shuttle vector
pVZ322 (self replicating plasmid).
[1060] The construct used, named pVZ321-PpetJ-phk, has the
structure presented in FIG. 25B, and the nucleotide sequence of its
insert is presented in FIG. 25C.
k) Construction of DNA-Vector for Overexpression of
Phosphoacetyltransacetylase
[1061] The open reading frame (ORF) slr2132 encodes a
phosphoacetyltransacetylase (pta), EC 2.3.1.8, Ac No. P73662. The
amino acid sequence of this protein is presented in FIG. 26A.
[1062] A construct was generated for overexpression of
phosphoacetyltransacetylase under control of the inducible promoter
PpetJ.
[1063] The construct includes the pet) promoter, the 2094 bp coding
sequence from ORF slr2132 and 258 bp downstream of the gene
(terminator region). The phosphoacetyltransacetylase gene was
amplified by PCR using the following primer:
TABLE-US-00016 (SEQ ID NO: 207) #pta_pPETJ1-NdeI
5'-GTGCCTCATATGACGAGTTCCCTTTATTTA AGCAC-3' (SEQ ID NO: 208)
#pta_pPETJ2-XhoI 5'-CGGTTGCTCGAGCATCTGGAACGGTTGGGT AAAT-3'
[1064] Primers contain a NdeI or XhoI restriction site,
respectively, for cloning (marked in bold letters).
[1065] The PCR fragment was digested with NdeI/XhoI and ligated
downstream of the PpetJ promoter into plC-PpetJ. The entire
PpetJ-phosphoacetyltransacetylase fragment was cut out of this
plasmid with PstI/XhoI and ligated into the E. coli-Synechocystis
shuttle vector pVZ322 (self replicating plasmid).
[1066] The construct used, named pVZ322-PpetJ-pta, has the
structure presented in FIG. 26B, and the nucleotide sequence of the
insert for construct pVZ322-PpetJ-pta is presented in FIG. 26C.
l) Construction of DNA-Vector for Co-Overexpression of
Phosphoketolase and Phosphoacetyltransacetylase
[1067] One further construct was created in order to co-overexpress
the phosphoketolase and phosphoacetyltransacetylase from one
transcript. The protein sequences, EC and Accession numbers of the
enzymes are already described above. The expression of the genes is
under control of the inducible promoter PpetJ. The phosphoketolase
and phosphoacetyltransacetylase genes were amplified by PCR using
the following primers:
TABLE-US-00017 phosphoketolase (phk) (SEQ ID NO: 209) #phk1
5'-GTGTCTCATATGGTTACATCCCCCTTTTCCCTT-3' (NdeI site inserted) (SEQ
ID NO: 210) #phk-BglII-rev 5'-GGTCACAGATCTGTTGTCCCCCATGGCCTA
GCTA-3' phosphoacetyltransacetylase (pta) (SEQ ID NO: 211)
#pta-BglII-fw 5'-CCTTGCAGATCTGGATACGTTGAGGTTATTTAA ATTATGA-3' (SEQ
ID NO: 212) #pta_pPETJ2-XhoI 5'-CGGTTGCTCGAGCATCTGGAACGGTTGG
GTAAAT-3'
[1068] All primers contain restriction sites for cloning (marked in
hold letters).
[1069] PCR fragments were cut with the appropriate restriction
enzymes and ligated downstream of the PpetJ promoter into pIC-PpetJ
as followed:
TABLE-US-00018 5'-XhoI-pIC-PpetJ-NdeI-3' 5'-NdeI-phk-BglII-3'
5'-BglII-pta-XhoI-3'
[1070] The entire PpetJ-phK pta fragment was cut out of the cloning
plasmid pIC20H with SmaI/NruI and ligated into SmaI site of the E.
coli-Synechocystis shuttle vector pVZ322 (self replicating
plasmid).
[1071] The construct named pVZ322-PpetJ-phk-pta has the structure
presented in FIG. 26D, and the nucleotide sequence of the insert of
pVZ22-PpetJ-phk-pta is presented in FIG. 26E.
m) Construction of DNA-Vector for Overexpression of Aldehyde
Dehydrogenase
[1072] The open reading frame (ORF)slr0091 encodes a aldehyde
dehydrogenase (aldh), EC 1.2.1.3, Ac No. BAA10564 Q55811. The amino
acid sequence for the protein is presented in FIG. 27A
[1073] A construct was generated for overexpression of aldehyde
dehydrogenase under control of the inducible promoter PpetJ. The
construct includes the petJ promoter, the 1369 bp aldehyde
dehydrogenase fragment containing the entire coding sequence from
ORF slr0091 and 205 bp downstream of the gene (terminator region).
The aldehyde dehydrogenase (aldh) gene was amplified by PCR using
the following primer:
TABLE-US-00019 (SEQ ID NO: 213) #aldh1-NdeI-fw
5'-GTGCCTCATATGAATACTGCTAAAACTGTTGT TGC-3' (SEQ ID NO: 214)
#aldh2-XhoI-rev 5'-GATCTCCTCGAGGTAAAGAATCAGCATAGGT CTGG-3'
[1074] Primers contain a NdeI or XhoI restriction site,
respectively, for cloning (marked in bold letters).
[1075] The PCR fragment was digested with NdeI/XhoI and ligated
downstream of the PpetJ promoter into plC-PpetJ. The entire
PpetJ-aldehyde dehydrogenase fragment was cut out of this plasmid
with PstI/XhoI and ligated into the E. coli-Synechocystis shuttle
vector pVZ322 (self replicating plasmid).
[1076] The construct used, named pVZ322-PpetJ-aldh, has the
structure presented in FIG. 27B, and the nucleotide sequence of the
insert of construct pVZ322-PpetJ-aldh is presented in FIG. 27C.
n) Construction of DNA-Vectors for Overexpression of PEP
Carboxylase
[1077] The open reading frame (ORF) sll0920 encodes the
phosphoenolpyruvate carboxylase (EC 4.1.1.31), BAA18393. The amino
acid sequence for this protein is presented in FIG. 28A.
[1078] One construct was generated for overexpression of
phosphoenolpyruvate carboxylase under control of the inducible
promoter PpetJ.
[1079] The construct includes the pet) promoter, the 3105 bp
ppc-fragment containing the entire coding sequence from
phosphoenolpyruvate carboxylase (sll 0920) and 59 bp downstream of
the gene (terminator region) was amplified by PCR using the
following primer:
(SEQ ID NO: 215) #ppc.NdeI.fw: 5'-CTAGAGGTTCATATGAACTTGGC-3', this
primer contains a NdeI restriction site (CATATG) for cloning
(marked in bold letters) (SEQ ID NO: 216) #ppc.XhoI.rv:
5'-GTAAGCAGGCTCGAGGCAAG-3', this primer contains a XhoI restriction
site (CTCGAG) for cloning (marked in bold letters).
[1080] The PCR fragment was digested with NdeI/XhoI, subcloned into
K8 (using NdeI/XhoI), cut out of this plasmid with SalI/XhoI and
ligated into the E. coli/Synechocystis shuttle vector pVZ321 (self
replicating plasmid). The pVZ321vector has the GenBank accession
number AF100176.
[1081] The construct used, named pVZ321-PpetJ-ppc, has the
structure presented in FIG. 28B, and the nucleotide sequence for
the pVZ321-PpetJ-ppc insert is presented in FIG. 28C.
o) Construction of DNA-Vectors for Overexpression of
Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase (RubisCO)
[1082] Overexpression of the Synechocystis RuBisCO was reached by
integration of a conjugative, self-replicating pVZ plasmid into
Synechocystis containing either the rbcLXS operon alone or the
rbcLXS operon as transcriptional fusion together with the pyruvate
decarboxylase from Zymomonas mobilis.
[1083] The entire rbc operon from Synechocystis sp. PCC6803 was
amplified by PCR using the primer pairs:
[1084] SynRbc-BglII-fw and SynRbc-PstI-rev for the over-expression
from the rbcL-promoter, which are shown in FIGS. 28D and 28E,
respectively.
[1085] SynRbc-SacI-fw and SynRbc-PstI-rev for the over-expression
as transcriptional fusion with the Pdc from Zymomonas mobilis. The
sequence of SynRbc-SacI-fw is shown in FIG. 28F.
[1086] The database entry numbers for the CyanoBase, the genome
database for cyanobacteria (available on the world wide web at
bacteria.kazusa.or.jp/cyanobase/index.html) for the Synechocystis
rbcL X-rbcS coding sequences are slr0009 for the ribulose
bisphosphate carboxylase large subunit (rbcL), slr0011 for the
possible Rubisco chaperonin (rbcX) and slr0012 for the ribulose
bisphosphate carboxylase small subunit (rbcS). The DNA sequence
coding for the rbcLXS operon is depicted in FIG. 28G. The protein
sequence obtained by translation of the protein coding DNA sequence
is depicted in FIG. 28H for the rbcL large subunit; the rbcX
Rubisco chaperonin protein sequence is shown in FIG. 28I and the
protein sequence of the ribulose bisphosphate carboxylase small
subunit (rbcS) is shown in FIG. 28J.
[1087] Mutants were selected on streptomycin plates and grown in
BG11 medium containing the appropriate antibiotics (kanamycin 100
mg/l; streptomycin 10 mg/l).
[1088] In Synechocystis sp. PCC6803 mutants were generated by
transforming the cells with the plasmid pVZ321b-Prbc-SynRbcLXS
(FIG. 28K).
[1089] In the following the vectors, which were used are
described.
a) Plasmid pSK9 Structure and Sequence
[1090] The non-public pSK9 vector was generated in the lab of V. V.
Zinchenko (Moscow. Russia). The structure of this plasmid is
schematically represented in FIG. 29A, and its nucleotide sequence
is presented in FIG. 29B.
b) Self-Replicating Synechocystis Plasmid pVZ321 Structure and
Sequence
[1091] The pVZ321vector has the GenBank accession number AF100176.
This vector is presented schematically in FIG. 30A, and the pVZ321
nucleotide sequence is presented in FIG. 30B.
c) Self-Replicating Synechocystis Plasmid pVZ322 Structure and
Sequence
[1092] The pVZ322 vector has the GenBank accession number AF100175.
FIG. 31A presents a schematic of its structure, and FIG. 31B
presents its nucleotide sequence.
d) Construction of the Cloning Vector plC20H
[1093] For cloning procedures a plasmid was constructed harboring
promoter PpetJ in the multi-cloning site of cloning vector pIC20H,
Ac. No. L08912, (Marsh J. L., Erfle M., Wykes E. J.; "The plC
plasmid and phage vectors with versatile cloning sites for
recombinant selection by insertional inactivation"; Gene 32:481-485
(1984)). Promoter PpetJ was cut out of the herein described pSK9
plasmid with ClaI and KpnI and ligated into pIC20H (ClaI/KpnI),
resulting in plasmid plC-PeptJ.
[1094] The plasmid plC-PpetJ has the structure presented
schematically in FIG. 32A, and the nucleotide sequence of plC PpetJ
is presented in FIG. 32B.
Generation of Additional Knock-Out/Knock-Down Mutants of
Synechocystis Sp. PCC 6603: Methods and Results
[1095] The following Knock-Out construct sequences have been
conveniently described and provided herein: (a) alanine
dehydrogenase (ad), (b) ADP-glucose pyrophosphorylase (g glgC), (c)
pyruvate water dikinase (ppsA). (d) lactate dehyrogenase (Idh), (e)
acetate kinase (ack) and (f) phosphoacetyltransacetylase (pta). The
following Knock-Down construct sequence is described and provided
pyruvate dehydrogenase (pdhB). These constructs may be used singly
or sequentially in order to provide one or more mutations.
Mutagenesis
[1096] Host cells are mutagenized by transformation of the
DNA-vectors (knock-out-constructs) using the natural competence of
Synechocystis sp. PCC 6803 for DNA uptake and its system for
homologous recombination as previously described herein. The
transformation may comprise one or more steps in order to create
mutant cells having a single, double, triple, etc. knockout and/or
knockdown mutations. Additionally, knockdown/knockout mutants may
additionally be mutagenized by introducing one or more
overexpressing DNA constructs as described herein. As noted
previously herein, the concentration of the appropriate
antibiotic(s) is increased stepwise when the cells are transferred
onto another agar plate or into liquid culture (for kanamycin from
initially 5 to 150 .mu.g/ml BG11, for chloramphenicol from
initially 1 to 15 .mu.g/ml BG11 medium) in order to get fully
segregated (homozygous) mutants. Transfers are done every 2 weeks.
In case of kanamycin, the concentration in the range from 50 to 150
.mu.g/ml agar is increased gradually over the course of 4
weeks.
Molecular Analysis of Mutant Host Cells
[1097] In order to clearly demonstrate that a targeted homologous
recombination event occurred in the selected mutant(s) cell, a
variety of methods well known to one of ordinary skill in the art
may be utilized. A test for successful knockout mutagenesis will be
done initially by PCR amplifying a DNA fragment from the inserted
antibiotic resistance cassette into the gene that should be knocked
out. In addition, knockout mutants as well as knock-down mutants
will be also checked by the detection and non-detection
respectively of the target enzyme mRNA level in the mutant and wild
type cells by using different techniques known in the art, e.g.
RT-PCR, Northern blot or RNase protection assays. These recombinant
DNA/molecular biology methods are well known to one of ordinary
skill in the art; For example see: Methods in Enzymology, Vol. 167,
(L. Packer, A. N. Glazer, eds); For extraction of genomic DNA:
Franche C, Damerval T. in Methods of Enzymology, Vol. 167 p.
803-808; for extraction of total RNA David 1 Lane, Katherine G.
Field, Gary J. Olsen, and Norman R. Pace in Methods of Enzymology,
Vol. 167 p. 138-144; for Extraction of plasmid DNA: Grant R.
Lambert and Noel G. Carr, Rapid Small-Scale Plasmid Isolation by
Several Methods from Filamentous Cyanobacteria, Arch Microbiol
(1982) 133: 122-125; for Northern Blots: Axmann, I. M., Kensche,
P., Vogel, J., Kohl, S., Herzel, H. & Hess, W. R. (2005) Genome
Biol 6, R73; for RT-PCR: Emanuel C, von Groll U, Muller M, Borner
T, Weihe A. Development- and tissue-specific expression of the RpoT
gene family of Arabidopsis encoding mitochondrial and plastid RNA
polymerases. Planta. 2006 April; 223(5):998-1009; for RNase
protection assay W. R. Hess, B. Hoch, P. Zeltz, T. Hubschmann, H.
Kossel and T. Borner. Plant Cell 6 (1994), pp. 1455-1465. Academic
Press, Inc., 1988), which are incorporated herein by reference.
[1098] Also, sufficient nucleotide sequence information for all
enzymes is provided herein or available from known nucleotide
sequence databases for the selection of the appropriate
probes/primers for these analyses. With Northern Blot analysis, the
abundance and relative amount of a mRNA will be detected. The same
would be the case using a RNase protection assay but with a much
higher sensitivity. The abundance and also the absolute amount of a
mRNA can be determined with a high sensitivity using the
RT-PCR.
[1099] With the PCR analysis, one forward primer is derived from
the genetic sequence of the targeted enzyme and one reverse primer
is derived from the biocide gene sequence; the amplified hybrid DNA
fragment will be characterized and analyzed for predicted size
and/or nucleotide sequence content. Mutant(s) cells found not to be
expressing wildtype mRNA and found to have the above noted
characteristics will be selected for further analysis.
Characterization of Knock-Out/Knock-Down Mutants
Cultivation of Cyanobacterial Wild Type and Mutant Strains
[1100] For a knock-out or knock-down mutant(s) related to the
formation of reserve compounds such as glycogen, e.g., mutants of
further reserve metabolites syntheses as PHB or cyanophycin, wild
type and mutant strains of Synechocystis PCC 6803 are grown as
batch cultures in BG11 medium at 29.degree. C. under continuous
illumination with white light (intensity: 40 .mu.E m.sup.-2
s.sup.-1) and aeration with air. For cultivation of mutants, the
appropriate antibiotics are added to the medium (kanamycin 75 mg/l;
chloramphenicol 15 mg/l). Samples are analyzed briefly before the
nitrogen step down ("+N"), directly after resuspension of the cells
in BG11 medium lacking a nitrogen source ("-N", 0 h) and after 3, 6
and 24 hours.
[1101] All other knock-out or knock-down mutants will be grown
under standard culture conditions known in the art.
[1102] As provided below, mutants and wild type cells will be
characterized regarding their intra- and extracellular pyruvate
content using optical enzymatic tests and their profile of all
relevant metabolites respectively. (incl. 3-PGA, PEP, pyruvate,
acetyl-CoA, glycogen, PHB, cyanophycin, malate, oxaloacetate,
2-oxoglutarate, acetate, lactate, etc.) using appropriate
techniques for example, spectroscopic methods, chromatographic
methods such ion chromatography or optical or enzymatic methods or
combinations thereof. The analysis will always be done in
comparison to the wild type.
[1103] Also the growth and pigmentation properties of mutant(s) wil
be compared to the wild type cell using standard protocols well
known in the art.
[1104] The example presented here will provide a graphic depiction
of growth properties for wild type and mutant cells as change in X
vs. time, wherein X is ideally dry weight or biovolume.
Alternatively, optical density, cell count and chlorophyll could be
used as reference parameters. Alternatively, pigmentation could be
quantified spectrophotometrically as another parameter.
Protocol for Characterization of Metabolic Mutants Containing at
Least One First and/or One First and One Second Genetic
Modification
[1105] Generation of knock-out and over-expression mutants with
single, double, triple, etc. knock-out and/or knock-down and/or
over-expression mutations as a first genetic modification and the
molecular analysis of such mutant cells in general is already
described above.
Characterization of Metabolic Mutants
[1106] Metabolic mutant strains having a first genetic modification
were characterized regarding their growth properties and certain
extra- and intracellular metabolites in comparison to wild type
strains. In addition the afore described metabolic mutants were
also transformed with PDC and ADH as a second genetic modification
and were characterized regarding growth properties, extra- and
intracellular metabolites and ethanol production rates in
comparison to the appropriate reference strain(s) expressing PDC
and ADH, but lacking the metabolic mutation (first genetic
modification).
Cultivation of Cyanobacterial Wild Type and Mutant Strains
[1107] Wild type and mutant strains of Synechocystis PCC 6803 were
grown as batch cultures in BG11 medium at 28-29.degree. C. For
cultivation of mutants the appropriate antibiotics were added to
the medium (kanamycin 75 ma/l; chloramphenicol 10 ma/l; gentamycin
3 mg/l or streptomycin 10 mg/l). In order to avoid premature
induction of gene expression in mutants having constructs with
PpetJ or PisiA promoter, these mutants were grown in culture medium
supplemented with excess copper or iron (5.times. Cu for PpetJ;
3.times. Fe for PisiA).
[1108] Prior to characterization experiments, pre-cultures were
grown in BG11 medium (no excess of Cu or Fe) and aeration with 0.5%
CO.sub.2 in air.
[1109] For characterization experiments, wild type and mutant
strains were grown in BG11 medium. Mutants having constructs with
PpetJ or PisiA (overexpression, knock-down mutants or mutants
expressing PDC and ADH) were transferred to BG11 lacking Cu (PpetJ
or Fe (PisiA), respectively, in order to induce gene expression
(described in detail for PDC/ADH expressing mutants).
[1110] The total culture volume in characterization experiments was
300 mL in a 500 mL Schott-Flask; the initial OD.sub.750 was 1.
Cultures were aerated with 0.5% CO.sub.2 in air.
[1111] All mutants were characterized under constant light
conditions (75-100 .mu.E m.sup.-2 s.sup.-1). In fast growing
cultures, the light intensity was increased during the growth
experiment (75-100 .mu.E m.sup.-2 s.sup.-1 up to OD5; then light
intensity was increased to 200 .mu.E m.sup.-2 s.sup.-1).
[1112] Knock-out mutants related to fermentative pathways such as
lactate dehydrogenase, acetate kinase or
phosphoacetyltransacetylase were additionally characterized under
day/night conditions (12 h 100 .mu.E m.sup.-2 s.sup.-1/12 h dark).
Knock-out mutants related to the formation of reserve compounds
such as glycogen or PHB were additionally examined after
transferring the cells in BG11 medium lacking a nitrogen source
(nitrogen starvation conditions) as previously described
herein,
Principle of Ethanol Quantification:
[1113] Ethanol is oxidized by nicotinamide-adenine dinucleotide
(NAD.sup.+) to acetaldehyde in a reaction which is catalyzed by the
enzyme alcohol dehydrogenase (ADH) (reaction 1). The acetaldehyde,
which is formed in the reaction, is quantitatively oxidized to
acetic acid by the enzyme aldehyde dehydrogenase (AI-DH) (reaction
2).
##STR00001##
[1114] In reactions (1) and (2) reduced nicotinamide-adenine
dinucleotide (NADH) is formed. The amount of NADH formed is
proportionate to the amount of ethanol in the sample. NADH is
easily quantified by means of its light absorbance. The absorbance
is usually measured at 340 nm, Hg 365 nm or Hg 334 nm.
Procedure:
[1115] Preparation of solutions: Solution 1:1.3 mg/ml NAD and 0.27
U aldehyde dehydrogenase in potassium diphosphate buffer, pH 9.0.
Solution 2: Suspension of alcohol dehydrogenase (ADH) with approx.
4000 U/ml. Alternatively, the chemicals and solutions of the
ethanol determination kit of Boehringer Mannheim/R-Biopharm (Cat.
No. 10 176 290 035) can be used. Sample and solution 1 are mixed in
a ratio of 3 ml solution 1 and 0.1 ml sample (if necessary the
sample is diluted with water). After approx. 3 min the absorbance
is measured (A.sub.1). The reaction is then started by the addition
of ADH suspension (solution 2, 0.050 ml for 3 ml solution 1 and 0.1
nm sample). After completion of the reaction (approx. 5 to 10 min)
the absorbance is measured again (A.sub.2). The absorption
measurements can be performed using a photometer or a microplate
reader. For plate reader measurements all volumes are
downscaled.
[1116] From the measured absorbance difference
.DELTA.A=(A.sub.2-A.sub.1) the ethanol concentration in the sample
is calculated with the equation:
c = V .times. MG .times. d .times. v .times. 2 .times. 1000 .times.
.DELTA. A ##EQU00001##
c, ethanol concentration [g/L]; V, total volume [mL]; MG, molecular
weight of ethanol (46.07 g/mol); e, extinction coefficient (6.3
L.times.mmol.sup.-1.times.cm.sup.-1 at 340 nm); d, light path [cm];
v, sample volume [mL]
Literature:
[1117] Protocol of the kit Ethanol, UV method for the determination
of ethanol in foodstuff and other materials, Cat. No. 10176290035,
R-Biopharm AG, Darmstadt, Germany.
[1118] H.-O. Beutler (1984) in Methods in Enzymatic Analysis
(Bergmeyer, H. U. ed.) 3.sup.rd ed. Vol. VI, pp. 598-606. Verlag
Chemie, Weinheim, Germany.
Growth Properties:
[1119] For characterization experiments, metabolic mutant and the
appropriate reference strains were cultured as described. Growth
was followed for about 14 days by measuring optical density (daily)
and chlorophyll (every second day). Photosynthetic O.sub.2
production was determined several times during exponential growth
phase using a Clark electrode as followed:
Measurement of Photosynthetic Oxygen Evolution
[1120] Cell are washed 2.times. with fresh growth medium by
centrifugation (3000.times.g, 10 min, room temperature) and
resuspension. The cells are finally resuspended in growth medium to
a chlorophyll concentration of 10 to 15 .mu.g chlorophyll/m.
Chlorophyll is measured as described by [N. Tandeau De Marsac and
J. Houmard]. The cells are filled into the chamber of a Rank
Brothers oxygen electrode (Digital Model 10, Rank Brothers,
Cambridge, England) and sodium bicarbonate is added to a final
concentration of 25 mM.
[1121] The excitation light for photosynthesis experiments is
provided by a slide projector with a 150-watt lamp (Osram, Xenophot
HLX Germany).
[1122] The oxygen concentration in the chamber is recorded
continuously with chart recorder (REC 112, Amersham Pharmacla
Biotech) connected to the electrode. The chamber of the oxygen
electrode is maintained at 25.degree. C. with a circulating,
temperature-controlled water bath (RM6, Lauda Brinkmann). For the
calibration of the electrode the signal difference of air-saturated
water (100% saturation) and oxygen free water (zero point) is
measured. Oxygen free water is obtained by adding sodium dithionite
(approximately 1 mg/ml). The measured amplitude is equated with the
solubility of oxygen in water at 25.degree. C. and a pressure of 1
bar (8.11 mg oxygen/L). Literature: N. Tandeau De Marsac and J.
Houmard in: Methods in Enzymology, Vol. 169, 318-328. L. Packer,
ed., Academic Press. 1988
Determination of Ethanol Production
[1123] For characterization of mutants expressing PDC and ADH or
only PDC or other ethanologenic enzymes as a second genetic
modification, ethanol was measured daily during the growth
experiment according to the afore described optical enzymatic
method ("Ethanol UV method" test kit by Boehringer
Mannheim/R-Biopharm, Darmstadt, Germany). Ethanol production of
metabolic mutants expressing PDC and ADH were compared to the
appropriate reference strain expressing PDC and ADH as a second
genetic modification, but lacking the respective metabolic
mutation, the first genetic modification.
[1124] The cells were cultured over a period of time of 14 days.
These cell cultures were further characterized during their
logarithmic growth phase at certain time points with regard to
their ethanol production rate, their chlorophyll content and
photosynthetic capacity (oxygen evolution in .mu.mol O2/mg Ch1*h).
These three values were measured in a period of time of
approximately 2 hours as described below. In the following these
measurements are referred to as "short term measurements" or "short
term experiments".
Simultaneous Measurement of Photosynthetic Oxygen Evolution and
Ethanol Production (Short Term Experiment)
[1125] For the comparison of ethanol production and photosynthesis,
ethanol production rates and rates of photosynthetic oxygen
evolution are measured simultaneous in a single assay.
[1126] Cells are washed 2.times. with fresh growth medium by
centrifugation (3000.times.g, 10 min, room temperature) and
resuspension. Cells are resuspended in growth medium to a
chlorophyll concentration of 10 to 15 .mu.g chlorophyll/mL.
Chlorophyll is measured as described in [N. Tandeau De Marsac and
J. Houmard in: Methods in Enzymology, Vol. 169, 318-328. L. Packer,
ed., Academic Press, 1988]. 1.9 mL of the cells and 0.1 mL of 500
mM sodium bicarbonate for carbon dioxide supply are filled into the
chamber of the oxygen electrode (Digital Model 10, Rank Brothers,
Cambridge, England), and the rate of the photosynthetic oxygen
evolution is measured as described herein (Measurement of
photosynthetic oxygen evolution). (for example with a chart
recorder REC 112, Amersham Pharmacia Biotech connected to the
electrode). The chamber of the oxygen electrode is maintained at a
constant temperature (in most cases 25.degree. C.) with a
circulating, temperature-controlled water bath (RM6, Lauda
Brinkmann). The chamber is translucent and illuminated from the
outside. The excitation light for photosynthesis experiments is
provided by a slide projector with a 150-watt lamp (Osram, Xenophot
HLX Germany). For measurements under standard conditions the light
intensity was adjusted to 300 .mu.m.sup.-2 s.sup.-1. Light
intensities at the oxygen electrode were determined and the
distance between light source and the chamber of the oxygen
electrode were adjusted accordingly in order to obtain the desired
light intensity of 300 .mu.m.sup.-2 s.sup.-1 at the oxygen
electrode. When the illumination is switched on, photosynthesis
starts and an increase of oxygen concentration in the chamber can
be observed. After a short period of time the plotted curve is
linear. From the linear part of the plotted curve the rate
(=photosynthetic oxygen evolution vs. time) is determined. The
entire measurement of oxygen is finished after not more than 10
minutes. After completion of this measurement illumination of the
sample in the chamber is continued under unchanged conditions Over
a period of one hour samples of 0.15 ml are taken in defined
intervals (in most cases every 10 minutes). Immediately after
removal samples are centrifuged (14,000.times.g, 10 min, 4.degree.
C.) and the supernatant is stored on ice. After completion of the
sampling the ethanol concentration in the supernatants is measured
as described herein. The ethanol concentration versus time is
plotted. Using the linear equation the rate of the increase of the
ethanol content in v/v in the assay per hour is calculated. The
rate of ethanol production is usually given in the dimension
.mu.mol ethanol*h.sup.-1*mg chlorophyll, the chlorophyll content
measured at the beginning of the experiment is then used.
Determination of Itra- and Extracellular Metabolites
[1127] Two different methods were used for the extraction of cells
to determine the level of intracellular metabolites. They are
described here as "Protocol for extraction of intracellular
metabolites" and "Extraction of metabolites using a Retsch mil".
The method "Extraction of metabolites using ice cold methanol (snap
shot extraction)" extracts the intracellular metabolites but seizes
also the metabolites in the medium. For the determination of
extracellular metabolites an extraction of the cells is not
necessary. Those metabolites were measured directly in the
media.
Protocol for Extraction of Intracellular Metabolites
[1128] use 5 ml culture.
[1129] Centrifuge for 10 min 4500 rpm.
[1130] Resuspend the pellet in 1 ml dd water.
[1131] Centrifuge 5 min with 14000 rpm. Discard the
supernatant.
[1132] Resuspend the pellet in 1 ml double distilled water
[1133] Centrifuge 5 min, 14000 rpm, 4.degree. C. Discard the
complete supernatant.
[1134] Continue or store the pellet by -20.degree. C. under Argon
atmosphere.
[1135] Add 600 ml of extraction buffer.
[1136] Extraction buffer: 10:3:1--methanol:chloroform:water
[1137] Vortex briefly.
[1138] Shake at 4 degrees for .gtoreq.10 min.
[1139] Centrifuge 5 min with 14000 rpm.
[1140] Transfer 500 .mu.l to a new tube.
[1141] Add 200 .mu.l chloroform and 200 .mu.l water.
[1142] Centrifuge 5 min with 14000 rpm.
[1143] Transfer 500 .mu.l of the upper phase to a new tube and
speed vac to dry.
[1144] Resuspend the pellet in 100 .mu.l double distilled
water.
[1145] Shake at 4 degrees for .gtoreq.20 min. Centrifuge 5 min with
14000 rpm.
[1146] Transfer 95 .mu.l to a vial for IC.
Extraction of Metabolites Using a Retsch Mill:
[1147] The protocol for extraction of intracellular metabolites was
designed by Dr. M. Grundel.
Protocol:
[1148] Cells (150 ml cell culture) are harvested by centrifugation
and resuspended in 400 .mu.l buffer (100 mM Tris/HCl, pH 7.5) to
which 200 .mu.l of glass beads (0.1 mm diameter) are added. Cell
lysis is performed using a Retsch mill model MM 301 (treatment for
10 minutes, 4.degree. C.). After removal of glass beads, remaining
intact cells and cell debris was removed by centrifugation (10
minutes, 4.degree. C.). The whole procedure is repeated once.
Proteins in the combined supernatants are precipitated by
deoxycholate/trichloroacetic acid treatment (Bensadoun and
Weinstein. 1976. Anal. Biochem. 70:241-250) and removed by
centrifugation. The supernatant, containing the soluble
metabolites, is neutralized with 2 M K.sub.2CO.sub.3 and adjusted
to a volume of 1.5 ml with 100 mM Tris/HCl buffer, pH 7.5. In order
to determine the concentration of metabolites, aliquots of 100-500
.mu.l are used in the optical tests.
Extraction of Metabolites Using Ice Cold Methanol (Snap Shot
Extraction):
Literature Describing the Method:
[1149] According to R. P. Maharjan, T. Ferenci. 2002. Global
metabolite analysis: the influence of extraction methodology on
metabolome profiles of Escherichia coli. Anal. Biochem.
313:145-154.
[1150] This method allows for the immediate freezing of
intracellular metabolite pools and the extraction of numerous
intra- and extracellular metabolites at the same time.
Protocol:
[1151] Batches of cyanobacterial cultures are dropped into an equal
volume of methanol, cooled by dry ice, and incubated on dry ice
until completely frozen. After thawing in ice/water (10 min) the
samples are centrifuged for 5 min (>=17.000.times.g, temperature
as low as possible). The pellet is extracted a second time with
cold 50% methanol (-20.degree. C.). Supernatants are combined.
Methanol is removed by evaporation at 35.degree. C. under vacuum
using a rotavapor apparatus. The remaining solution is lyophilized,
the residue is resuspended in a minimal volume of water.
[1152] The efficiency of extraction of bacterial cells with cold
methanol is similar to that with hot ethanol or hot methanol. But
the method is very simple, rapid and changes in the stability and
reactivity in metabolites are minimized.
[1153] When extracellular pyruvate and oxoglutarate are assayed, an
extraction is not necessary since both metabolites are detectable
directly in the media. Quantification of Intracellular and
extracellular pyruvate and oxoglutarate levels before and after
nitrogen deprivation is done as previously described herein.
[1154] Pyruvate and phosphoenolpyruvate are quantified using an
optic enzymatic test of Hausler et al. (2000), Anal. Biochem,
281:1-. This method allows for the quantification of pyruvate and
phosphoenolpyruvate in one test
Protocol:
[1155] The quantifications are based on the reduction of pyruvate
to lactate by lactate dehydrogenase (LDH) at the expense of NADH
which is oxidized to NAD+. In the first step, pyruvate was assayed.
After completion of this reaction, pyruvate kinase is added.
Pyruvate kinase converts phosphoenolpyruvate to pyruvate and thus
allows for determination of phosphoenolpyruvate.
[1156] To 450 .mu.l master mix (9 .mu.l 120 mM NADH, 12 .mu.l 1 M
MgCl2, 46 .mu.l 1 M KCl, 12 .mu.l 100 mM ADP, 360 .mu.l 100 mM
HEPES, 10 .mu.l H.sub.2O) 520 .mu.l sample (if necessary diluted
with H.sub.2O) are added. Add 2 .mu.l LDH to start the reaction.
The oxidation of NADH is observed as decrease of absorbance at 340
nm. Either the difference of the absorbances at 340 nm minus 380 nm
is measured by difference spectroscopy (turbid or colored samples;
.epsilon.340-380=4.83 l.times.cm.times.mmol-1) or the absorbance at
540 nm is measured against water (.epsilon.340=6.28
l.times.cm.times.mmol-1). After complete reaction of pyruvate, 2
.mu.l pyruvate kinase are added to the assay. NADH oxidation is
measured as before. From the differences of the absorbances at the
start and the end of the reactions, the amount of oxidized NADH
(=amount of pyruvate, and phosphoenolpyruvate, respectively) is
calculated.
Chemicals and Solutions:
[1157] 1. Lactate dehydrogenase suspension from bovine heart
(L-LDH, Sigma L2625-2.5KU, suspension with 5629.5 U/ml), diluted
1:10 2. Pyruvate Kinase from rabbit muscle (P K, Serve 34085,
suspension with 4000 U/ml), diluted 1:20
3. 100 mM HEPES/NaOH (pH 7.5)
4. 1 M MgCl2
5. 100 mM ADP
6. NADH (Sigma, N6005) 20 mM in H.sub.2O
7. 1 M KCl
[1158] Photometric Quantification of Pyruvate (and/or Lactate) in
an Enzymatic Cycling System
Method:
[1159] According to E. Valero & F. Garcia-Carmona. 1996.
Optimizing Enzymatic Cycling Assays: Spectrophotometric
Determination of Low Levels of Pyruvate and L-Lactate. Anal.
Biochem. 239:47-52
[1160] This method allows for the quantification of pyruvate
(and/or lactate) with a 10-fold higher sensitivity than the
pyruvate quantification method described before.
Protocol:
[1161] In a cyclic reaction pyruvate is reduced to lactate under
consumption of NADH, the lactate is oxidized by lactate oxidase to
pyruvate. The rate of NADH consumption, monitored
spectrophotometrically at 340 nm is proportional to the amount of
pyruvate (plus lactate if present) in the sample. For calibration
curves, different amounts of pyruvate are added to the master mix
(end volume 1000 .mu.l) consisting of 50 mM TRIS-buffer, pH 7.5,
256 .mu.M NADH, 18 .mu.g lactate dehydrogenase and 60 .mu.g lactate
oxidase. The reaction is started by addition of lactate
dehydrogenase and the time course of the reaction at 340 nm is
followed for some minutes. Samples with unknown amounts of pyruvate
and lactate are treated identically and quantified using the
calibration curve. Detection limit is about 1 nmol pyruvate and/or
lactate.
Chemicals and Solutions:
1. 50 mM TRIS/HCl (pH 7.5)
2. 20 mM NADH in H.sub.2O
[1162] 3. 025 mg/ml lactate dehydrogenase in 50 mM TRIS/HCl (pH
7.5) 4. 2.6 mg/ml lactate oxidase in 50 mM TRIS/HCl (pH 7.5).
Spectrophotometric Quantification of 2-Oxoglutarate Using an
Enzymatic Test
Method:
[1163] The method used is an adaptation of a fluorimetric method
(P. J. Senior. (1975). J. Bacteriol. 123:407-418) for
spectrophotometry. The oxidation of NADH, followed by the
absorption change at 340 nm, is proportional to the concentration
of 2-oxoglutarate.
Protocol:
[1164] Cuvettes contained a final volume of 1000 .mu.l: 100-500
.mu.l sample; 10 .mu.l ammonium sulfate; 10 .mu.l NADH; 10 .mu.l
ADP; 10 .mu.l glutamate dehydrogenase solution; TRIS buffer added
to a final volume of 1000 .mu.l. The reaction is started by the
addition of glutamate dehydrogenase.
Chemicals and Solutions:
[1165] 1. 1 M ammonium sulfate
2. 20 mM NADH
3. 0.1 M ADP
[1166] 4. 2.6 enzyme units per ml glutamate dehydrogenase (from
bovine liver; 104 [1159] enzyme units per mg Serva lot no.
22904)
5. 0.1 M TRIS/HC pH 8.0
[1167] Acetaldehyde was quantified by a modification of the
protocol of a kit for ethanol quantification (Ethanol kit,
R-Biopharm AG). Acetaldehyde is converted by aldehyde dehydrogenase
under formation of NADH, which is quantified by its absorption at
340 nm. The amount is proportionate to the acetaldehyde content of
the sample.
[1168] All mutant strains were characterized regarding their
profile of relevant intracellular metabolites using ion
chromatography always in comparison to the wild type or appropriate
reference strain, respectively.
[1169] Short description of the UV-method for the determination of
acetic acid in foodstuff and other materials from Boehringer
Mannheim/R-Biopharm, Darmstadt, Germany
[1170] Principle: Acetic acid (acetate) is converted to acetyl-CoA
in the presence of the acetyl-CoA synthetase (ACS),
adenosine-5'-triphosphate (ATP) and coenzyme A (CoA) (1).
(1) Acetate+ATP+CoA ACS acetyl-CoA+AMP+PP
[1171] Acetyl-CoA reacts with oxaloacetate to citrate in the
presence of citrate synthase (CS) (2).
(2) Acetyl-CoA+oxaloacetate+H.sub.2O CS citrate+CoA
[1172] The oxaloacetate required for reaction (2) is formed from
L-malate and nicotineamide-adenine dinucleotide (NAD) in the
presence of L-malate dehydrogenase (L-MDH) (3). In this reaction
NAD is reduced to NADH.
(3) L-malate+NAD+L-MDH oxaloacetate+NADH+H+
[1173] The determination is based on the formation of NADH measured
by the increase in light absorbance at 340, 334 or 365 nm. Because
of the equilibrium of the preceding indicator reaction, the amount
of NADH formed is not linearly (directly) proportional to the
acetic acid concentration (this fact is been taken into
consideration in the calculation of acetic acid
concentrations).
[1174] The above described methods for the quantification of
acetate, pyruvate, acetaldehyde and 2-oxoglutarate can detect
changes in the static steady state levels of these metabolic
intermediates. As mentioned above the first genetic modification
can result in a change of the metabolic flux of these metabolic
intermediates, which is hard to detect by assays, which are able to
detect the steady state level of a metabolite, but not the changes
in the flux of the metabolite. In particular, these enzymatic
assays might not properly show the changes in the metabolic
activity of a photoautotrophic host cell, induced by the first
genetic modification
[1175] An overview of alternative assay methods, which can be used
to detect the change in the metabolic activity of a
photoautotrophic host cell of this invention is shown in the Review
of Shimizu, "Metabolic Engineering-Interating Methodologies of
Molecular Breeding and Bioprocess Systems Engineering", Journal of
Bioscience and Bioengineering, Vol. 94, No. 6: 563-573 (2002),
which is hereby incorporated by reference. These methods are more
time-consuming and complex than the above described enzymatic
assays and are for example metabolic flux analysis (MFA), cell
capability analysis, metabolic control analysis (MCA) or
.sup.13C-NMR and gas chromatography. Mass spectroscopy (GCMS)
measurements.
[1176] Wild type (WT) and mutant metabolite (pyruvate, acetaldehyde
or acetyl-CoA or precursors thereof) measurements will be obtained
as previously described herein and presented in the tables
below.
TABLE-US-00020 Metabolite Intracellular Metabolite Extracellular
level in mmol per liter level in mmol per liter OD.sub.750 wt
mutant wt mutant 1.0 +N A A + .DELTA. F F + .DELTA. -N, B B +
.DELTA. G G + .DELTA. 0 h -N, C C + .DELTA. H H + .DELTA. 3.5 h -N,
D D + .DELTA. I I + .DELTA. 6 h -N, E E + .DELTA. J J + .DELTA. 24
h
[1177] Data will be verified by repetitions.
A-J represent wild type values for the indicated conditions .DELTA.
represents an increment relative to the wt measurement
[1178] The table shows an example for such an experiment. In other
experiments the optical density (OD.sub.750) at the beginning of
the experiment and the time points can be different
TABLE-US-00021 Metabolite Intracellular level in mmol per liter
Metabolite Extracellular (calculated per packed level in mmol per
Time of cell volume.sup.1) liter culture volume cultivation wt
mutant wt mutant T1 A A + .DELTA. E E + .DELTA. T2 B B + .DELTA. F
F + .DELTA. T3 C C + .DELTA. G G + .DELTA. T4 D D + .DELTA. H H +
.DELTA.
[1179] Data will be verified by repetitions.
[1180] A-H represent wild type values
[1181] .DELTA. represents an increment relative to the wt
measurement
[1182] Parameters such as OD.sub.750nm, Chorophyll content, protein
content and cell number will also be measured in standardizing and
evaluating metabolite values at different time points.
[1183] In addition, measurements can be obtained for variations in
culture conditions such as light intensity, growth in darkness and
in day/night cycles respectively, CO.sub.2 supplementation and
temperature. Also, further variations might concern the composition
of the growth medium (e.g. concentration of nitrate, ammonium,
phosphate, sulfate or microelements (e.g. Cu, Fe)). All these
variations in culture conditions are known to one of ordinary skill
in the art
[1184] The data will be analyzed and presented graphically as
previously described herein.
Analysis of Ethanol Production
[1185] In order to discover whether the enhanced level of
biosynthesis of pyruvate, acetaldehyde or acetyl-CoA in the
mutant(s) cells also leads to a higher production of ethanol,
Synechocystis sp. PCC 6803, both wildtype as well as the mutant(s)
cells are transformed with the plasmid pVZ containing the Zymomonas
mobilis Pdc and AdhII enzymes or other plasmids encoding
ethanologenic genes under the control of the iron dependent isiA
promoter or other promoters.
[1186] Analysis of ethanol production is done as previously
described herein. Synechocystis sp. PCC 6803 with and without Pdc
and Adh and Synechocystis sp. PCC 6803 mutant(s) cells with and
without Pdc and Adh will be compared. This example will present a
graphic depiction of these results that clearly demonstrate that
increased ethanol production is provided by the mutant(s) cells
when compared to the wild type cell.
Generation of Overexpression Mutants of Synechocystis Sp. PCC 6803:
Methods and Results
[1187] The following overexpression construct sequences have been
conveniently described and provided herein: (a) malic enzyme, (b)
malate dehydrogenase. (c) pyruvate kinase 1, (d) pyruvate kinase 2,
and (e) pyruvate kinase, enolase and phosphoglycerate mutase. These
constructs may be used singly or sequentially in order to provide
one or more mutations. Also, constructs contain either the natural
promoter for the enzyme gene of interest or an inducible
promoter.
Mutagenesis
[1188] Host cells are mutagenized by transformation of the
overexpression DNA-vectors using the natural competence of
Synechocystis sp. PCC 6803 for DNA uptake. In case of integrative
overexpression mutants, the system of Synechocystis sp. PCC 6803
for homologous recombination as previously described herein is
used. In addition, self-replicating constructs may also be used.
The transformation may comprise one or more steps in order to
create mutant cells having a single, double, triple, etc.
overexpression mutations. Additionally, one or more
knockdown/knockout mutations (as described herein) may be
introduced. As noted previously herein, the concentration of the
appropriate antibiotic(s) is increased stepwise when the cells are
transferred onto another agar plate or into liquid culture (for
kanamycin from initially 5 to 150 .mu.g/ml BG11, for
chloramphenicol from initially 1 to 15 .mu.g/ml BG11 medium) in
order to get fully segregated (homozygous) mutants. Transfers are
done every 2 weeks. In case of kanamycin, the concentration in the
range from 50 to 150 .mu.g/ml agar is increased gradually over the
course of 4 weeks.
Molecular Analysis of Mutant Host Cell
[1189] In order to establish that the selected mutant(s) cell is
overexpressing the target enzyme, RNA will be extracted from wild
type and mutant cells and will be examined by using different
techniques known in the art, e.g. RT-PCR, Northern blot or RNase
protection assays. These recombinant DNA/molecular biology methods
are well known to one of ordinary skill in the art; For example
see: Methods in Enzymology, Vol. 167, (L. Packer, A. N. Glazer,
eds) Academic Press, Inc., 1988); For extraction of genomic DNA:
Franche C, Damerval T. in Methods of Enzymology, Vol. 167 p.
803-808; for extraction of total RNA David 1. Lane, Katherine G.
Field, Gary J. Olsen, and Norman R. Pace in Methods of Enzymology,
Vol. 167 p. 138-144; for Extraction of plasmid DNA: Grant R.
Lambert and Noel G. Carr, Rapid Small-Scale Plasmid isolation by
Several Methods from Filamentous Cyanobacteria, Arch Microbiol
(1982) 133: 122-125; for Northern Blots: Axmann, I. M., Kensche,
P., Vogel, J., Kohl, S., Herzel, H. & Hess, W. R. (2005) Genome
Biol 6, R73; for RT-PCR: Emanuel C, von Groll U, Muller M, Borner
T, Weihe A. Development- and tissue-specific expression of the RpoT
gene family of Arabidopsis encoding mitochondrial and plastid RNA
polymerases. Planta. 2006 April; 223(5):998-1009; for RNase
protection assay. W. R. Hess, B. Hoch, P. Zeltz, T. Hubschmann, H.
Kossel and T. Borner. Plant Cell 6 (1994), pp. 1455-1465., which
are incorporated herein by reference.
[1190] Also, sufficient nucleotide sequence information for all
enzymes is provided herein or available from known nucleotide
sequence databases for the selection of the appropriate
probes/primers for these analyses. With Northern Blot analysis, the
abundance and relative amount of a mRNA will be detected. The same
would be the case using a RNase protection assay but with a much
higher sensitivity. The abundance and also the absolute amount of a
mRNA can be determined with a high sensitivity using the RT-PCR.
Mutant(s) cells found to be overexpressing the target mRNA will be
selected for further analysis.
Characterization of Overexpression Mutants
Cultivation of Cyanobacterial Wild Type and Mutant Strains
[1191] wild type (WT) and mutant strains will be grown under
standard culture conditions.
[1192] Nitrogen step-down conditions will be as previously
described herein.
[1193] Conditions for the induction of inducible promoters is
provided herein through the teachings of the specification and by
way of reference to specific publications. See also D. A. Los, M.
K. Ray and M. Murata, Differences in the control of the
temperature-dependent expression of four genes for desaturases in
Synechocystis sp. PCC 6803, Mol. Microbiol. 25 (1997), pp.
1167-1175.
[1194] As provided below, mutants and wild type cells will be
characterized regarding their intra- and extracellular pyruvate
content using optical enzymatic tests and their profile of all
relevant metabolites respectively. (incl. 3-PGA, PEP, pyruvate,
acetyl-CoA, glycogen, PHB, cyanophycin, malate, oxaloacetate,
2-oxoglutarate, acetate, lactate, etc.) using ion chromatography
always in comparison to the wild type.
[1195] Also the growth and pigmentation properties of mutant(s)
will be compared to the wild type cell using standard protocols
well known in the art.
[1196] The example presented here will provide a graphic depiction
of growth properties for wild type and mutant cells as change in X
vs. time, wherein X is ideally dry weight or biovolume.
Alternatively, optical density, cell count and chlorophyll could be
used as reference parameters. Alternatively, pigmentation could be
quantified spectrophotometrically as another parameter.
[1197] Wild type (WT) and mutant metabolite (pyruvate, acetaldehyde
or acetyl-CoA or precursors thereof) measurements will be obtained
as previously described herein and presented in the table
below.
TABLE-US-00022 Metabolite Intracellular Metabolite Extracellular
level in mmol per liter level in mmol per liter OD.sub.750 wt
mutant wt mutant 1.0 +N A A + .DELTA. F F + .DELTA. -N, B B +
.DELTA. G G + .DELTA. 0 h -N, C C + .DELTA. H H + .DELTA. 3.5 h -N,
D D + .DELTA. I I + .DELTA. 6 h -N, E E + .DELTA. J J + .DELTA. 24
h
[1198] Data will be verified by repetitions.
[1199] A-J represent wild type values for the indicated
conditions
[1200] .DELTA. represents an increment relative to the wt
measurement
[1201] The table shows an example for such an experiment. In other
experiments the optical density (OD.sub.750) at the beginning of
the experiment and the time points can be different
TABLE-US-00023 Metabolite Intracellular level in mmol per liter
Metabolite Extracellular (calculated per packed level in mmol per
Time of cell volume.sup.1) liter culture volume cultivation wt
mutant wt mutant T1 A A + .DELTA. E E + .DELTA. T2 B B + .DELTA. F
F + .DELTA. T3 C C + .DELTA. G G + .DELTA. T4 D D + .DELTA. H H +
.DELTA.
[1202] Data will be verified by repetitions.
[1203] A-H represent wild type values
[1204] .DELTA. represents an increment relative to the wt
measurement
[1205] Parameters such as OD.sub.750nm, Chlorophyll content,
protein content and cell number will also be measured in
standardizing and evaluating metabolite values at different time
points.
[1206] In addition, measurements can be obtained for variations in
culture conditions such as light intensity, growth in darkness and
in day/night cycles respectively. CO.sub.2 supplementation and
temperature. Also, further variations might concern the composition
of the growth medium (e.g. concentration of nitrate, ammonium,
phosphate, sulfate or microelements (e.g. Cu, Fe)). All these
variations in culture conditions are known to one of ordinary skill
in the art.
[1207] The data will be analyzed and presented graphically as
previously described herein.
Analysis of Ethanol Production
[1208] In order to discover whether the enhanced level of
biosynthesis of pyruvate, acetaldehyde or acetyl-CoA in the
mutant(s) cells also leads to a higher production of ethanol,
Synechocystis sp. PCC 6803, both wildtype as well as the mutant(s)
cells are transformed with the plasmid pVZ containing the Zymomonas
mobilis Pdc and AdhII enzymes or other plasmids encoding
ethanologenic genes under the control of the iron dependent isiA
promoter or other promoters.
[1209] Analysis of ethanol production is done as previously
described herein. Synechocystis sp. PCC 6803 with and without Pdc
and Adh and Synechocystis sp. PCC 6803 mutant(s) cells with and
without Pdc and Adh will be compared. This example will present a
graphic depiction of these results that dclearly demonstrate that
increased ethanol production is provided by the mutant(s) cells
when compared to the wild type cell.
X. Experimental Data for Characterization of Metabolic Mutants
Containing at Least One First or One First and One Second Genetic
Modification
[1210] In the following available experimental data regarding
pyruvate secretion are discussed for photoautotrophic cells
harboring at least one first genetic modification. Furthermore
ethanol production rate, if available, are also discussed for
photoautotrophic cells containing in addition to the at least one
first genetic modification at least one second genetic
modification.
X.1 Metabolic Mutant Harbouring a Glycogen Synthase Double Knock
Out Mutation as a First Genetic Modification
[1211] Characterization of the glycogen deficient glycogen synthase
double knock out mutants of Synechocystis PCC 6803:
Nomenclature:
TABLE-US-00024 [1212] Enzyme: Glycogen synthase 1 Glycogen (starch)
synthase 2 EC no.: EC 2.3.1.21 EC 2.3.1.21 Gene name: glgA1 glgA2
Gene in s110945 s111393 Synechocystis PCC 6803:
[1213] background: Diverting the production of storage reserves
into an enhanced production of pyruvate/ethanol [1214]
Manipulation: double knockout by insertion of a chloramphenicol
cassette (.DELTA.glgA1) and kanamycin cassette (.DELTA.glgA2)
M8-mutant Cm, Km Complete segregation: yes
Characterization of the Mutants Harboring the Glycogen Synthase
Double Knock Out Mutation as a the First Genetic Modification, but
Lacking the Second Genetic Modification (Ethanologenic
Enzymes).
[1215] Determination of intracellular Glycogen before and after a N
step down
[1216] The procedure is an adaptation of the method described by
Ernst et al. (A. Ernst, H. Kirschenloher, J. Diez, P. Boger. 1984.
Arch. Microbiol. 140120-125). Glycogen is isolated by alkaline
hydrolysis of cells followed by precipitation of glycogen with
ethanol. Isolated glycogen is digested with amylolytic enzymes to
glucose, which is quantified in a standard optical test.
[1217] Protocol:
[1218] Spin down 1-4 ml of Synechocystis culture before and after N
step down resp. at RT and remove the supernatant
[1219] Add 200 .mu.l KOH (30% w/v) to the pellet and incubate 90
minutes at 95.degree. C. in a heating block
[1220] Add 600 .mu.l cold ethanol (96%) and incubate 90 min on
ice
[1221] Spin down and discard the supernatant
[1222] Wash once with ethanol (70%) and once with ethanol (96%)
[1223] Dry the pellet in a vacuum centrifuge
[1224] Dissolve the pellet in 45-90 .mu.l acetate buffer
[1225] Add 5-10 .mu.l enzyme mix (amyloglucosidase+alpha-amylase
from Bacillus amyloliquefaciens, purchased from Roche) and incubate
90 min at 45.degree. C.
[1226] Use 10-40 .mu.l of the resulting sample for the
determination of glucose after manufacture's instruction (Infinity
glucose hexokinase liquid stable reagent for optical test at 340
nm; Cat No. TR15421 Thermo Electron Corporation)
Reaction:
##STR00002##
[1228] Chemicals and solutions:
1. aqueous solution of KOH (30% w/v) 2. ethanol 96% v/v 3. 100 mM
acetate buffer, adjusted to pH 5.0 with NaOH 4. enzyme mixture of
amylo glucosidase (26.7 mg/ml; Boehringer, lot 1490306) plus
alpha-amylase (1.0 mg/ml; Boehringer, lot 84874220-34) in 100 mM
acetate buffer pH 5.0)
[1229] Quantification of intracellular and extracellular pyruvate
and oxoglutarate levels before and after nitrogen deprivation ("N
step down")
Explanation for "N Step Down":
[1230] This means sedimentation of cyanobacterial cells by
centrifugation, decantation of the nitrate-containing (+N) medium
and resuspension of the culture in nitrate-free (-N) medium.
Cultivation Under Continuous Light (40 .mu.E m.sup.-2 s.sup.-1),
BG11, 29.degree. C.:
[1231] Growth properties: no difference between wild type (wt) and
mutant (M8) (the growth of M8 is impaired under High Light
conditions [130 .mu.E m.sup.-3 s.sup.-1] and low inoculi [initial
OD.sub.750<0.1])
[1232] Pigmentation: no difference between wt and mutant
[1233] Storage substances: no glycogen production by the mutants in
contrast to the wt Continuous Light (40 .mu.Em.sup.-2 s.sup.-1),
BG11 without nitrogen (24 h, 48 h), 29.degree. C.: (N
starvation)
[1234] Growth properties: wt and mutant stopped growing. After
passage to BG11 medium containing nitrogen, wt started to grow
again whereas the mutant M8 gradually lost the ability to grow,
depending on duration of nitrogen depletion.
[1235] Pigmentation: After withdrawal of nitrogen, wt started to
degrade phycobilisomes (measured as absorbance at 625 nm): yellow
color; M8-mutant did not degrade phycobilisomes: still blue-green
color; unchanged chlorophyll levels (absorbance at 681 nm) in both
wt and mutant M8.
Pyruvate Level:
TABLE-US-00025 [1236] Intracellular level in mmol per liter
Extracellular (calculated per packed level in mmol per cell
volume.sup.1) liter culture volume OD.sub.750 wt M8 wt M8 1.0 +N
0.8 0.8 0.007 0.018 -N, nd nd 0 h -N 0.005 0.038 3.5 h -N, 0.004
0.08 6 h -N, 0.9 1.6 0.007 0.470 24 h
Data were verified by repetitions. nd, not detectable The packed
cell volume is less than 1% of the culture volume
[1237] Growth properties and extracellular pyruvate levels of the
.DELTA.glgA1/.DELTA.glgA2 double mutant (M8) under nitrogen replete
and nitrogen starved conditions are presented in FIG. 32C.
[1238] The glycogen deficient mutant M8 was grown up to an
OD.sub.750 of 0.6. After a centrifugation step, the cells were
washed twice with nitrogen deficient BG11 medium and transferred to
medium with nitrogen (+N, control) and without nitrogen (-N),
respectively. After 24 h incubation, nitrogen was added to the
nitrogen deficient cultures (black arrow). The growth of the
cultures was estimated by measurement of chlorophyll.
Abbreviations: Chl, chlorophyll a; Pyr, pyruvate
Oxoglutarate Level:
TABLE-US-00026 [1239] Intracellular level in mmol per liter Hours
after (calculated per packed Extracellular level in mmol nitrogen
cell volume.sup.1) per liter culuture volume step down wt M8 wt M8
0.5 0.036 0.038 nd nd 2 0.17 0.22 nd nd 5 0.18 0.26 nd 0.01 24 0.22
0.53 nd 0.14 .sup.1The packed cell volume is less than 1% of the
culture volume nd, not detectable
Light/Dark Cycle (16 h/8 h), BG11, 29.degree. C.:
[1240] Growth properties: no difference between wt and mutants M1
and M8
[1241] Further mutant characterization of the glycogen deficient
mutant M8 in comparison with the wild type strain of Synechocystis
sp. PCC6803
Culture Conditions:
[1242] Continuous light (150 .mu.E m.sup.-2 s.sup.-1), 28.degree.
C.:
[1243] Aeration with air (no additional CO.sub.2
supplementation)
[1244] Culturing in glass flasks with 5 cm diameter, 400 ml culture
volume
[1245] Media: BG11 buffered with TES buffer (Sigma-Aldrich Inc.) at
pH 8
Storage Substances:
[1246] No glycogen production by the mutants in contrast to the
wild type.
[1247] Pyruvate Concentrations in the Media Determined by Using an
Optical Enzymatic Test:
TABLE-US-00027 Pyruvate Pyruvate Pyruvate Pyruvate 0 h after N 3.5
h after N 6 h after N 24 h after N OD.sub.750 Chlorophyll step down
step down step down step down WT 1.2 6.18 .mu.g/ml 0 .mu.M 5.1
.mu.M 4.0 .mu.M 2.5 .mu.M M8 mutant 1.1 3.60 .mu.g/ml 0 .mu.M 37
.mu.M 79 .mu.M 473 .mu.M
[1248] Pyruvate Concentrations in the Media Determined by Ion
Chromatography:
TABLE-US-00028 Pyruvate Pyruvate 0 h after 24 h after OD.sub.750
Chlorophyll N step down N step down WT 1.2 6.18 .mu.g/ml 0 .mu.M
13.4 .mu.M M8 mutant 1.1 3.60 .mu.g/ml 0 .mu.M 511 .mu.M
Pyruvate Concentrations in the Media Plus Cells (Snap Shot
Extraction) Determined by Ion Chromatoarahy:
TABLE-US-00029 [1249] Pyruvate Pyruvate 0 h after 24 h after
OD.sub.750 Chlorophyll N step down N step down WT 1.2 6.18 .mu.g/ml
0 .mu.M 6.12 .mu.M M8 mutant 1.1 3.60 .mu.g/ml 0 .mu.M 523
.mu.M
[1250] Wildtype and mutant were transferred into a medium without
combined nitrogen and gown for 24 hours. Subsequently the amount of
pyruvate in the culture medium was determined in with an optical
enzymatic method and by ion chromatography. The sum of intra- and
extracellular pyruvate was determined by ion chromatography after
snapshot extraction
[1251] Shown is the conductimetric detection of pyruvate in
methanol extracts (snapshot) of cultures of wildtype and a glycogen
synthase deficient mutant after 24 h under N-deficient conditions.
The area of the pyruvate peak corresponds to 523 pmoles.
[1252] Data results are presented graphically in FIGS. 32D and
32E.
Summary Pertaining to Ethanol Production:
[1253] The loss of the two functional glycogen synthases in
Synechocystis PCC 6803 mutant M8 resulted in a two-times increased
intracellular pyruvate level and an at least 10-times increased
extracellular pyruvate level after nitrogen depletion (24 h). In
dense cultures (OD.sub.750 1.0), the extracellular pyruvate level
is actually increased up to 500 times. In the wild type, these
concentrations remained unchanged and much lower. The enhanced
pyruvate level is used for ethanol production.
[1254] Glycogen is made during the day and would therefore compete
with ethanol production in the light it is degraded during the
night and may thus support ethanol production by a quasi continuous
production.
Possible Advantages of Glycogen Deficiency:
[1255] Glycogen synthesis requires energy (ATP):
[1256] Photosynthesis.fwdarw.glucose phosphate
[1257] glucose phosphate+ATP.fwdarw.ADP-glucose+pyrophosphate
[1258] m ADP-glcose.fwdarw.glycogen+n ADP
[1259] During the night, glycogen will be degraded:
[1260] glycogen+n phosphate.fwdarw.n glucose phosphate
[1261] gluose phosphate.fwdarw..fwdarw.pentose
phosphate+CO.sub.2.uparw.
[1262] pentose phosphate.fwdarw..fwdarw.pyruvate pyruvate
ethanol+CO.sub.2.uparw.
Conclusions:
[1263] Ethanol production via glycogen requires more energy and
releases 50% more CO.sub.2 than direct production.
[1264] A further advantage may be that glycogen-deficient mutants
degrade photosynthetic pigments at a much lower rate than the wild
type under conditions of nitrogen deficiency. Thus, growth could be
retarded during ethanol production by lowering nitrogen supply.
[1265] In order to find out whether the pyruvate produced by the
glycogen synthase double knock out mutant in Synechocystis can be
used for ethanol production, the glycogen synthase double knock out
mutant cells (denoted as M8 in the below two graphs) were
transformed with the plasmid pVZ321b-PnblA-pd/adh containing the
alcohol dehydrogenase and pyruvate decarboxylase genes under the
transcriptional control of the nblA promoter inducible by nitrogen
starvation (denoted as M8 PnblA in the below two graphs). The
concentration of pyruvate in the growth medium was determined for
the M8 mutant without the pVZ321b-PnblA-pdc/adh plasmid after
having induced pyruvate secretion into the medium by nitrogen
starvation (indicated by M8-N in the below graphs). In addition the
concentration of pyruvate and ethanol in the growth medium was also
determined for the M8 mutant including the pVZ321b-PnblA-pdc/adh
plasmid after having induced pyruvate production by nitrogen
starvation (Indicated by M8 PnblA-N in the below graphs). For the
reason of comparison the respective pyruvate concentrations are
also shown for the uninduced cells (denoted with M8 PnblA+N and
M8+N, respectively).
[1266] Both graphs depict on the Y-axis the concentrations of
pyruvate and ethanol in .mu.M normalized to the cell density
measured at 750 nm (mnm). The x-axes denote the course of the
experiments in hours.
[1267] As can be seen in FIG. 32F the graph shows the pyruvate
concentrations. It can clearly be seen that the pyruvate
concentration in the growth medium is higher for the M8 mutant
without Adh and Pdc enzymes than for the M8 mutant including both
ethanol forming enzymes under the conditions of nitrogen
starvation. In the case that the cells are not subjected to
nitrogen starvation pyruvate could not be detected in the growth
medium.
[1268] FIG. 32G depicts the ethanol concentration determined in the
growth medium for the M8 mutant with the Adh and Pdc enzymes under
the conditions of nitrogen starvation and without nitrogen
starvation. The graph shows that the ethanol concentration is
higher for the M8 mutant under the conditions of nitrogen
starvation than without nitrogen starvation. By comparing both
graphs it can be observed that nearly all pyruvate produced by the
M mutant can be converted into ethanol by the Adh and Pdc enzymes:
The M8 mutant without the Adh and Pdc enzymes secretes high amounts
of pyruvate into the growth medium, but the M8 including both
enzymes only excretes small amounts of pyruvate but a high amount
of ethanol into the growth medium.
[1269] Furthermore the glycogen deficient glycogen synthase double
knock out mutants of Synechocystis PCC 6803 were transformed with
the plasmid pVZ containing ZmPdc and ADHII under the control of the
iron starvation inducible promoter isiA using the standard
protocols described above. Ethanol production rates and the
OD.sub.750nm were determined over the course of 15 days. Results
are depicted graphically in FIG. 32H.
[1270] Further, short term measurements of ethanol production rates
were carried out for the glycogen synthase double knock out mutant
in Synechocystis PCC 6803 with and without a second genetic
modification of at least one overexpressed enzyme for ethanol
formation and these production rates were compared to the ethanol
production rates of the corresponding Synechocystis cells only
harboring the second genetic modification.
.DELTA.glgA1/.DELTA.glgA2 mutant
TABLE-US-00030 .mu.mol .mu.mol .mu.mol % of O.sub.2/mg EtOH/mg
EtOH/ theoretical Chl* h Chl* h .mu.mol O.sub.2 fixed CO.sub.2 S.
PCC6803 pVZ321b- 98.3 5.0 0.051 15.4 PisiA-PDC-ADHII
.DELTA.glgA1/A2 pVZ321b- 34.8 5.4 0.154 46.2 PisiA-PDC-ADHII
[1271] The above table shows the ethanol production rates
normalized either to the chlorophyll content, the maximal
photosynthetic capacity as determined by the oxygen evolution and
the percentage of theoretical fixed CO.sub.2 which is diverted to
ethanol production for a Synechocystis strain without the glycogen
synthase double knock out mutation, the first genetic modification
(S. PCC6803 pVZ321b-PisiA-PDC-ADHII), and for Synechocystis strains
having both the first and second genetic modification
(.DELTA.glgA1/A2 pVZ321b-PisiA-PDC-ADHII). The data show that the
overall photosynthetic capacity of the cells harboring the double
knock out mutation is reduced. The results also indicate that a
higher percentage of carbon fixed via photosynthesis can be
diverted to ethanol production via a reduction of the enzymatic
affinity or activity of glycogen synthase for example by
introducing a knock out mutation of both genes glgA1/glgA2 coding
for glycogen synthase into cyanobacteria) cells such as
Synechocystis.
X.2 Metabolic Mutant Harbouring a Knock Out of
ADP-Glucose-Pyrophosphorylase (.DELTA.GLGC) as a First Genetic
Modification
[1272] Construction of the DNA-vector pGEM-T/.DELTA.glgC-KM, which
was used for generation of .DELTA.glgC mutant, was already
described herein. The obtained .DELTA.glgC mutant was partially
segregated and was grown in BG11 medium containing 75 mg/l
kanamycin. The segregation status was checked by southern blot
analysis using a radio-labeled glgC probe. Approximately 80% of the
wild-type gene copies were replaced by the introduced mutant gene
copy.
[1273] The partially segregated mutant .DELTA.glgC was examined in
comparison to Synechocystis wild-type strain under constant light
conditions as described herein.
Growth Characteristics Under Constant Light Conditions
[1274] The .DELTA.glgC mutant is generally more sensitive to tight
at low concentrated inoculi than the wild type strain
(Synechocystis PCC6803). During further batch culturing no
significant differences were detected in cell growth and
chlorophyll content between the mutant and the Synechocystis
PCC6803 wild type. However, the photosynthetic capacity of the
.DELTA.glgC mutant was about 35% lower compared to the
Synechocystis PCC6803 wild type. This finding is consistent with
data reported by Miao et al., 2003 (Miao, X, Wu, Q C, Wu, G. &
Zhao, N. (2003) Changes in photosynthesis and pigmentation in an
agp deletion mutant of the cyanobacterium Synechocystis sp.;
Biotechnol Lett. 25, 391-396).
[1275] Like in the .DELTA.glgA mutant described above, in the
.DELTA.glgC mutant the extracellular pyruvate level is strongly
increased. Data from one representative experiment are shown in the
following table:
TABLE-US-00031 4 days 7 days 9 days pyru- pyru- pyru- vate vate
vate OD.sub.750 [mM] OD.sub.750 [mM] OD.sub.750 [mM] PCC6803 Wt 1.7
0.009 2.0 0.001 2.4 0.003 .DELTA.glgC 1.1 0.087 2.0 0.093 2.2
0.199
[1276] In wild type cells glycogen synthesis is increased during
nitrogen starvation. Therefore, in the .DELTA.glgC mutant, that is
not able to produce glycogen, an additional increase of the
pyruvate level was achieved by a nitrogen step down.
[1277] After 9 days of culturing under standard conditions, the
culture was split into two parts. With one half of the culture a
nitrogen step down was performed (as described for the .DELTA.glgA
mutant) and cells were grown on BG11 lacking combined nitrogen (-N)
for two days. The second half of the culture was grown in full BG11
medium (+N) as a control. Two days after the nitrogen step-down,
the excretion of pyruvate into the medium was measured.
TABLE-US-00032 +N -N OD.sub.750 pyruvate [mM] OD.sub.750 pyruvate
[mM] PCC 6803 Wt 1.7 0.012 1.2 0.10 .DELTA.glgC 1.3 0.295 1.2
0.361
ADP Glucose Pyrophosphorylase (GlgC) Knock-Out Mutant Expressing
PDC and ADH
[1278] The DNA-vector pGEM-T/.DELTA.glgC-KM was transformed into
the PDC-ADHII expressing mutant Synechocystis PCC6803
pSKIO-PpetJ-PDC-ADHII. The obtained mutant .DELTA.glgC
pSK10-PpetJ-PDC-ADHII was fully segregated and was grown in BG11
medium containing 100 mg/l kanamycin and 10 mg/l streptomycin.
[1279] Ethanol production was induced by copper starvation and
compared to that of Synechocystis wild-type
pSK10-PpetJ-PDC-ADHII.
[1280] In short term experiments under optimal conditions (light,
CO.sub.2) the overall as well as the relative (to photosynthetic
activity) ethanol production rate of the .DELTA.glgC
pSK-PpetJ-PDC-ADHII mutant was higher compared to that of the
reference strain S. PCC6803 pSK-PpetJ-PDC-ADHII. Therefore the
short term experiments performed at the beginning of the log phase
(day 5 and 6 during the growth experiment) indicate a higher
potential for ethanol production for the .DELTA.glgC
pSK-PpetJ-PDC-ADHII mutant (Data are the mean of 2
measurements)
.DELTA.glgC Mutant
TABLE-US-00033 .mu.mol .mu.mol .mu.mol % of O.sub.2/mg EtOH/mg
EtOH/ theoretical Chl* h Chl* h .mu.mol O.sub.2 fixed CO.sub.2 S.
PCC6803 pSK- 250 4 0.016 4.8 PpetI-PDC-ADHII .DELTA.glgC pSK-PpetI
125 9 0.072 21.6 PDC-ADHII
[1281] Similar to the glycogen synthase double knock out mutation,
these results indicate that by reducing the enzymatic affinity or
activity of ADP-glucose-pyrophosphorylase for example by a knock
out mutation of the gene encoding ADP-glucose-pyrophosphorylase a
higher percentage of carbon fixed via photosynthesis can be
redirected to ethanol production. In the case that the
photoautotrophic host cells do not have a second genetic
modification, a drastic increase of pyruvate secretion into the
growth medium can be detected.
X.3 Metabolic Mutant Harbouring a Knock Out of Pyruvate Water
Dikinase (.DELTA.PpsA) as a First Genetic Modification
[1282] Knock out of phosphoenolpyruvate synthase or pyruvate
water-dikinase (PpsA) was accomplished by insertion of a kanamycin
resistance cassette into gene slr0301. Construction of the
DNA-vector pGEM-T/.DELTA. ppsA, which was used for generation of
the ppsA knock-out mutant, was already described herein. The
obtained ppsA knock-out mutant was fully segregated and cultivated
in BG11 medium containing 75 mg/l kanamycin.
[1283] The mutant .DELTA.ppsA was characterized in comparison to
the Synechocystis wild-type strain under constant light conditions
as described herein.
[1284] No significant differences could be detected in cell growth,
chlorophyll content and photosynthetic oxygen production between
Synechocystis PCC680 wild-type and the .DELTA.ppsA mutant. However,
In several independent growth experiments the extracellular
pyruvate level of the .DELTA.ppsA mutant was increased at the end
of the log-phase. Data from one representative experiment are shown
in the following table:
TABLE-US-00034 4 days 10 days 14 days pyru- pyru- pyru- vate
OD.sub.750 vate vate OD.sub.750 [mM] [mM] [mM] OD.sub.750 [mM]
PCC6803 Wt 2.0 0 12.8 0.009 13.3 0.010 .DELTA.ppsA 1.8 0.014 8
0.010 10.8 0.073
X.4 Metabolic Mutant Harbouring a Knock Out of Either Acetatekinase
(.DELTA.ack) or a Double Knock Out of Acetatekinase and
Phosphoacetyltransacetylase (.DELTA.ack/pta) as a First Genetic
Modification
[1285] The following knock-out mutants were generated: the
single-mutants .DELTA.ack and .DELTA.pta and the double mutant
.DELTA.ack/.DELTA.pta. Knock-out of acetatekinase (ack) was
accomplished by replacement of a 0.65 kb fragment of slr1299 (ack
gene) by a kanamycin resistance cassette. As described herein,
plasmid pBlue-ack-Kan was used to generate the .DELTA.ack mutant.
Knock-out of phosphoacetyltransacetylase (pta) was accomplished by
replacement of a 0.45 kb fragment of slr2132 (pta gene) by a
chloramphenicol resistance cassette. The construction of plasmid
pUC-pta-Cm, which was used for generation of .DELTA.pta mutant is
described above. The double knock-out mutant .DELTA.ack/.DELTA.pta
was generated by transformation of pBlue-ack-Kan into the
.DELTA.pta mutant.
[1286] All mutants were fully segregated. Mutants were grown in
BG11 medium containing the appropriate antibiotics (kanamycin 75
mg/l; chloramphenicol 10 mg/l).
[1287] Mutants .DELTA.ack, .DELTA.pta, .DELTA.ack/pta and
Synechocystis wild-type strains were examined under constant light
conditions as described.
Results:
[1288] No significant differences could be detected in cell growth,
chlorophyll content and photosynthetic oxygen production between
the Synechocystis PCC6803 wild type and mutants .DELTA.ack,
.DELTA.pta and double mutant .DELTA.ack/.DELTA.pta.
[1289] Excretion of pyruvate into the medium could be detected at
the end of the log phase and was increased in the mutants compared
to the wild type. Data from representative experiments are shown in
the following tables. The optical density at 750 nm (OD.sub.750nm)
and the concentration of pyruvate in the medium are given at two
time points at the end of the log phase.
TABLE-US-00035 10 days 14 days pyruvate pyruvate OD.sub.750 [mM]
OD.sub.750 [mM] PCC6803 wt 4.6 0.006 6.2 0.012 .DELTA.ack 6.6 0.009
7.0 0.025 .DELTA.pta 6.7 0.010 6.3 0.019
TABLE-US-00036 10 days 12 days pyruvate pyruvate OD.sub.750 [mM]
OD.sub.750 [mM] PCC6803 wt 8 0.003 8 0.011 .DELTA.ack/.DELTA.pta 6
0.004 7 0.026
Acetatekinase (ack) and Acetatekinase
(ack)/Phosphoacetyltransacetylase (pta) Knock-Out Mutants
Expressing PDC and ADH
[1290] The self-replicating plasmid pVZ321b-PpetJ-PDC-ADHII was
conjugated into each of the mutants: hack, and double mutant
.DELTA.ack/pta, resulting in mutants .DELTA.ack
pVZ321b-PpetJ-PDC-ADHII, and .DELTA.ack/pta
pVZ321b-PpetJ-PDC-ADHII. Mutants were grown in BG11 medium
containing the appropriate antibiotics (kanamycin 75 mg/l;
chloramphenicol 10 mg/l; streptomycin 10 mg/l). Ethanol production
was induced by copper starvation under constant light and compared
to Synechocystis wild-type harboring pVZ321b-PpetJ-PDC-ADHII as
described above.
Results:
[1291] In several independent growth experiments, the double mutant
.DELTA.ack/pta, harboring pVZ321b-PpetJ-PDC-ADHII, exhibited
significantly higher ethanol production rates compared to the
reference strain S. PCC6803 pVZ321b-PpetJ-PDC-ADHII. In the single
mutant .DELTA.ack, harboring pVZ321b-PpetJ-PDC-ADHII, ethanol
production was increased compared to the reference strain S.
PCC6803 pVZ321b-PpetJ-PDC-ADHII. However, this effect was not
apparent, when given relative to cell growth.
[1292] Data from one representative experiment are shown in the
following table. FIGS. 32I and 32J depict a graphical presentation
of these data.
TABLE-US-00037 Time [days] 0 6 d 11 d 13 d PCC6803 pVZ321b-
OD.sub.750 1.2 2.5 3.2 3.9 PpetI-PDC-ADHII EtOH [%] 0.000 0.030
0.060 0.072 .DELTA.ack/pta pVZ321b- OD.sub.750 1.2 2.3 2.6 2.7
PpetI-PDC-ADHII EtOH [%] 0.000 0.044 0.098 0.121 .DELTA.ack
pVZ321b- OD.sub.750 1.3 2.8 3.9 4.8 PpetI-PDC-ADHII EtOH [%] 0.000
0.034 0.082 0.094
[1293] The following table shows the ethanol concentration in the
medium at the end of a growth experiment and the ethanol production
rate relative to cell growth (given as the slope of ethanol
production [%] per OD.sub.750nm and day.
TABLE-US-00038 EtOH [%] EtOH after 13 production days of rate
growth [%/OD.sub.750*d] PCC6803 pVZ321b-PpetI-PDC- 0.072 0.001
ADHII .DELTA.ack/pta pVZ pVZ321b-PpetI- 0.121 0.0039 PDC-ADHII
.DELTA.ack pVZ321b-PpetI-PDC- 0.094 0.001 ADHII
[1294] When mutants .DELTA.ack pVZ321b-PpetJ-PDC-ADHII, and
.DELTA.ack/pta pVZ321b-PpetJ-PDC-ADHII and the reference strain S.
PCC6803 pVZ321b-PpetJ-PDC-ADHI were grown under day/night cycle
conditions, similar results were obtained. After induction of PDC
and ADHII by copper starvation, strains .DELTA.ack/pta
pVZ321b-PpetJ-PDC-ADHII and .DELTA.ack pVZ321b-PpetJ-PDC-ADHII
showed higher ethanol production rates compared to the reference
strain S. PCC6803 pVZ321b-PpetJ-PDC.
[1295] At three consecutive days during the logarithmic growth
phase, photosynthetic capacity and ethanol production was measured
in the oxygen electrode as described.
[1296] In these short-term measurements photosynthetic activity is
measured under optimized conditions (saturating light and carbon
supply). Results represent the maximal photosynthetic capacity of
cells rather than the real photosynthetic activity during
cultivation.
[1297] Following the reaction equation of photosynthesis 6
CO.sub.2+12 H.sub.2O.fwdarw.C.sub.6H.sub.12O.sub.6+6 O.sub.2+6
H.sub.2O, the photosynthetic capacity [.mu.mol O.sub.2/mg Chl*h] is
equivalent to the maximal carbon fixation [.mu.mol CO.sub.2/mg
Chl*h]. Therefore the factor (.mu.mol EtOH per/.mu.mol O.sub.2)
given in the following table puts EtOH production into perspective
of carbon fixation/photosynthesis.
[1298] Values are the mean of three consecutive measurements.
TABLE-US-00039 PS capacity EtOH production .mu.mol [.mu.mol
O.sub.2/mg [.mu.mol EtOH/mg EtOH/ Chl* h] Chl* h .mu.mol O.sub.2
PCC6803 pVZ321b- 221 3.6 0.016 PpetI-PDC-ADHII .DELTA.ack/pta
pVZ321b- 241 6.1 0.025 PpetI-PDC-ADHII .DELTA.ack pVZ321b-PpetI-
301 7.2 0.024 PDC-ADHII
Conclusions:
[1299] Ethanol production in the double mutant .DELTA.ack/pta,
harboring pVZ321b-PpetJ-PDC-ADHII, was significantly enhanced
compared to the reference strain (wt) and also in comparison to the
single mutant .DELTA.ack pVZ321b-PpetJ-PDC-ADHII. For the single
mutant .DELTA.ack pVZ321b-PpetJ-PDC-ADHII, high ethanol production
rates were obtained in short term experiments.
X.5 Metabolic mutant harbouring a knock down of pyruvate
dehydrogenase E1 Component (Beta Subunit) (pdhBanti) as a First
Genetic Modification
[1300] Knock-down of Pyruvate dehydrogenase (PdhB) was accomplished
by regulated expression (PpetJ) of the corresponding antisense RNA
(sll1721-pdhB). Construction of the DNA-vector
pSK9/PpetJ-pdhB.sub.anti, which was used for the generation of a
pdhB knock down mutant, was already described herein. The obtained
pdhB knock-down mutant was fully segregated and was grown in BG11
medium containing 14 mg/l chloramphenicol. The mutant pdhB.sub.anti
was characterized in comparison to the Synechocystis wild-type
strain under constant light conditions as described herein.
Expression of anti-sense RNA was induced by copper starvation as
described for induction experiments with the promoter PpetJ.
Expression of anti-sense RNA was verified by northern blot
analysis.
Results:
[1301] No significant differences could be detected in cell growth,
chlorophyll content and photosynthetic oxygen production between
Synechocystis PCC6803 wild type and pdhB.sub.anti mutant. After
induction of the pet promoter, the level of extracellular pyruvate
was slightly increased in the pdhB.sub.anti mutant compared to the
wild-type. This effect was verified in three independent growth
experiments, data from one representative experiment are shown.
TABLE-US-00040 7 days 9 days pyruvate pyruvate OD.sub.750 [mM]
OD.sub.750 [mM] PCC6803 wt 3.6 0 5.3 0.004 pdhb.sub.anti 3.9 0.004
6.1 0.015
X.6 Metabolic Mutant Harbouring an Overexpressed
Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase (Rubisco) as a
First Genetic Modification
[1302] Mutant and Synechocystis wild-type strains were grown at
28.degree. C., under constant light (70 .mu.E m.sup.-2 s.sup.-1)
and aerated with CO.sub.2-enriched air (0.5% CO.sub.2). The initial
OD.sub.250 was about 1 in a total culture volume of 200 ml in a 250
ml Schott-flask. For comparison of the ethanol production an
integrative ethanol producing mutant (6803 pSK10-PisiA-PDC/ADHII)
was compared to the isogenic, ethanologenic mutant containing
moreover the RubisCO overexpressing plasmid
(pVZ321b-Prbc-SynRbc).
Methods:
[1303] The rate of oxygen evolution was measured with a Clark-type
oxygen electrode (Rank Brothers, UK). Prior to the measurement
cells were washed 2.times. and resuspended in BG-11 medium
supplemented with 25 mM NaHCO.sub.3. Light intensity was saturating
with approx. 500 .mu.E/s*m.sup.2.
[1304] For preparation of cell extracts, cells were pelleted,
washed two times with 20 mM HEPES/KOH, pH 7.5, 5 mM EDTA, 2 mM DTT,
dissolved in this buffer and broken with a beadbeater (2.times.10
min). The supernatant of a centrifugation (15 min, 14000 rpm,
4.degree. C., Micro 200R, Hettich) was used for the experiments.
The protein content of cell extracts was measured with the method
of Lowry.
[1305] RubisCO activity was measured similar as described in Iwaki
et al. (2006) Photosynth Res. 2006 June; 88(3):287-97. Epub 2006
May 12. Expression of foreign type I ribulose-1,5-bisphosphate
carboxylase/oxygenase stimulates photosynthesis in cyanobacterium
Synechococcus PCC7942 cells:
[1306] 5 .mu.l to 15 .mu.l of cell extracts were mixed with 750
.mu.l of 50 mM HEPES/KOH, pH 7.5, 20 mM MgCl.sub.2, 50 mM
KHCO.sub.3, 0.15 mM NADH, 5 mM ATP, 2.5 mM Phosphocreatine, 1.5
.mu.l carbonic anhydrase (10 U/.mu.l in 50 mM HEPES, pH 7.5), 7.5
.mu.l creatine kinase (0.5 U/.mu.l) 3.75 .mu.l of
glyceraldehyde-3-phosphate dehydrogenase (12.5 mg/ml),
phosphoglycerate kinase (suspension with 10 mg/ml). The assay was
incubated at 30.degree. C. for 10 min. Then the reaction was
started by the addition of 7.5 .mu.l of 250 mM
ribulose-1,5-bisphosphate and the absorption of 340 nm was
monitored.
Results and Conclusions:
[1307] The mutant with RuBisCO over-expression (6803
pVZ321b-Prbc-SynRbcLXS) grows as fast as the Synechocystis wild
type and shows no phenotypical differences except for the
chlorophyll content that is reduced by 20-30% compared to wild type
(see FIG. 32K). Interestingly, at the same time the mutant produces
significant more biomass observed by dry weight determination at
several time points during the cultivation experiment (Tab.1). At
the end point the difference in dry weight accounts to about 30%.
This means although both cultures are indistinguishable by the
optical density the mutant seems to build up more biomass. Either
the cells are larger in size or the cells are denser packed by
biomass (eg. with carbohydrates like glycogen or fatty acids).
[1308] FIG. 32L shows the growth parameter (OD at 750 nm and
Chlorophyll content) of Synechocystis wild type and a mutant that
over-express the endogenous RuBisCO operon.
TABLE-US-00041 TABLE 1 Biomass (dry weight, mean value of
triplicates) during the (in FIG. 50-1A shown) cultivation
experiment of Synechocystis wild type cells and cells
overexpressing RuBisCO. Prbc-SynRbcLXS WT 6803 time CHl a Dry
weight CHl a Dry weight [d] OD.sub.750 nm [mg/l] [g/l] OD.sub.750
nm [mg/l] [g/l] 0 0, 96 3, 82 0, 23 0, 91 3, 69 0, 18 7 6, 09 22,
60 1, 01 6, 36 29, 07 1, 02 11 8, 14 21, 22 1, 51 7, 99 33, 89 1,
30 16 10, 17 18, 30 1, 70 10, 01 24, 97 1, 32
[1309] Measurements of the RuBisCO activity from the mutant with
RuBisCO over-expression revealed an about 2-fold increase in the
activity compared to the wild type (see Tab.2). This was confirmed
by semi-quantitative Western blot analyses, too (data not shown).
Furthermore for this mutant and the wild type the oxygen evolution
was determined. Based on the wild-type level a slight increase
(about 15%) in the oxygen evolution was detectable for the cells
overexpressing the Synechocystis RuBisCO.
TABLE-US-00042 TABLE 2 RuBisCO activity and photosynthetic oxygen
evolution of Synechocystis wild type and a mutant overexpressing
the endogenous RuBisCO operon. RuBisCO activity [.mu.mol oxygen
evolution RBP/min * mg protein] [.mu.mol O.sub.2/h * mg chl]
PCC6803 wild type 0.23 (100%) 107.8 (100%) pVZ321b-Prbc- 0.48
(209%) 124.6 (115%) SynRbcLXS
[1310] In a further experiment the potential positive effect of the
detected increased RubisCO activity for the ethanol production was
analyzed. For this purpose growth and ethanol production of an
integrative ethanol producing mutant (6803 pSK10-PisiA-PDC/ADHII)
was compared to the Isogenic, ethanologenic mutant containing
moreover the RubisCO overexpressing plasmid
(pVZ321b-Prbc-SynRbc).
[1311] FIGS. 32L, 32M and 32N, respectively show the OD.sub.750,
the ethanol production and the ethanol production normalized to the
OD.sub.750 for the mutant Synechocystis PCC6803 harboring the
pSK10-PisiA-PDC/ADHII plasmid and the mutant additionally
containing the vector pVZ321b-Prbc-SynRbc.
[1312] Both ethanologenic Synechocystis mutants exhibit a similar
ethanol production rate of about 0.017% (v/v) per day for 14 days
under continuous light illumination (see FIG. 32-4C). Over the
whole time-scale the mutant with the RubisCO over-expression
produces a bit more ethanol (about 8% compared to the reference).
Also when the ethanol production is normalized to the cell density
(OD at 750 nm as indicator for the growth) this difference in the
ethanol production remains. This indicates that an elevated RubisCO
activity can lead to an increased ethanol formation. The potential
to direct additional carbon fixed via photosynthesis into ethanol
production might be further improvable by optimization of the
RubisCO expression level as well as by combination with other
metabolic mutations, enhancing the level of substrates for the
ethanologenic enzymes.
X.7 Metabolic Mutant Harbouring an Overexpressed Pyruvate Kinase 2
as a First Genetic Modification
[1313] Construction of the DNA-vector pVZ321-PpetJ-pyk2, which was
used for the generation of a pyk2 overexpression mutant, was
already described herein.
[1314] The obtained mutant Synechocystis PCC6803 pVZ321-PpetJ-pyk2
was cultivated in BG11 medium containing 14 mg/l chloramphenicol
and characterized in comparison to the Synechocystis wild-type
strain under constant tight conditions as described herein.
Expression of pyruvate kinase gene was induced by copper
starvation.
Results:
[1315] No significant differences could be detected in cell growth,
chlorophyll content and photosynthetic oxygen production between
Synechocystis PCC680 wild type and mutant PCC6803 PpetJ-pyk2.
[1316] After induction of the pets promoter, the level of
extracellular pyruvate was slightly increased in the PCC680
PpetJ-pyk2 mutant compared to the wild-type.
TABLE-US-00043 6 days 9 days 14 days pyru- pyru- pyru- vate vate
vate OD.sub.750 [mM] OD.sub.750 [mM] OD.sub.750 [mM] PCC6803 Wt 1.3
0.018 1.9 0.005 2.5 0.009 PpetI-pyk2 0.8 0.016 1.3 0.051 1.9
0.064
Pyruvatekinase 2 Overexpression Mutant Expressing PDC and ADH
[1317] Pyruvate kinase 2 was also expressed from self-replicating
plasmid pVZ321 under control of its endogeneous promoter Ppyk2 in
the ethanol producing strain S. PCC6803 pSK-PisiA-PDC-ADHII.
Generation of plasmid pVZ-Ppyk2-pyk2, which was conjugated into
Synechocystis pSK-PisiA-PDC-ADHII, was already described
herein.
[1318] The ethanol production rates and the oxygen evolution for
the photosynthetic capacity of Synechocystis strains S. PCC6803
pSK-PisiA-PDC-ADHII harboring plasmid pVZ-Ppyk2-pyk2 and reference
strain S PCC6803 pSK-PisiA-PDC-ADHII were determined as mentioned
above.
(data are mean of two measurements)
TABLE-US-00044 .mu.mol .mu.mol .mu.mol O.sub.2/mg EtOH/mg EtOH/
Chl* h Chl* h .mu.mol O.sub.2 S. PCC6803 pSK-PisiA- 164.50 9.5
0.058 PDC-ADHII Ppyk2-pyk2 pSK- 134.3 10.0 0.074
PisiA-PDC-ADHII
X.8 Metabolic Mutant Photoautotrophic Cells Harbouring an
Overexpressed Pyruvate Kinase (Pyk) Enolase (Eno) and
Phosphoglycerate Mutase (Pgm) as First Genetic Modifications
[1319] Two mutants have been created for overexpression of the
three glycolytic genes pyruvate kinase (pyk), enolase (eno) and
phosphoglycerate mutase (pgm).
[1320] In one mutant expression of pyruvate kinase 1 (from E.
coli), enolase and phosphoglycerate mutase (both from Zymomonas
mobilis) is controlled by the ribulose-1,5-bisphosphate
carboxylase/oxygenase (RubisCO) promoter (Prbc) from Synechococcus
PCC7942. Construction of the DNA-vector pVZ321-p67, which was
conjugated into Synechocystis PCC6803 to generate mutant PCC6803
Prbc-pyk-eno-pgm, was already described herein.
[1321] In the other mutant the expression of additional copies of
the endogenous genes pyruvate kinase 2, enolase and
phosphoglycerate mutase from Synechocystis PCC6805 is controlled by
the PpetJ promoter. DNA-vector pVZ322-PpetJ-pyk2-eno-pgm, which was
conjugated into Synechocystis PCC6803 to generate mutant PCC6803
PpetJ-pyk2-eno-pgm, was already described herein.
[1322] The obtained mutants PCC6803 pVZ321-Prbc-pyk-eno-pgm and
PCC6803 pVZ322-PpetJ-pyk2-eno-pgm were cultivated in BG11 medium
containing 14 mg/l chloramphenicol or 3 mg/l gentamycin,
respectively, and characterized in comparison to the Synechocystis
wild-type strain under constant light conditions as described
herein.
Results:
[1323] No significant differences could be detected in cell growth,
chlorophyll content and photosynthetic oxygen production between
Synechocystis PCC6803 wild type and mutants PCC6803
Prbc-pyk-eno-pgm and PCC6803 PpetJ-pyk2-eno-pgm.
[1324] Excretion of pyruvate was increased in mutant PCC6803
Prbc-pyk-eno-pgm compared to wild-type, as shown in the following
table:
TABLE-US-00045 10 days 14 days pyruvate pyruvate OD.sub.750 [mM]
OD.sub.750 [mM] PCC6803 (BG11) 4.6 0.006 6.2 0.012 PCC6803
Prbc-pyk-eno- 3.0 0.017 6.1 0.032 pgm
[1325] In mutant PCC6803 PpetJ-pyk2-eno-pgm the level of
extracellular pyruvate was increased after induction of the
glycolytic genes by copper starvation.
TABLE-US-00046 7 days 14 days pyruvate pyruvate OD.sub.750 [mM]
OD.sub.750 [mM] PCC6803 (BG11-Cu) 1.6 0 3 0.006 PCC6803
Prbc-pyk-eno- 3.1 0.013 3.7 0.024 pgm
Expression of Pyruvate Kinase, Enolase and Phospho-Glycerate Mutase
in Synechocystis Strains Expressing Pdc Enzyme Alone as a Second
Genetic Modification.
[1326] Plasmids pVZ321-p67 and pVZ322-PpetJ-pyk2-eno-pgm were each
conjugated into the ethanol producing strain Synechocystis PCC6803
pSK10-PpetJ-pdc expressing only PDC. (Construct pSK10-PpetJ-pdc is
a derivate of pSK10-PpetJ-pdc-adhII, from that the adhII gene was
cut out with SacI and PstI.) The resulting mutants were cultured in
BG11 containing 10 nm/l streptomycin and 7 mg/l chloramphenicol or
2 mg/l gentamycin, respectively. Expression of pdc (and in mutant
PpetJ-pyk2-eno-pgm also of the glycolytic genes) was induced by
copper starvation (PpetJ).
[1327] In short term measurements both mutants expressing the
glycolytic enzymes showed a better ethanol production rate
(relative to photosynthetic activity) than the reference strains.
Data in the following table are means of two consecutive
measurements within one cultivation.
TABLE-US-00047 .mu.mol O.sub.2/ .mu.mol EtOH/ .mu.mol EtOH/ mg Chl
* h mg Chl * h umol O.sub.2 PCC 6803 psK-PpetI-PDC 130 1.8 0.014
PCC 6803 pSK-PpetI-PDC 148 3.2 0.022 pVZ-PpetI-pyk2-eno-pgm PCC
6803 pSK-PpetI-PDC 197 2.5 0.012 PCC 6803 pSK-PpetI-PDC 104 2.8
0.028 pVZ-PpetI-pyk2-eno-pgm
Conclusions
[1328] These data suggest that overexpression of the glycolytic
enzymes pyruvate kinase, enolase and phosphoglycerate mutase leads
to a higher flux from CO.sub.2 towards pyruvate which results in a
higher ethanol production rate, relative to the photosynthetic
capacity.
X.9 Metabolic Mutant Photoautotrophic Cells Harbouring an
Overexpressed Malic Enzyme (Me) and Malate Dehydrogenase (Mdh) as
First Genetic Modifications
[1329] Overexpression of malic enzyme (Me) and malate dehydrogenase
(Mdh) were accomplished by regulated expression of the
corresponding genes (slr0721-me; sll0891-mdh) via the PpetJ
promoter. Construction of DNA-vectors pSK9/PpetJ-me and
pSK9/PpetJ-me-mdh, which were used for generation of me- and
me/mdh-overexpression mutants, was already described herein. The
obtained overexpression mutants were fully segregated and were
grown in BG11 medium containing 14 ml/l chloramphenicol. Mutants
PpetJ-me and PpetJ-me/mdh were examined in comparison to the
Synechocystis wild-type strain under constant light conditions as
described herein. Expression of me and mdh genes was induced by
copper starvation and successfully proven by northern blot analysis
via a radio-labeled me- and mdh-probe, respectively (data not
shown).
Results:
[1330] No significant differences could be detected in cell growth,
chlorophyll content and photosynthetic oxygen production between
Synechocystis PCC6803 wild type and PpetJ-me and PpetJ-me/mdh
mutant, respectively.
[1331] An enhanced extracellular pyruvate level was detected in the
medium of the PpetJ-me and the PpetJ-me/mdh mutants after induction
by copper starvation. The following table shows the extracellular
pyruvate concentrations measured 10 days after induction in
comparison with values measured in medium from non-induced
cells.
TABLE-US-00048 Not induced (BG11) Induced (BG11-Cu) 10 days 10 days
pyruvate pyruvate OD.sub.750 [mM] OD.sub.750 [mM] PCC6803 Wt 8.3
0.010 9.3 0.011 PpetI-me 10.1 0.005 8.3 0.032 PpetI-me-mdh 7.8
0.005 8.5 0.024
[1332] The higher extracellular pyruvate levels measured in the
induced PpetJ-me and PpetJ-me/mdh mutants (compared to wildtype and
non-induced cells) suggest, that overexpression of malic enzyme or
malic enzyme in combination with malate dehydrogenase leads to a
higher pyruvate level within the cyanobacterial cells.
X.10 Metabolic Mutant Cells of Nostoc/Anabaena PCC7120 and Anabaena
variabilis ATCC Harbouring a Knockout of the
ADP-Glucose-Pyrophosphorylase as a First Genetic Modification
[1333] In the following the EtOH production in Anabaena PCC7120
transformed with the integrative PpetE-PDC-ADHII and PpetE-PDC
constructs will be discussed.
[1334] In a first test experiment EtOH production in Anabaena
PCC7120 with PpetE-pdc-adhII or PpetE-pdc inserted in
ADP-glucose-pyrophosphorylase gene, agp, was measured of the
following mutants: A.7120 .DELTA.agp
(a114645)::C.K3-PpetE-pdc-adhII, named "PpetE-pdc-adhII" and A.7120
.DELTA.agp (a114645)::C.K3-PpetE-pdc named "PpetE-pd". Mutant
A.7120 .DELTA.agp (a114645)::C.K3, named .DELTA.agp, served as
control.
[1335] Cultures of all mutants were grown at 28.degree. C., under
continuous light conditions (40 .mu.E/m2 s.sup.1) in batches of 50
ml in 100 ml Erlenmeyer flasks with shaking. Precultures were grown
in BG11 medium lacking copper sulfate (BG11-Cu), supplemented with
neomycin (100 .mu.g/ml). It should be noted here, that the petE
promoter might not be fully repressed under this BG11-Cu
conditions, as the glassware was not treated to remove trace
amounts of copper from it. The petE promoter seems to be smoothly
regulated in Anabena PCC7120 [Buikema, W., and R. Haselkorn. 2001
Expression of the Anabaena hetR gene from a copper-regulated
promoter leads to heterocyst differentiation under repressing
conditions. PNAS USA 98:2729-2734], therefore trace amounts of
copper coming from the glassware might be sufficient to induce
expression.
[1336] Expression of the ethanologenic genes was induced by
addition of 1.times. copper (0.32 .mu.M CuSO.sub.4). This
corresponds to the copper concentration present in BG11 medium.
[1337] As a measure of growth, chlorophyll was determined at
several time points and ethanol was measured using the already
described enzymatic method.
TABLE-US-00049 TABLE 1 Growth and ethanol production of Anabaena
mutants expressing ethanologenic genes under control of petE
promoter. CHl, chlorophyll in [.mu.g/ml] and EtOH [%]. time 0
(start) 5 days 9 days 14 days Chl EtOH Chl EtOH Chl EtOH Chl EtOH
"PpetE- 1 0.002 3 0.014 6 0.022 8 0.037 pdc- adhII" "PpetE- 3 0.006
5 0.015 6 0.028 8 0.044 pdc" .DELTA.agp 8 0 16 0.0001 20 0.0001 25
0.0001 (con- trol)
[1338] Ethanol was produced by both integrative mutants, while in
the control strain (mutant .DELTA.agp) no ethanol production was
detected. The similar ethanol production rates obtained in mutants
"PpetE-pdc-adhII" and "PpetE-pdc" clearly indicate that also in
Anabaena PCC7120 expression of PDC alone is sufficient for ethanol
production. Thus it appears that this strain constitutively
expresses an endogenous ADH enzyme converting acetaldehyde into
ethanol. Several open reading frames are annotated as alcohol
dehydrogenases in Anabaena PCC7120 (available on the world wide web
at bacteria.kazusa.or.jp/cyanobase/), however all genes show only
little similarity (less than 30% identical amino acids) to
SynADH.
Detailed Discussion of the Embodiments Involving Overexpressed
ZN.sup.2+ Dependent Alcohol Dehydrogenase and Pdc and/or ADH
Enzymes Under the Control of Various Inducible Promoters
[1339] In the following further embodiments of the invention
related to for example an overexpressed Zn.sup.2+ dependent alcohol
dehydrogenase, an overexpressed alcohol dehydrogenase, directly
converting acetyl-CoA to ethanol, or promotes that can be induced
by nutrient starvation, cold shock, heat shock, salt stress, light
exposure or stationary growth of the host cell will be explained in
more detail.
[1340] Construction of selfreplicating (extrachromosomal) and
chromosome-integrative vectors for the inducible overexpression of
ethanologenic enzymes in cyanobacteria
[1341] Construction of extrachromosomal pVZ-vectors for inducible
overexpression of pyruvate decarboxylase (ZmPdc) and alcohol
dehydrogenase (ZmAdhII) from Zymomonas mobilis
[1342] The construction of the certain vectors including the
different promoters were done by using the following general
protocol:
[1343] EcoRI/BamHI restriction of the pCB4-LR(TF)pa shuttle vector
in order to cut off the pdc and adh genes. This shuttle vector was
constructed by Dr. John Coleman, University of Toronto, Toronto,
Canada.
[1344] ligation of the pdc/adh containing EcoRI/BamHI fragment into
the cloning vector pDrive (EcoRI/BamHI). The pDrive vector (Qiagen,
Hilden, Germany, GenBank no.: DQ996013) was already described
above.
[1345] amplification of the isiA-, nblA- and ntcA-promoter using
chromosomal DNA from Synechocystis sp. PCC 6803 and the following
primers (all amplified promoters have a length of about 600 bp and
include the ribosome binding site of the corresponding genes):
TABLE-US-00050 (SEQ ID NO: 217) isiA-fw-SalI
5'-GTCGACCTTCCAGCACCACGTCAAC-3' (SEQ ID NO: 218) isiA-rev-EcoRI
5'-GAATTCACAGAATTGCCTCCTTAATTGAG-3' (SEQ ID NO: 219) nblA-fw-SalI
5'-ACGCGTCGACTTATGGTTGATTCGCATTG-3' (SEQ ID NO: 220) nblA-rev-EcoRI
5'-CGGAATTCATAGCTGTTGCCCTCCAAG-3' (SEQ ID NO: 221) ntcA-fw-SalI
5'-GTCGACAACGACGGAGGTTTAAGGG-3' (SEQ ID NO: 222) ntcA-rev-EcoRI
5'-GAATTCATGCACGTTCACGGTAATGG-3'
[1346] All forward primer included the SalI restriction site, all
reverse primer included a EcoRI restriction site for cloning
(marked bold).
[1347] ligation of the SalI/EcoRI cut promoter fragments into the
pDrive-pdc/adh (SalI/EcoRI) generating the constructs
pDrive-PisiA-pdc/adh, pDrive-PnblA-pdc/adh and
pDrive-PntcA-pd/adh
[1348] SalI/PstI restriction of pDrive-PisiA-pdc/adh,
pDrive-PnblA-pdc/adh and pDrive-PntcA-pdc/adh and ligation of the
corresponding promoter-pdc/adh fusions into the self replicating
broad-host range vector pVZ321b (SalI/PstI), a derivate of the
pVZ321 (constructed by V. V. Zinchenko Moscow, Russia; described
above) with an additional streptomycin resistance
cassette/cartridge introduced into the XbaI site of pVZ321. The
pVZ321b vector was constructed by Anne Karradt,
Humboldt-Universitaet Berlin, Plant Biochemistry Department (Prof.
Lockau) and was used as a cargo plasmid for conjugation pVZ321 Gen
Bank no: AF100176 available in the NCBI data base (available an the
world wide web at ncbi.nlm.nih.gov).
[1349] End products of the cloning procedure are the pVZ-vectors:
FIG. 33A presents a schematic diagram of pVZ-PisiA-pdc/adh; FIG.
33B presents a schematic diagram of pVZ-PnblA-pdc/adh; and FIG. 33C
presents a schematic diagram of pVZ-PntcA-pdc/.
[1350] FIG. 33D presents the nucleotide sequence of adhII and pdc
genes from Zymomonas mobilis. The source of this polynucleotide is
the shuttle vector pCB4-LR(TF)pa, a kind gift from John Coleman.
FIG. 33E presents a schematic diagram of some restriction sites
occurring within the adhII and pdc gene sequences. FIGS. 33F and
33G presents the amino acid sequence of ZmPdc and ZmAdhII,
respectively
[1351] Various gene promoter elements were utilized to control
constitutive and/or induced gene expression. Sequences for these
elements are presented herein. As known to those skilled in the
art, other genetic elements may serve the same purpose.
[1352] Remark: in all following nucleotide sequences of promoters
restriction sites for clonings are marked (colored).
[1353] The isiA promoter (Synechocystis sp. PCC6803) element
nucleotide sequence is presented in FIG. 34A. This genetic element
induces gene expression under conditions of iron starvation.
[1354] The nblA promoter (Synechocystis sp. PCC6803) element
nucleotide sequence is presented in FIG. 34B. This genetic element
induces gene expression under conditions of nitrogen
starvation.
[1355] The ntcA promoter (Synechocystis sp. PCC6803) element
nucleotide sequence is presented in FIG. 34C. This genetic element
induces gene expression under conditions of nitrogen
starvation.
[1356] The pVZ321b cloning vector (derivate of pVZ321 was
constructed by Anne Karradt, Humboldt-Universitaet Berlin, Plant
Biochemistry Department (Prof. Lockau), Berlin. The nucleotide
sequence for pVZ321b is presented in FIG. 35A, and the structure of
this plasmid is presented schematically in FIG. 35B.
[1357] Introduction of further well suited inducible promoters into
the existing pVZ-expression constructs (point 1).
[1358] In order to create expression constructs as described above
(point 1) but under control of a different promoter, the promoter
sequence was cut out by SalI/EcoRI digestion of the corresponding
pVZ-Pxxx-pdc/adh construct (xxx for isiA, ntcA, nblA). The new
promoter sequence containing the restriction sites SalI/EcoRI as
described for the isiA-, nblA- and ntcA-promoter was ligated into
the "promoter free" pVZ construct resulting in a pdc/adh expression
construct under control of the new promoter.
[1359] Representative new promoters include, but are not limited
to, the following:
[1360] (1) FIG. 36A depicts the nucleotide sequence of the petJ
promoter (Synechocystis sp. PCC 6803) (petJ gene: sll1796 (encoding
for cytochrome c553; induced expression under copper
starvation);
REFERENCES
[1361] J Bio. Chem. 2004 Feb. 20; 279(8):7229-33. Epub 2003
December [1362] The efficient functioning of photosynthesis and
respiration in Synechocystis sp. PCC 6803 strictly requires the
presence of either cytochrome c6 or plastocyanin. [1363] Duran R V,
Hervas M, De La Rosa M A, Navarro J A. [1364] A plasmid created
with this promoter element is presented schematically in FIG. 36B.
[1365] (2) FIG. 36 C depicts the nucleotide sequence of the sigB
promoter (Synechocystis sp. PCC 6803) sigB gene: sll0306 (encoding
for RNA polymerase group 2 sigma factor) induced expression after
heat shock, in stationary growth phase/nitrogen starvation and
darkness)
REFERENCES
[1365] [1366] Arch Microbial, 2006 October; 186(4):273-86. Epub
2006 Jul. 26. [1367] The heat shock response in the cyanobacterium
Synechocystis sp. Strain PCC 6803 and regulation of gene expression
by HrcA and SigB. [1368] Singh A K, Summerfield I C, Li H, Sherman
L A [1369] FEBS, Lett. 2003 Nov. 20; 554(3):357-62. [1370]
Antagonistic dark/light-induced SigB/SigD, group 2 sigma factors,
expression through redox potential and their roles in
cyanobacteria. [1371] Imamura S, Asayama M, Takahashi H, Tanaka K,
Takahashi H, Shirai M [1372] J Biol. Chem. 2006 Feb. 3;
281(5):2668-75. Epub 2005 Nov. 21. [1373] Growth phase-dependent
activation of nitrogen-related genes by a control network of group
1 and group 2 sigma factors in a cyanobacterium. [1374] Imamura S,
Tanaka K, Shira M, Asayama M. [1375] A plasmid created with this
promoter element is presented schematically in FIG. 36D. [1376] (3)
FIG. 36 E depicts the nucleotide sequence of the htpG promoter
(Synechocystis sp. PCC 6803) htpG gene: sll0430: (encoding for heat
shock protein 90, molecular chaperone) induced expression after
heat shock
REFERENCES
[1376] [1377] Plant Physiol. 1998 May; 117(1):225-34.
Transcriptional and posttranscriptional control of mRNA from IrtA,
a light-repressed transcript in Synechococcus sp PCC 7002. [1352]
Smartzidou H, Widger W R [1378] A plasmid created with this
promoter element is presented schematically in FIG. 36F. [1379] (4)
FIG. 36 G shows the nucleotide sequence of the IrtA promoter
(Synechocystis sp. PCC 6803) IrtA gene:sll0947 (encoding the light
repressed protein A homolog induced expression after light to dark
trnsition)
REFERENCES
[1379] [1380] Plant Physiol. 1998 May; 117(1):225-34. [1381]
Transcriptional and posttranscriptional control of mRNA from IrtA,
a light-repressed transcript in [1382] Synechococcus sp. PCC 7002.
[1383] Samartzdou H, Widger W R [1384] A plasmid created with this
promoter element is presented schematically in FIG. 36H. [1385] (5)
the nucleotide sequence of the psbA2 promoter (Synechocystis sp.
PCC 6803) (FIG. 36I) psbA2 gene: slr1311 (encoding the photosystem
II D1 protein) induced expression after dark to light
transition
REFERENCES
[1385] [1386] Biochem Biophys Res Commun. 1999 Feb. 5;
255(1):47-53. [1387] Light-dependent and rhythmic psbA transcripts
in homologous/heterologous cyanobacterial cells. [1388] Agrawal G
K, Asayamna M, Shirai M. [1389] A plasmid created with this
promoter element is presented schematically in FIG. 36J. [1390] (6)
FIG. 36K shows the nucleotide sequence of the rbcL promoter
(Synechocystis sp. PCC 6803) rbcL gene: slr0009 (encoding the
ribulose biphosphate carboxylase/oxygenase large subunit
constitutive strong expression under continuous light
conditions
REFERENCES
[1390] [1391] Plant Mol. Biol. 1989 December; 13(6):693-700 [1392]
Influence of light on accumulation of photosynthesis-specific
transcripts in the cyanobacterium Synechocystis 6803. [1393]
Mohamed A, Jansson c. [1394] A plasmid created with this promoter
element is presented schematically in FIG. 36L. [1395] (7) FIG. 36M
depicts the nucleotide sequence of the psaA promoter (Synechocystis
sp. PCC6803); PsaA gene: slr1834 (encoding P700 apoprotein subunit
Ia) induced expression under low white light and orange light, low
expression level under high light and red light, repressed in
darkness
REFERENCES
[1395] [1396] Plant Cell Physiol. 2005 September; 46(9):1484-93.
Epub 2005 Jun. 24. [1397] Regulation of photosystem I reaction
center genes in Synechocystis sp. strain PCC 6803 during Light
acclimation. [1398] Herranen M. Tyystjarvi T. Aro E M. [1399] Plant
Cell Phys. 2006 July; 47(7):878-90. Epub 2006 May 16. [1400]
Characterization of high-light-responsive promoters of the psaAB
genes in Synechocystis sp. PCC 6803. [1401] Muramatsu M, Hihara Y.
[1402] A plasmid created with this promoter element is presented
schematically in FIG. 36N. [1403] (8) FIG. 36O shows the nucleotide
sequence of the ggpS promoter (Synechocystis sp. PCC6803); ggpS
gene: sll1566 (encoding glucosylglycerolphosphate synthase) induced
expression after salt stress
REFERENCES
[1403] [1404] Plant Physiol. 2004 October; 136(2):3290-300. Epub
2004 Sep. 10. Gene expression profiling reflects physiological
processes in salt acclimation of Synechocystis sp. strain PCC 6803.
[1405] Marin K, Kanesaki Y, Los D A, Murata N, Suzuki I, Hagemann
M. [1406] J. Bacterial, 2002 June; 184(11):2870-7. [1407]
Salt-dependent expression of glucosylglycerol-phosphate synthase,
involved in osmolyte synthesis in the cyanobacterium Synechocystis
sp. strain PCC 6803. [1408] Marin K, Huckauf J. Fulda S, Hagemann
M. [1409] A plasmid created with this promoter element is presented
schematically in FIG. 36P. [1410] (9) FIG. 36Q depicts the
nucleotide sequence of the nirA promoter (Synechocystis sp.
PCC6803); nirA gene: slr0898 (encoding ferredoxin-nitrite
reductase) induced expression after transition from ammonia to
nitrate
REFERENCES
[1410] [1411] Appl Environ Microbiol. 2005 October; 71(10):678-84.
[1412] Application of the Synechococcus nirA promoter to establish
an inducible expression system for engineering the Synechocystis
tocopherol pathway. [1413] Qi Q, Hao M, Ng W O, Slater S C, Baszis
S R, Weiss J D, Valentin H E. [1414] J. Bacterial. 1998 August;
180(16):4080-8 [1415] cis-acting sequences required for
NtcB-dependent, nitrite-responsive positive regulation of the
nitrate assimilation operon in the cyanobacterium Synechococcus sp.
strain PCC 7942. [1416] Maeda S, Kawaguchi Y, Ohe T A, Omata T.
[1417] A plasmid created with this promoter element is presented
schematically in FIG. 36R. [1418] (10) FIG. 36S depicts the
nucleotide sequence of the petE promoter (Anabaena sp. PCC7120);
petE gene: all0258 (encoding plastocyanin precursor) induced
expression at elevated copper concentrations
REFERENCES
[1418] [1419] Microbiology, 1994 May; 140 (Pt 5):1151-9. [1420]
Cloning, sequencing and transcriptional studies of the genes for
cytochrome c-553 and plastocyanin from Anabaena sp. PCC 7120.
[1421] Ghassemian M, Wong B, Ferreira F, Markley J L, Straus N A.
[1422] Proc Natl Acad Sci USA. 2001 Feb. 27; 98(5):2729-34. Epub
2001 Feb. 20. [1423] Expression of the Anabaena hetR gene from a
copper-regulated promoter leads to heterocyst differentiation under
repressing conditions. [1424] Buikema W J, Haselkorn R. [1425] A
plasmid created with this promoter element is presented
schematically in FIG. 36T [1426] (11) FIG. 36U shows the nucleotide
sequence of the hspA promoter (Synechocystis sp. PCC6803); hspA
gene: sll1514 16.6 kDa small heat shock protein, molecular
chaperone multi-stress responsible promoter (heat, cold, salt and
oxidative stress)
REFERENCES
[1426] [1427] Curr Microbial, 2004 September; 49(3):192-8. [1428]
Expression of the heat shock gene hsp16.6 and promoter analysis in
the cyanobacterium. Synechocystis sp. PCC 6803. [1429] Fang F,
Barnum S R. [1430] J. Exp Bot, 2005; 57(7):1573-8. Epub 2006 Mar.
30. [1431] The heat shock response of Synechocystis sp. PCC 6803
analyzed by transcriptomics and proteomics. [1432] Suzuki I, Simon
W J, Slabas A R. [1433] A plasmid created with this promoter
element is presented schematically in FIG. 36V. [1434] (12) FIG.
36W depicts the nucleotide sequence of the hliB promoter
(Synechocystis sp. PCC6803); hliB gene: ssr2595: high
light-inducible polypeptide HliB, CAB/ELIP/HLIP superfamily
multi-stress responsible promoter (heat, cold, salt and oxidative
stress)
REFERENCES
[1434] [1435] J Biol. Chem. 2001 Jan. 5; 276(1):306-14. [1436] The
high light-inducible polypeptides in Synechocystis PCC6803.
Expression and function in high light. [1437] He Q, Dolganov N,
Bjorkman O. Grossman A R. [1438] Arch. Microbiol, 2007 April;
187(4):337-42. Epub 2007 Feb. 10. [1439] The response regulator
RpaB binds the high light regulatory 1 sequence upstream of the
high-light-Inducible hliB gene from the cyanobacterium
Synechocystis PCC 6803. [1440] Kappell A D, van Waasbergen L G.
[1441] A plasmid created with this promoter element is presented
schematically in FIG. 36X [1442] (13) FIG. 36Y shows the nucleotide
sequence of the clpB1 promoter (Synechocystis sp-PCC6803); clpB1
gene: slr1641: ATP-dependent Clp protease, Hsp 100, ATP-binding
subunit Clp8 multi-stress responsible promoter (heat, cold, salt
and oxidative stress)
REFERENCES
[1442] [1443] Microbiology, 2004 May; 150 (Pt 5):1271-81. [1444]
Effects of high light on transcripts of stress-associated genes for
the cyanobacteria Synechocystis sp. PCC 6803 and Prochlorococcus
MED4 and MIT9313. [1445] Mary I, Tu C J. Grossman A, Vaulot D.
[1446] J Exp Sot. 2006; 57(7):1573-8. Epub 2006 Mar. 30. [1447] The
heat shock response of Synechocystis sp. PCC 6803 analysed by
transcriptomics and proteomics. [1448] Suzuki I, Simon W J, Slabas
A R. [1449] A plasmid created with this promoter element is
presented schematically in FIG. 36Z. [1450] Introduction of
Alternative Ethanologenic Genes to ZmPdc and ZmAdhII Into the
Existing pVZ-Expression Constructs (Point 1)
[1451] In order to create expression constructs as described above
(point 1) but with different alcohol dehydrogenases, the adh
encoding sequence was cut out by SacI/PstI digestion of the
corresponding pVZ-Pxxx-pcd/adh construct (xxx for isiA, nblA,
ntcA). The new adh sequence containing the restriction sites
SacI/PstI (introduced by used primer) was ligated into the "adh
free" pVZ construct resulting in a construct that expresses the
ZmPdc together with new Adh.
[1452] Remark: In all following nt sequences of genes restriction
sites (marked in yellow or blue) for clonings as well as
translation starts (start codons, marked in green) and translation
stops (stop codons, marked in red) are color coded.
[1453] In this context, new alcohol dehydrogenases include the
following:
[1454] (1) FIG. 37A presents the nucleotide sequence for ZmADHI
(adhA gene from Zymomonas mobilis ZM4) and FIG. 378 presents the
amino acid sequence for ZmAdhI AAV89860 FIG. 37C presents a
schematic representation of the plasmid pVZ321b-PisiA-PDC-ZmADH1.
FIG. 370 presents a schematic representation of the plasmid
pVZ321b-PntcA-PDC-ZmAH1. FIG. 37E presents a schematic
representation of the plasmid pVZ321b-PnblA-PDC-ZmADH1.
[1455] (2) The nucleotide sequence of SynAdh (adh gene (slr1192)
Synechocystis sp. PCC 6803) is presented in FIG. 38A. The amino
acid sequence of this protein (SynAdh protein sequence BAA18840) is
presented in FIG. 38B.
[1456] FIG. 38C presents a schematic representation of the plasmid
pVZ321b-PisiA-PDC-SynADH. FIG. 38D presents a schematic
representation of the plasmid pVZ321b-PntcA-PDC-SynADH. FIG. 38E
presents a schematic representation of the plasmid
pVZ321b-PnblA-PDC-SynADH.
[1457] In order to create expression constructs as described above
(point 1) but with AdhE-type alcohol dehydrogenases, the pdc/adh
encoding sequence was cut out by EcoRI/BamHI and EcoRI/PstI
digestion resp. of the corresponding pVZ-Pxxx-pdc/adh construct
(xxx for isiA, ntcA, nblA). The adhE sequence of E. coli and
Thermosynechococcus elongatus resp. containing the restriction
sites EcoRI/BamHI and EcoRI/PstI resp. (introduced by used primer)
were ligated into the "pdc/adh free" pVZ construct resulting in
constructs that express the AdhE-type alcohol dehydrogenases.
[1458] (3) The nucleotide sequence for EcAdhE (adhE gene from E.
coli K12) is presented in FIG. 39A. The amino acid sequence for
this protein (EcAdhE protein sequence NP.sub.--415757) is presented
in FIG. 39B.
[1459] FIG. 39C presents a schematic representation of the plasmid
pVZ321b-PisiA-PDC-EcAdhE FIG. 39D depicts a schematic
representation of the plasmid pVZ321b-PntcA-PDC-EcAdhE. FIG. 39E
presents a schematic representation of the plasmid
pVZ321b-PnblA-PDC-EcAdhE.
[1460] (4) The nucleotide sequence for the ThAdhE gene (adhE gene
(tlr0227) from Thermosynechococcus elongatus BP-1) is presented in
FIG. 40A, and the amino acid sequence for this protein (ThAdhE
protein sequence BAC07780) is presented in FIG. 40B.
[1461] FIG. 40C presents a schematic representation of the plasmid
pVZ321b-PisiA-PDC-ThAdhE. FIG. 40D presents a schematic
representation of the plasmid pVZ321b-PntcA-PDC-ThAdhE. FIG. 40E
presents a schematic representation of the plasmid
pVZ321b-PnblA-PDC-ThAdhE.
[1462] In order to create expression constructs as described above
(point 1) but with an alternative pyruvate decarboxylase to the
Zymomonas mobilis enzyme, the Pdc encoding sequence was cut out by
EcoRI/SacI digestion of the corresponding pVZ-Pxxx-pdc/adh
construct (xxx for isiA, ntcA, nblA). The pdc sequence from
Zymobacter palmae containing the restriction sites EcoRI/SacI
(introduced by used primer) was ligated into the "pdc free" pVZ
construct resulting in a construct that express the Pdc from
Zymobacter palmae together with the preexisting Adh.
[1463] FIG. 41A presents the nucleotide sequence for ZpPdc (pdc
gene from Zymobacter palmae ATCC 51623), and the amino acid
sequence for this protein (ZpPdc protein sequence AAM49566) is
presented in FIG. 41B.
Construction of Chromosome Integrative pSK-Vectors
[1464] In order to create plasmids for stable chromosome
integration in cyanobacteria the whole inserts from the described
pVZ constructs (point 1 and 3) containing the promoter sequence and
the coding region of the ethanologenic enzymes (Pdc and Adh) were
cut out by SalI/PstI digestion. The resulting inserts were ligated
into the pSK10, a derivate of the pSK9 (a kind gift of V. V.
Zinchenko and described in Sobotka et al., 2008, JBC) using the
SalI/PstI restriction sites. In some cases other restriction sites
were used, e.g. in case of pVZ321b-Pxxx-pdc-adh/the restriction
sites XbaI/PstI were used, in case of pVZ321b-Pxxx-Ecdhe the
restriction sites XbaI/BamHI were used.
[1465] FIG. 42A presents the nucleotide sequence of the pSK10
cloning vector (derivate of pSK9 [V. V. Zinchenko, Moscow, Russia;
unpublished]). FIG. 425 presents a schematic representation of this
plasmid.
[1466] Several pSK10 constructs with ZmPdc/ZmAdhII were
obtained
[1467] FIG. 42C presents a schematic diagram of
pSK10-PisiA-PDC-ADHII.
[1468] FIG. 42D presents a schematic diagram of
pSK10-PnblA-PDC-ADHII.
[1469] FIG. 42E presents a schematic diagram of
pSK10-PntcA-PDC-ADHII
[1470] Several pSK10 constructs with ZmPdc/ZmAdhI were
obtained.
[1471] FIG. 42F presents a schematic diagram of
pSK10-PisiA-PDC-ADHII.
[1472] FIG. 42G presents a schematic diagram of
pSK10-PnblA-PDC-ADHI.
[1473] FIG. 42H presents a schematic diagram of
pSK10-PntcA-PDC-ADHI.
[1474] Several pSK10 constructs with ZmPdc/SynAdh were
obtained.
[1475] FIG. 42I presents a schematic diagram of
pSK10-PisiA-PDC-SynADH.
[1476] FIG. 42J presents a schematic diagram of
pSK10-PnblA-PDC-SynADH.
[1477] FIG. 42K presents a schematic diagram of
pSK10-PntcA-PDC-SynADH.
[1478] Several pSK10 constructs with EcAdhE were obtained.
[1479] FIG. 42L presents a schematic diagram of
pSK10-PisiA-PDC-EcAdhE.
[1480] FIG. 42M presents a schematic diagram of
pSK10-PnblA-PDC-EcAdhE
[1481] FIG. 42N presents a schematic diagram of
pSK10-PntcA-PDC-EcAdhE.
[1482] Several pSK10 constructs with ThAdhE were obtained.
[1483] FIG. 42O presents a schematic diagram of
pSK10-PisiA-PDC-ThAdhE.
[1484] FIG. 42P presents a schematic diagram of
pSK10-PnblA-PDC-ThAdhE.
[1485] FIG. 42Q presents a schematic diagram of
pSK10-PntcA-PDC-ThAdhE
Expression of Pdc and Adh in the Filamentous; Diazotropic
Cyanobacteria Nostoc/Anabaena Spec. PCC7120 and Anabaena variabilis
ATCC 29413
[1486] In order to generate ethanol producing Anabaena strains,
different constructs were created for conjugation into Anabaena
PCC7120 and Anaboena variabilis ATCC29413.
[1487] Nostoc/Anabena spec. PCC7120 and Anabaena variabilis ATCC
29413 were transformed using Self-replicating plasmids.
[1488] The ethanologenic genes were cloned into self-replicating
plasmids for conjugation into Anabaena PCC7120. In these constructs
different promoters were used to control expression of pdc and
adhII.
pRL1049 Constructs
[1489] Genes encoding pdc and adhII from Zymomonas mobilis were
cloned into the self-replicating plasmid pRL1049, which is known to
replicate in Nostoc strains. Nucleotide and amino acid sequences of
adhII and pdc genes from Zymomonas mobilis are already described
herein.
[1490] The promoter-pdc-adhII fragment was cut out of the herein
described pSK10-PpetJ-pdc-adhII plasmid with ClaI and BamHI and
ligated into pRL1049. Promoter sequences were exchanged via EcoRI
and SalI. Different promoters were used: promoters originating from
PCC 6803: PisiA, PpetJ and PrbcL (nucleotide sequences are already
described herein) and promoters originating from PCC 7120: PcrhC
and PpetE.
[1491] Promoter sequences of PcrhC and PpetE are shown in FIGS. 42R
and 42S, respectively (SalI and EcoRI restriction sites for cloning
are marked in bold letters):
[1492] FIG. 42R depicts the crhC promoter (Anabaena sp. PCC7120)
(crhC gene: alr4718, RNA helicase crhC cold shock inducible)
[1493] FIG. 42S shows the petE promoter (Anabaena sp. PCC7120) petE
gene a110258, plastocyanin precursor (petE) induced by addition of
Cu
[1494] The structure of plasmid pRL1049-PpetE-PDC-ADHII is shown in
FIG. 42T.
[1495] The sequence of the plasmid pRL1049-PpetE-PDC-ADHII is shown
in FIG. 42U.
pRL593 Construct
[1496] In addition to pRL1049 the broad range plasmid pRL593 was
used for expression of pdc and adhII in Anabaena PCC7120. The
structure of plasmid pRL593-PisiA-PDC-ADHII is presented in FIG.
42V and the DNA sequence is depicted in FIG. 42W.
EtOH Production in Anabaena PCC7120 Harboring Self-Replicating
Plasmid pRL593-PisiA-PDC-ADHII
[1497] EtOH production in Anabaena PCC7120 harboring
pRL593-PisiA-PDC-ADHII following induction by iron starvation was
measured in BG11 medium (+N) and in medium lacking combined
nitrogen (-N) in day (12 h)/night (12 h) cycle. The results of this
measurement is presented in FIGS. 42X and 42Y.
[1498] Ethanol production in medium +N appeared higher than under
condition lacking combined nitrogen (-N); but this effect was not
very pronounced when calculated per OD.sub.750nm. The best EtOH
production rate in Anabaena PCC7120/pRL593-PisiA-pdc-adhII achieved
was 0.0076% EtOH per day, constant for 19 days. This rate is lower
compared to Synechocystis strains expressing pdc-adhII under
control of P isiA, but continues for a longer time.
Characterization of Generated Ethanologenic Synechocystis
Cyanobacteria
P.1 Experimental Data for Characterization of Genetically Modified
Photoautotrophic Host Cells Containing at Least One Second Genetic
Modification
Expression Levels of ZmPdc/ZmAdhII in Generated Synechocystis
Cyanobacterial Mutants:
[1499] In order to quantify the induction rate of the used
promoters, Pdc/AdhI protein levels in cultures with and without
nutrient starvation were estimated by Western blot analysis.
[1500] In the case of the mutant with the isiA-promoter cultures
were grown with and without addition of iron for about 48 hours. In
the case of the mutants with the ntcA- and nblA-promoter cultures
were grown with and without addition of nitrogen to the media. To
get more comparable signals in the immunodetection from the
cultures under induced conditions, different dilutions of the
prepared crude extracts were used.
Activities of ZmPdc/ZmAdhII in Cyanobacterial Mutants:
[1501] In order to compare the enzymatic activities of Pdc/AdhII
with the estimated expression level, activities of Adh and Pdc were
measured in crude extracts of the corresponding cultures.
[1502] In the case of the mutant with the isiA-promoter, cultures
were grown with and without addition of iron for about 48 hours.
The mutant with the ntcA-promoter was grown in standard BG11.
Estimated activities were calculated on the corresponding protein
concentration of the used crude extracts. It should be noted that
Pdc activities were estimated in the presence of added thiamine
pyrophosphate (cofactor for Pdc enzyme).
[1503] Results are presented in FIGS. 43A and 43B.
Ethanol Generation Rates in Cyanobacterial Mutants
[1504] In general the inducible promoters used therein can be
induced by medium exchange or by letting the cyanobacterial mutants
grow into starvation conditions in the case of promoters which are
inducible by nutrient starvation for example iron or copper
starvation.
[1505] The use of inducible promoters for the over-expression of
ethanologenic enzymes in cyanobacteria allow for switch on or
switch off ethanol production on demand. Several promoters that are
used for this purpose are inducible by the nutrient status, e.g.
Iron or copper availability. To reach these inducible conditions
either a medium exchange or growth into these starvation conditions
are possible.
Induction by Medium Exchange
[1506] Mutants and Synechocystis wild-type strains were grown at
28.degree. C., under constant light (50 .mu.E m.sup.-2 s.sup.-1)
either on a shaker (100 rpm) or in aerated culture vessels, bubbled
with Co.sub.2-enriched air (0.5% CO.sub.2). The initial OD.sub.750
was between 2 and 3 in a total culture volume of 50 ml in
Erlenmeyer flasks or 100 ml in the aerated culture vessels.
[1507] When an optical density of 2-3 was reached the culture was
harvested by centrifugation and the supernatant was discarded. The
cell pellet was washed with the new medium (e.g. without iron,
without copper, without nitrate and thereafter resuspended in the
respective medium for promoter induction. If iron starvation is
needed (isiA-promoter) the ferric ammonium citrate in the BG11 was
omitted, in the case of copper starvation (petJ-promoter) the trace
metal mix used was prepared without addition of copper sulfate, for
nitrogen starvation the sodium nitrate in the BG11 was omitted.
Induction by Letting the Cultures Grow into Starvation
Conditions:
[1508] Promoter induction by growing into starvation is based on
the consumption of nutrients due to the nutrient demand of a
culture. After nutrients are consumed the culture enters the
starvation condition which leads to the induction of the
appropriate promoter. The duration to reach such a starvation
condition can be influenced/limited by reduction of the amount of
the respective nutrient in the BG11 medium, e.g. 1/3 of the Ferric
ammonium citrate or copper sulfate concentration.
[1509] Furthermore, for repression of the nirA-promoter ammonia
(0.265 g/l corresponds to 5 mM NH.sub.4Cl) was added to the BG11
medium, which already contains nitrate. The culture induces itself
by consuming the ammonia as a preferred nitrogen source at first
(nirA promoter not induced) and upon complete consumption of
ammonia starts consuming the nitrate accompanied with induction of
the nirA-promoter.
B611 Media Recipe:
NaNO.sub.3: 1.5 g
K.sub.2HPO.sub.4: 0.04 g
MgSO.sub.4.7H.sub.2O: 0.075 g
CaCl.sub.2.2H.sub.2O: 0.036 g
[1510] Citric acid: 0.006 g Ferric ammonium citrate: 0.006 g EDTA
(disodium salt): 0.001 g
NaCO.sub.3: 0.02 g
[1511] Trace metal mix A5 1.0 ml (see below) Distilled water: 10
L
Trace Metal Mix A5:
H.sub.3BO.sub.3: 2.86 g
MnCl.sub.2.4H.sub.2O: 1.81 g
ZnSO.sub.4.7H.sub.2O: 0.222 g
NaMoO.sub.4.2H.sub.2O: 0.39 g
CuSO.sub.4. 5H.sub.2O: 0.079 g
Co(NO.sub.3).sub.2.6H.sub.2O: 49.4 mg
[1512] Distilled water: 1.0 L P.2 Ethanol Production Rates of
Genetically Modified Photoautotrophic Host Cells Containing
Zymomonas mobilis Pdc and AdhII as a Second Genetic
Modification
[1513] Ethanol production rates and OD.sub.750nm, values were
determined as described above and are shown in FIGS. 44A, 44B and
44C.
[1514] The concentration of ethanol in the growth medium was
determined using a standard UV-ethanol assay purchased from
R-Biopharm AG. In particular the assay is based on the UV detection
of NADH at 340 nm. It is based on the detection of generated NADH
according to the following enzymatic reaction catalyzed by alcohol
dehydrogenase and aldehyde dehydrogenase:
Ethanol+NAD+.fwdarw.acetaldehyde+NADH+H.sup.+
acetaldehyde+NAD.sup.++H.sub.2O.fwdarw.acetic acid+NADH+H.sup.+
P.3 Ethanol Production Rates of Genetically Modified
Photoautotrophic Host Cells Containing Zymomonas mobilis Pdc and
Synechocystis Adh as a Second Genetic Modification
[1515] Further the ethanol production rates of Synechocystis
cultures transformed with Zymomonas mobilis Pdc and an endogenous
Synechocystis Adh were also determined as described above. Results
are presented in FIG. 440.
P.4 Ethanol Production Rates of Genetically Modified
Photoautotrophic Host Cells Containing Zymomonas mobilis Pdc and
Various Wildtype as Well as Mutant AdhE Enzymes as a Second Genetic
Modification
Background:
[1516] The use of so called AdhE-type alcohol dehydrogenases (Adh),
which contain two enzymatic activities, namely a CoA-dependent
aldehyde dehydrogenase and an iron-dependent alcohol dehydrogenase
activity would allow the production of ethanol in genetically
modified cyanobacteria without requirement of a pyruvate
decarboxylase (Pdc). The substrate for this dual enzyme is
acetyl-CoA that is converted via two steps (by forming acetaldehyde
as transient intermediate) into ethanol. AcetylCoA is similar to
pyruvate a central metabolite in the cell which might be a well
convertible precursor for the ethanol production, too.
Interestingly, besides the group of enterobacteria where an AdhE is
very common, also some cyanobacteria contain such an AdhE enzyme,
e.g. Thermosynechococcus elongates BP-1, Microcystis aeruginosa and
some Aponinum species.
[1517] Therefore, besides the approach to use the Pdc together with
a conventional Adh, the over-expression of AdhE could also be
convenient for ethanol production in cyanobacteria. For this
purpose, the well characterized AdhE from E. coli and the
corresponding enzyme from Thermosynechococcus were chosen.
Mutant Generation:
[1518] Several plasmids to over-express both AdhE's were
constructed and respective mutants in Synechocystis 6803 were
created (see above described plasmid maps). Furthermore specific
activity-enhancing point-mutations were created in the adhE-gene
from E. coli K.sub.12 wild-type strain, which lead to specific
amino acid exchanges.
[1519] The AdhEs were over-expressed on a self-replicating
extra-chromosomal plasmid, pVZ321b, under control of the
copper-dependent petJ-promoter. Mutants were selected on
streptomycin plates and grown in BG11 medium containing the
appropriate antibiotics (kanamycin 100 mg/l and streptomycin 10
mg/l).
[1520] The following pVZ321b mutants were generated:
[1521] 6803 pVZ321b-PpetJ-EcAdhE (wt)
[1522] 6803 pVZ321b-PpetJ-EcAdhE (E568K, exchange from glutamic
acid at position 568 to lysine)
[1523] 6803 pVZ321b-PpetJ-EcAdhE (A267T/E568K, exchange of alanine
at position 267 to threonine and in addition E568K)
[1524] 6805 pVZ321b-PpetJ-ThAdhE (AdhE from
Thermosynechococcus)
Growth Conditions:
[1525] Mutants and Synechocystis wild-type strains were grown at
28.degree. C., under constant light (50 .mu.E m-2 s-1) on a shaker
(100 rpm). The initial OD.sub.750 was about 5 in a total culture
volume of 50 ml in a 100 ml Erlenmeyer flask. The ethanol
concentration was determined as described.
[1526] Results are presented in FIG. 45, wherein ethanol production
of Synechocystis mutants that express AdhE of E. coli (3 different
variants) are compared to Synechocystis wild type.
Results and Conclusions:
[1527] Exemplarily shown are ethanol production rates of the AdhEs
of E. coli. Compared to the wild type over the cultivation time of
about 5 weeks significant amounts of ethanol were produced by the
mutants. All overexpression mutants showed a significant ethanol
production. The exchange from glutamic acid at position 568 to
lysine (E568K), which shall reduce the oxygen sensitivity seems to
enhance the efficiency of the E. coli AdhE (EcAdhE) in
Synechocystis compared to the E. coli wild-type enzyme. The further
exchange of alanine at position 267 to threonine (A267T) did not
lead to an additional improvement of the first point mutation
(E568K), although it is might increase the acetaldehyde
dehydrogenase activity of the E. coli enzyme. But for both modified
EcAdhE variants an about 3-fold increase in ethanol accumulation
was observed. Therefore, it is possible to improve the AdhE enzyme
by site-directed mutations in order to reach better production
rates in cyanobacteria.
[1528] Synechocystis mutants that express the cyanobacterial
thermophilic AdhE (ThAdhE) from Thermosynechococcus show a similar
ethanol production rate to the mutants, which express the improved
variants of the EcAdhE (data not shown). Thus, if this enzyme can
be optimized in the same way, it might be even better than the E.
coli enzyme. In general the application of AdhE-type alcohol
dehydrogenases to produce ethanol in cyanobacteria is possible. The
potential to improve this kind of enzymes as shown for the E. coli
enzyme may allow for a large scale application for future ethanol
production processes.
P.5 Characterization of Genetically Modified Photoautotrophic Host
Cells Containing Zymomonas mobilis Pdc and Different Adh Enzymes as
a Second Genetic Modification
Background:
[1529] The introduction of a pyruvate decarboxylase (Pdc) and an
alcohol dehydrogenase (Adh) into cyanobacteria enables a light
driven production of ethanol in these phototrophic bacteria by
directing carbon fixed via photosynthesis into ethanol production.
The substrate for the Pdc enzyme is pyruvate that is converted by
decarboxylation into acetaldehyde and CO.sub.2. The generated
acetaldehyde is then converted by an Adh enzyme into the
end-product ethanol. In contrast to the Pdc almost all organisms
contain Adhs leading a huge number of Adh enzymes with quite
different characteristics. Interestingly, in Zymomonas mobilis two
different Adhs are present, which are not related to each other and
originate from different ancestors. The AdhI from Zymomonas mobilis
is a so-called Zn-dependent, oxygen insensitive alcohol
dehydrogenase, whereas the AdhII is Fe-dependent and
oxygen-sensitive. Both are quite effective with high affinities for
their substrates, acetaldehyde and NADH and outstanding due to
their high maximum velocities. Therefore both Adhs from Zymomonas
seem to be well suited, however the AdhI exhibits substrate
inhibition at elevated ethanol concentrations and the AdhII might
be partially inactive in cyanobacteria, since they produce large
amounts of oxygen by photosynthesis.
[1530] Therefore three different Adhs were analyzed for their
suitability for the ethanol production in cyanobacteria. Besides
the well characterized Zymomonas Adhs, a Zn-dependent Adh from
Synechocystis PCC6803 (SynAdh), which is not yet characterized in
the literature, but which was characterized by the inventors for
the first time, was chosen, since this enzyme should be also
oxygen-insensitive and therefore active in cyanobacteria.
Mutant Generation:
[1531] Several plasmids to overexpress all three Adhs together with
the Pdc from Zymomonas mobilis (Zm) were constructed and the
respective mutants were created in Synechocystis 6803 (see above
described plasmid maps).
[1532] To over-express each Pdc/Adh combination a self-replicating
extra-chromosomal plasmid, the pVZ321b, was used on which the
regarding pdc/adh-genes are expressed under control of the
copper-dependent pet-promoter. Mutants were selected on
streptomycin plates and grown in BG11 medium containing the
appropriate antibiotics (kanamycin 100 mg/l and streptomycin 10
mg/l).
[1533] The following pVZ321b mutants were generated:
6803 pVZ321b-PpetJ-ZmPdc/ZmAdhI 6803 pVZ321b-PpetJ-ZmPdc/ZmAdhII
6803 pVZ321b-PpetJ-ZmPdc/SynAdh
Growth Conditions:
[1534] Mutants were grown in BG11 medium without copper at
28.degree. C. and constant light conditions (100 .mu.E m-2 s-1).
The initial OD.sub.250 was about 15 in a total culture volume of
about 150 ml in a culture vessel aerated with CO.sub.2-enriched air
(0.5% CO.sub.2). The ethanol concentration was determined as
described above and the growth was determined by measurements of
the optical density at 750 nm. At the 11th day the cultures were
diluted by addition of 1 volume of new BG11 medium without
copper.
[1535] FIGS. 46A, 46B and 46C present results of growth, ethanol
accumulation and ethanol production per growth of Synechocystis
mutants that express ZmPdc/ZmAdhI (squares), ZmPdc/ZmAdhII
(diamonds) and ZmPdc/SynAdh (triangles), respectively.
Results and Conclusion:
[1536] All three PDC/ADH expressing Synechocystis mutants were able
to produce ethanol efficiently with similar production rates (FIGS.
46A, 46B and 46C). Thus, all three Adh enzymes seem to convert the
generated acetaldehyde, produced by the PDC into ethanol. In
general each of the three Adhs can be used for the ethanol
production in cyanobacteria.
[1537] Interestingly, the growth rate of the different mutants is
very similar at least for the first 10 days of cultivation, then
after addition of new BG11-medium the mutant expressing Pdc/SynAdh
looks more healthy and seems to grow faster than the mutants
expressing the Zymomonas mobilis Adhs, which rather have stopped
growing (although new nutrients were added). This is probably due
to the decreased vitality of respective ethanol producing cells
(visible by yellow pigmentation and bleaching as well as by the
reduced oxygen evolution), since a small amount of the generated
ethanol is reconverted to acetaldehyde by both Zymomonas Adhs. This
back-reaction decreased the yield of ethanol on one hand and on the
other hand is harmful for the cells, because of the toxicity of the
accumulating acetaldehyde. The Adh of Synechocystis does not
exhibit this back-reaction (at least under the tested growth
conditions), since in contrast to mutants expressing ZmAdhI or
ZmAdhII no acetaldehyde was detectable in the gas-phase of a SynAdh
expressing mutant culture (determined by gas chromatography, see
FIG. 46D). FIG. 46D presents measurements for outgas samples of
Synechocystis mutants that express ZmPdc/ZmAdhI (dashed line).
ZmPdc/ZmAdhI (solid line) and ZmPdc/SynAdh (dotted line) analyzed
by gas chromatography. The grey arrow indicates the acetaldehyde,
the black arrow the ethanol peak. This finding makes the
ZmPdc/SynAdh expressing mutant a more efficient ethanol producer,
because this mutant is healthier during the period of ethanol
production and is able to maintain the initial ethanol production
rate over a longer time scale as visible in FIGS. 46A, 46B and
46C.
[1538] Due to the fact that the ZmPdc/SynAdh expressing mutants do
not convert the produced ethanol back into acetaldehyde, there is
no toss in the production process. This is clearly visible in the
increased ethanol accumulation of these mutants. Both mutants
expressing the respective Zymomonas Adhs exhibit a lower ethanol
yield. Already after 10 days of cultivation there is a significant
difference in the ethanol content of the cultures, which indicates
that the loss by the back-reaction is not marginal.
[1539] Taken together, each of the three Adhs is applicable for the
ethanol production in cyanobacteria, in particular the
Synechocystis Adh enzyme. But with the aim of long-term ethanol
production with maximal yields it can be summarized the Adh of
Synechocystis is obviously advantageous and well suited for the
production process because of the lack of the observed
disadvantageous back-reaction.
[1540] Further experiments were prepared in which the acetaldehyde
formation in presence of different amounts of ethanol was
monitored. These experiments showed that cells expressing Pdc and
Adh I of Zymomonas mobilis produced more acetaldehyde when more
ethanol was added to the growth medium. It is therefore concluded,
that the acetaldehyde is formed by a back reaction from ethanol and
is not formed by a Pdc enzyme, which produces too much acetaldehyde
to be completely further converted into ethanol by the Adh
enzyme.
[1541] Analysis of ethanol and acetaldehyde by gas chromatography
(GC) was performed under following conditions. Gas chromatograph:
Shimadzu GC-2014; column: SGE ID-BP634 3.0, 30 m.times.0.53 mm;
carrier gas: helium; temperature: 40.degree. C. constant. An
acetaldehyde standard eluted under this conditions at 3.2 min. For
the standard, acetaldehyde (Cart Roth) was diluted to 1 mg/ml in
water, 25 .mu.l were injected into a 250 ml gas sampling tube, the
acetaldehyde was vaporized (30 min, 60.degree. C.). After cooling
different volumes were analyzed by GC. A calibration curve was
obtained by plotting the integrated peak area against the amount of
acetaldehyde.
[1542] The gas phase over the cultures was sampled with a gas tight
syringe pierced into the tubing at the outlet and 250 .mu.l were
injected into the GC.
[1543] For measurement of the acetaldehyde production from ethanol
Synechocystis cells were pelleted, repeatedly washed with BG-11 and
dissolved to 10 .mu.g Chl/ml in BG-11 medium. 2 ml of the cultures
were mixed with ethanol in clear gas vials (4 ml total volume)
closed with rubber seals. The samples were incubated at room
temperature for defined time periods in the light (approx. 1000
.mu.E/s*m2). 250 .mu.l of the gas phase were sampled with a gas
tight syringe and analyzed. Chlorophyll was determined as in
described in Tandeau De Marsac, N. and Houmard, J. in: Methods in
Enzymology, Vol. 169, 318-328. L. Packer, ed., Academic Press,
198.
TABLE-US-00051 TABLE 1 Ethanol and acetaldehyde in the gas phase
above ethanol producing strains. The gas phase above transgenic
strains of Synechocystis PCC6803 expressing different Pdcs and Adhs
using the plasmid pVZ323 PpetI was analyzed for ethanol and
acetaldehyde content. As a control the ethanol was also quantified
in the culture medium. ethanol acetaldehyde ethanol gas medium gas
phase [.mu.mol/L] phase [.mu.mol/L] [.mu.mol/L] PCC6803 wild type
n.d. n.d. n.d. ZmPdc/ZmADH I 0.70 4.5 8670 ZMPdc/ZmADH II 0.62 3.5
5134 ZpPdc/ZmADH II 0.33 3.3 -- ZmPdc/native ADH n.d. 4.0 7777
ZpPdc/native ADH n.d. 2.8 -- Pdc/SynADH n.d. 5.1 9757 ZmPdc, Pdc of
Zymomonas mobilis; ZpPdc, Pdc of Zymobacter palmae; ZrnAdh I, Adh I
of Zymomonas mobilis; ZmAdh II, AdhII of Zymomonas mobilis; native
Adh, no expression of an heterologous Adh, the native Adh of
Synechocystis is present; SynAdh, Adh of Synechocystis is
overexpresseds; n.d. not detectable; --, not measured
[1544] FIG. 46E shows the acetaldehyde production after addition of
ethanol in different concentrations. Wild type and ethanol
producing transgenic cells Synechocystis PCC6803, overexpressing
different Pdc and Adh enzymes (see text) were incubated for 30 min
under illumination with 0.05% to 0.4% (v/v) of ethanol. The y-axis
of FIG. 46E denotes the acetaldehyde concentration in the gas phase
(in .mu.mol/l) and the x-axis shows the ethanol concentration in %
(v/v).
[1545] FIG. 46E shows that only for the Synechocystis strain
transformed with pVZ323 PpetJ Pdc/ZmADH I, the amount of
acetaldehyde in the gas phase could be increased by adding more
ethanol to the growth medium. For the Synechocystis PCC6803 strains
transformed with pVZ323 PpetJ Pdc/SynAdh no increase in
acetaldehyde could be detected upon addition of ethanol.
[1546] The Adh enzyme from Synechocystis was further characterized
by preparing crude cell extracts from Synechocystis PCC6803
overexpressing SynAdh. For the reason of comparison crude cell
extracts from Synechocystis cells overexpressing Zymomonas mobilis
Adh it were prepared as well.
[1547] For preparation of crude extracts, cells were pelleted,
dissolved in buffer supplemented with 1 mM DTT and broken
(beadbeater, 2.times.10 min, glass beads with 100 .mu.m diameter).
The supernatant of a centrifugation (15 min, 14000 rpm, 4.degree.
C., Micro 200R, Hettich) was used for the experiments.
[1548] Synechocystis or Zymomonas mobilis Adh enzyme activity was
measured either as ethanol oxidation or as acetaldehyde reduction,
i.e. In the direction of ethanol formation. The assays for ethanol
oxidation contained in a total volume of 800 .mu.l 30 mM Tris/HCl
(pH 8.5), 1 mM NAD+ or 1 mM NADP+, 1 M ethanol and the crude
extract. The Adh activity was measured as rate of the increase of
the absorbance at 340 nm. For measurement of the acetaldehyde
reduction, the assays contained 30 mM MES/KOH (pH 6.2), 0.3 mM NADH
or 0.3 mM NADPH, and crude extracts. The reaction was started by
addition of an acetaldehyde solution to a final concentration of
0.125 M and the rate of decrease of the absorbance at 340 nm was
measured. For the measurements of the pH-dependency of the Adh 40
mM MES adjusted with Tris base (pH 6.5 to 8.0) and with NH3 (pH 8.5
and 9.0) was used as buffer. Protein was determined by the method
of Lowry.
TABLE-US-00052 TABLE 2 ADH activities measured as ethanol
oxidation. Crude extracts of Synechocystis wild type, Synechocystis
cells expressing Adh II of Zymomonas mobilis, or the AHD of
Synechocystis were analyzed. The assays contained NAD+ and/or
NADP.sup.+ in the given concentrations. Shown are specific
activities in nMol min.sup.-1 rng.sup.-1 of total protein. with Adh
II Z. with Adh Wild type mobilis Synechocystis 1 mM NAD.sup.+ 0.4
85.2 1.4 1 mM NADP.sup.+ 1.6 3.3 6.8 0.1 mM NADP.sup.+ 2.4 3.4 8.9
1 mM NAD.sup.+ + 0.1 mM 2.2 65.7 8.7 NADP.sup.+ 1 mM NAD.sup.+ + 1
mM 1.3 25.5 6.4 NADP.sup.+
[1549] This table 2 shows that Adh II from Zymomonas mobilis has a
higher enzymatic activity than Synechocystis Adh enzyme for the
unwanted backreaction, the oxidation of ethanol back to
acetaldehyde if NAD.sup.+ or mixtures of NAD.sup.+ and NADP.sup.+
are used as a cosubstrates.
TABLE-US-00053 TABLE 3 ADH activities measured in the direction of
ethanol production. The assays contained NADH or NADPH or a
combination of NADH and NADP.sup.+. Shown are the specific
activities in nMol min.sup.-1 mg.sup.-1 of total protein, with ADH
II with ADH Wild type Z. mobilis Synechocystis 0.3 mM NADH 13.7
62.8 53.3 0.3 mM NADPH 9.0 71.4 55.4 0.3 mM NADPH + 1 2.9 3.7 2.8
mM NADP.sup.+
[1550] The pH-dependency of the acetaldehyde reduction by crude
extracts containing the Synechocystis Adh is shown in the next FIG.
46F. Surprisingly very different results were found for NADH and
NADPH. With NADH as cosubstrate a steady decrease of activity at
higher pH values was measured (maximum activity at pH 6.1), whereas
the NADPH dependent reduction had a broad pH optimum. This FIG. 46F
shows the acetaldehyde reduction rates of a crude extract
containing Synechocystis Adh enzyme with NADH and NADPH,
respectively (0.15 nM final concentration) at different pH-values.
The activities are given in dE/min.
[1551] This finding is particularly interesting because according
to literature the amount of NADPH In Synechocystis exceeds the
amount of NADH approximately 10 times. Therefore Synechocystis Adh
enzyme is expected to have a broad pH-optimum in transformed
Synechocystis cells or other cyanobacterial strains.
[1552] The Adh enzyme of Synechocystis also has different kinetic
constants for NADH and NADPH. FIG. 46G summarizes the acetaldehyde
reduction rates at different cosubstrate concentrations.
Measurements were performed at pH 6.1. Using Lineweaver-Burk plots,
which depict the reciprocal of the rate of acetaldehyde reduction
versus the reciprocal of the concentration of NADH (squares) or
NADPH (rhombi), respectively (FIG. 46H) K.sub.m and v.sub.max for
NADH were calculated with 1 mM and 1.6 .mu.Mol min.sup.-1 m.sup.-1
crude extract. For NADPH K.sub.m and v.sub.max were 15 .mu.M and
0.4 .mu.Mol min.sup.-1 m.sup.-1 for the crude extract. The K.sub.m
for the NADH-dependent reaction of the Synechocystis Adh enzyme was
calculated to be approximately 1 mM.
Further Characterization of the Purified SynAdh Enzyme
[1553] In order to study the properties of the SynADH in more
detail, a number of different measurements with the purified enzyme
were performed. Experiments with cell extracts can be problematic
in some circumstances, e.g. they could contain inhibiting
substances or enzymes competing for the substrates.
Methods
[1554] SynADH was overexpressed as fusion protein with glutathione
S-transferase (GST) in E. coli. The fusion protein was purified by
affinity chromatography (Glutathione Sepharose.TM. 4, GE
Healthcare). The GST part of the fusion protein was then removed by
proteolytic digestion with PreScission Protease (GE
Healthcare).
Heterologous Expression and Purification of the SynAdh
[1555] ORF slr1192 from Synechocystis was amplified by PCR using
the primers:
TABLE-US-00054 (SEQ ID NO: 223) 5' CTCTAGGATCCATGATTAAAGCCTACG 3'
and (SEQ ID NO: 224) 5' CACGGACCCAGCGGCCGCCTTTGCAGAG 3'.
[1556] The primers contain nucleotide exchanges, which were
introduced into the primers to obtain a BamHI and a NotI
restriction site (the restriction sites are underlined in the
sequences). Phusion High fidelity DNA polymerase was used for the
PCR, which was performed according to the protocol of the
manufacturer (New England BioLabs Inc.).
The PCR resulted in an DNA fragment of 1010 bps, which was ligated
into the PCR cloning vector pJET1.2 blunt (GeneJETT.TM. PCR Cloning
Kit, Fermentas) and E. coli cells (.alpha.-Select Chemical
Competent Cells, Bioline) were transformed with the ligation assay.
Plasmidic DNA was isolated (GeneJET.TM. Plasmid. Miniprep Kit,
Fermentas) from positive clones, the DNA. was digested with BamHI
and NotI and the 1010 bps fragment containing slr1192 was
recovered. The fragment was ligated into pGEX-6P-1 (GE Healthcare)
which had been digested with BamHI and NotI. E. coli was
transformed and plasmidic DNA was prepared as before. The
correctness of the construct was verified by digestion with
different restriction enzymes and by complete sequencing of the
1010 bps insert
[1557] For the expression of the fusion protein chemical competent
BL21 E. coli cells were transformed with the construct. A single
colony was cultured in LB-medium complemented with ampicillin (125
.mu.g/ml) and glucose (1% w/v). The culture volume was stepwise
increased to 200 ml. Cells were finally harvested by centrifugation
(4500 rpm, 10 min, Rt, Rotina 420R Hettich) resuspended in 200 ml
LB-medium with ampicillin (125 .mu.g/ml) and IPTG (isopropyl
thiogalactoside, 0.5 mM) and cultured under shaking at 20.degree.
C. over night. Cells were then harvested, washed with buffer A (20
mM Tris/HCl, pH 7.5, 150 mM KCl, 1 mM Dithiotreitol) and
resuspended in this buffer. Cells were disrupted by sonication (UW
2070, Bandelin) under ice cooling and the lysate was cleared by
centrifugation (15 min, 14,000 rpm, 4.degree. C., Micro 200R
Hettich). 4 ml column material Glutathione Sepharose.TM. 4 Fast
Flow (GE Healthcare) was washed 5 times with buffer A and added to
the cell lysate. After incubation. (2 hours at Rt under shaking)
the material was packed in a disposable plastic column (12 cm
length, 1 cm diameter). The column material was washed with 5
column volumes (20 ml) buffer A and subsequently resuspended in 1.5
ml buffer A supplemented with 80 .mu.l PreScission Protease (2
units/.mu.l). After incubation at 4.degree. C. over night, the
column was eluted with buffer A. Fractions of 1.5 ml or 1 ml were
collected.
[1558] SDS Polyacrylamide get electrophoresis was performed with
standard methods using 15% polyacrylamide gels. Page Ruler.TM.
unstained protein ladder (Fermentas) was the molecular weight
standard.
[1559] Alcohol dehydrogenase activity was measured in the direction
of acetaldehyde reduction. The assay contained in a total volume of
1000 .mu.l 30 mM MES/KOH, pH 6.0, 1 mM DTT, 0.3 mM NADPH and
different volumes of samples. The reaction was started by addition
of acetaldehyde to a final concentration of 100 mM, the rate of the
decrease of the absorbance at 340 nm was measured.
Results ad Discussion
[1560] The success of the purification was verified by SDS
Polyacrylamide gel electrophoresis (SDS/PAGE) analysis and by
measurement of the alcohol dehydrogenase activity. As shown in FIG.
46I the main protein in the eluate has a molecular weight of
approx. 36 kDa. This corresponds to the molecular weight of the
SynADH, which was calculated from the amino acid sequence with 35.9
kDa. The PreScission protease has a molecular weight of 46 kDa. The
GST-tag, if expressed alone, has a molecular weight of 29 kDa. The
SDS/PAGE analysis shows that SynADH was enriched, but not purified
to homogeneity.
[1561] The results for the measurement of the alcohol dehydrogenase
activity are given in table 1, wherein the activity of the cell
lysate was defined as 100% yield. As shown therein only 50% of the
SynADH in the cell lysate was bound to the column material. In the
finally obtained fractions of the eluate the enzyme was enriched
approximately 16-fold. This purification factor is not high but for
a one step purification this is not unexpected. Approx. 35% of the
activity was finally recovered in fractions 1 to 5 of the
eluate.
[1562] Fraction 2 of the purification was used for the measurement
of the kinetic parameters of the SynAdh as described in the
following.
TABLE-US-00055 protein activity/vol. activity/protein total volume
conc. [.mu.mol/min [.mu.mol/min Purification activity yield sample
[ml] [mg/ml] *ml] *mg] [-fold] [.mu.mol/min] [%] cell lystate 15
14.3 7.86 0.55 1 117.9 100 flow thorugh 15 11.7 3.86 0.33 57.9 49
wash solution 20 1.5 0.37 0.25 7.3 6 fraction 1 1.5 1.25 10.92 8.7
15.8 16.4 fraction 2 1 1.25 10.92 8.7 15.8 10.9 fraction 3 1 0.91
9.60 10.5 19.1 9.6 fraction 4 1 0.35 3.60 10.3 18.7 3.6 fraction 5
1 0.11 1.10 10.0 18.2 1.1 fractions 1-5 41.6 35 computed value
[1563] Adh enzyme activity was measured either as ethanol oxidation
(back reaction) or as acetaldehyde reduction min the direction of
ethanol formation, forward reaction). The ethanol oxidation and
acetaldehyde reduction were measured at room temperature as rate of
change of absorbance at 340 nm. Both ethanol oxidation and
acetaldehyde reduction were analyzed at different pH values.
Experiments were made at pH 7.5 in presence of high concentrations
of KCl in order to mimic the Intracellular conditions. In addition
ethanol oxidation rates were assayed at pH 8.5 and acetaldehyde
reduction rates at pH 6.0. This pH values were taken from the
literature, they account for the different pH-optima of forward and
backward reaction of ADH II of Zymomonas mobilis.
Ethanol Oxidation:
[1564] The assays for the determination of the K.sub.m values for
NAD.sup.+ and NADP.sup.+ contained in a total volume of 1000 .mu.l
30 mM HEPES/KOH (pH 7.5), 150 mM KC, 1 mM DTT, 1.5 M ethanol,
purified enzyme and NAD.sup.+ or NADP.sup.+ in different
concentrations. For measurements at pH 8.5 HEPES/KOH was
substituted by 30 mM Tris/HC (pH 8.5), KCl was omitted. The assays
for the determination of the Km value for ethanol contained in a
total volume of 1000 .mu.l 30 mM HEPES/KOH (pH 7.5), 150 mM KCl, 1
mM DTT, 1 mM NADP*, purified enzyme and ethanol in different
concentrations. For measurements at pH 8.5 HEPES/KOH was
substituted by 30 mM Tris/HCl (pH 85), KCl was omitted.
Acetaldehyde Reduction:
[1565] The assays for the determination of the K.sub.m values for
NADH and NADPH contained in a total volume of 1000 .mu.l 30 mM
HEPES/KOH (pH 7.5), 150 mM KCl, 1 mM DTT, 2 mM acetaldehyde,
purified enzyme and NADH or NADPH in different concentrations. For
measurements at pH 6.0 HEPES/KOH was substituted by 30 mM MES/KOH
(pH 6.0), KCl was omitted. The assays for the determination of the
Km value for acetaldehyde contained in a total volume of 1000 .mu.l
30 mM HEPES/KOH (pH 7.5), 150 mM KCl, 1 mM DT, 0.32 mM NADPH,
purified enzyme and acetaldehyde in different concentrations. For
measurements at pH 6.0 HEPES/KOH was substituted by 30 mM MES/KOH
(pH 6.0), KCl was omitted.
Results
[1566] The K.sub.m and v.sub.max values of SynAdh for the different
substrates were determined with Lineweaver-Burk plots. The K.sub.m
values are summarized in table 1 and table 2.
TABLE-US-00056 TABLE 1 K.sub.m values of SynAdh for the different
substrates of the acetaldehyde reduction. Shown are the K.sub.m
values for NADH, NADPH and acetaldehyde at two different conditions
(see Methods); --, not measured. pH 7.5, 150 mM KCl pH 6.0 NADH
1000 .mu.M -- NADPH 15 .mu.M 20 .mu.M acetaldehyde 180 .mu.M 200
.mu.M
TABLE-US-00057 TABLE 2 K.sub.m values of SynAdh for the dfferent
substrates of the ethanol oxidation. Shown are the K.sub.m values
for NAD.sup.+, NADP.sup.+ and ethanol at two different conditions
(see Methods). pH 7.5, 150 mM KCl pH 8.5 NAD.sup.+ 10 mM 10 mM
NADP.sup.+ 15 .mu.M 15 .mu.M ethanol 23 mM 59 mM
Discussion
[1567] The K.sub.m value is an inherent property of an enzyme. It
is defined as the substrate concentration necessary to obtain
half-maximal velocity of the enzymatic reaction. The lower the
K.sub.m value, the higher the "affinity" of the enzyme to the
substrate.
[1568] The K.sub.m values of SynAdh for the substrates of the
acetaldehyde reduction were determined in earlier experiments with
cell extracts. The results for the purified enzyme presented here
are nearly identical to those results. The affinity of the enzyme
for NADPH is relatively high (K.sub.m approx. 15 .mu.M), but the
affinity for NADH is very low (K.sub.m for NADH approx. 1000
.mu.M). This means, that the reaction is much more effectively
catalyzed with NADPH than with NADH, and NADPH will be the
cosubstrate preferred by SynADH, all the more as in cyanobacteria,
as in other photosynthetic organisms NADPH exceeds NADH by far. In
Synechocystis PCC 6803 the pool of NADP.sub.total
(NADP.sup.++NADPH) is approx. 10 fold higher than the pool of
NAD.sub.total (NAD.sup.++NADH) as described in Cooley & Vermas,
J. Bacteriol. 183(14) (2001) 4251-42589. The K.sub.m value of
SynAdh for acetaldehyde was determined with approx. 200 .mu.M. As a
comparison the Km value of ADH I and ADH II of Zymomonas mobilis
given in the literature are between 8 and 21 .mu.M for acetaldehyde
and 12 to 27 .mu.M for NADH as described in Hoppner & Doelle,
Eur. L Appl. Microbiol. Biotechnol. 17, (1983), 152-157 and
Kinoshita et al., Appl. Microbiol. Biotechnol. 22, (1985), 249-254,
respectively.
[1569] The affinities of SynAdh to the substrates of the
acetaldehyde reduction are more or less similar to those of ADH I
and ADH II of Zymomonas mobilis, but the properties of the back
reaction are totally different. The K.sub.m value of ADH I and ADH
II of Z. mobilis for ethanol are given in the literature with 24
.mu.M (ADH I) and 140 .mu.M (ADH II), the K.sub.m for NAD+ with 73
.mu.M (ADH I) and 110 .mu.M (ADH II) [6]. The affinity of SynAdh to
ethanol is by far lower, the K.sub.m value for ethanol was
determined with approx. 23 mM to 59 mM. This means that ADH I and
ADH II will catalyze the formation of acetaldehyde already at low
ethanol concentrations, while effective acetaldehyde formation with
SynAdh requires much higher ethanol concentrations. As for the
forward reaction the two co-substrates behave totally different in
the back reaction. The K.sub.m for NAD.sup.+ was determined with 10
mM, the K.sub.m for NADP.sup.+ with 15 .mu.M.
[1570] The finding that SynAdh has a very low affinity towards
ethanol is an explanation for the ineffectivity of the back
reaction. The missing or relatively small formation of acetaldehyde
may be the explanation for the increased vitality of cell strains
containing the SynAdh when compared to ethanol producing strains
with other Adhs, as acetaldehyde is toxic to cells.
Phylogenetic Analysis of the SynAdh Enzyme
[1571] Phylogenetic analysis shows that Adh is a member of the
family of Zinc-binding GroES-like domain alcohol dehydrogenases,
which is phylogenetically different from the family of short chain
Rossmann fold like Adh enzymes or the family of Fe-containing
Fe-Adh enzymes.
[1572] The FIG. 47A shows a in-depth phylogenetic analysis of
different alcohol dehydrogenase families. Within the clade of
Zinc-binding GroES-like domain alcohol dehydrogenases three
sub-clades denoted A to C can be found and furthermore a Zymomonas
Adh enzyme, which is only distantly related to the other members of
the Zinc-binding GroES-like domain alcohol dehydrogenases. The
values in parentheses indicate the average percentage of protein
sequence identity of the members of one respective sub-clade to
Synechocystis Adh enzyme NP 443028. It can clearly be seen that for
example the members of the sub-clade B including Synechocystis Adh
enzyme share an average sequence identity with SynAdh of 61.77%.
Each of the different families contain a number of representative
members, which are denoted by their respective National Center for
Biotechnology information (NCBI) database entry numbers (available
on the world wide web at ncbi.nlm.nih.gov/). In particular the
phylogenetic tree was constructed with protein sequences of
different Adh enzymes using Neighbor-joining method. Distinct
clades includes Adh enzymes with different metal-binding domains.
The table of FIG. 47B shows the annotations, the organisms and the
database accession codes for the protein sequences of the different
sub-clades A to C in the clade of Zinc-binding GroES-like domain
alcohol dehydrogenases shown in FIG. 47A.
[1573] Genes encoding the alcohol dehydrogenase (Adh) from
Synechocystis sp. PCC 6803 were compared to all proteins from the
NCBI non-redundant database (available on the world wide web at
ncbi.nlm.nih.gov) with BLAST (1) to retrieve top bacterial sequence
matches, including 40 from extant cyanobacteria. Protein sequences
of these adh genes were aligned with ClustalW (2). Phylogenetic
tree was constructed with MEGA version 3.1 (3) using the
neighbor-joining method with Poisson correction substitution model
and 100 bootstrap replicates assuming uniform heterogeneity among
sites. The detailed options are as following:
TABLE-US-00058 Method: Neighbor-Joining Phylogeny Test and options:
Bootstrap (100 replicates; seed = 64238) Include Sites:
============================== Gaps/Missing Data: Pairwise Deletion
Substitution Model: ============================== Model: Amino:
Poisson correction Substitutions to include: All Pattern among
Lineages: Same (Homogeneous) Rates among sites: Uniform rates No.
of Sites: 315 No Of Bootstrap Reps = 100
[1574] The above phylogenetic analysis revealed three clades of
structurally and catalytically different types of alcohol
dehydrogenases: 1) Zn-containing `long-chain` ADH with a GroES-like
(ADH-N) domain at the N.sup.+ terminal end; 2) Insect-type, or
`short-chain` ADH; and 3) Fe-containing ADH (FIG. 47). The
Zn-containing ADHs (4, 5) are dimeric or tetrameric enzymes that
bind two atoms of zinc per subunit. Both zinc atoms are coordinated
by either cysteine or histidine residues; the catalytic zinc is
coordinated by two cysteines and one histidine. The Zn-containing
ADH contains a GroES-like (ADH-N) domain at N.sup.+ terminal and a
Rossmann-fold NAD(P)+-binding (NADB_Rossmann) domain at C.sup.+
terminal. A number of other Zn-dependent dehydrogenases, including
the glutathione dependent formaldehyde dehydrogenase (homologous to
gene adhC in Zymomonas mobilis) and the NADP-dependent quinone
oxidoreductase (qor) are closely related to Zn-ADH (6) and are
included in this family.
[1575] The short-chain Adh's belong to the short-chain
dehydrogenases/reductases family (SDR) (7), most of which are
proteins of about 250 to 300 amino acid residues with a
Rossmann-fold NAD(P)+-binding domain. Little sequence similarity
has been found in this family although there is a large degree of
structural similarity.
[1576] The Fe-containing ADH's are distantly related to gene adhB
from Z. mobilis. This group shares sequence homology with glycerol
and butanol dehydrogenases.
REFERENCES
[1577] 1. S. F. Altschul et al., Nucleic Acids Res. 25, 3389
(1997). [1578] 2. J. Thompson, D. Higgins, T. Gibson, Nucleic Acids
Res. 22, 4673 (1994). [1579] 3. S. Kumar, K. Tamura, M. Nei,
Briefings in Bioinformatics 5, 150 (2004). [1580] 4. H. Jornvall,
B. Persson, J. Jeffery, Eur. J. Biochem. 167, 195 (1987). [1581] 5.
H. W. Sun, B. V. Plapp, J. Mol. Evol. 34, 522 (1992). [1582] 6. B.
Persson, J. Hallborn, M. Walfridsson, B. Hahn-Hagerdal, S. Keranen,
M. Penttila, H. Jornvall, FEBS Lett. 324, 9 (1993). [1583] 7. H.
Jornvall, B. Persson, M. Krook, S. Atrian, R. Gonzalez-Duarte, J.
Jeffery, D. Ghosh, Biochemistry 34, 6005 (1995).
[1584] The FIGS. 47C to 47I show the protein sequences of the Adh
enzymes of sub-clade B, which also included the Zinc-dependent
Synechocystis Adh enzyme. In particular, FIG. 47C presents the
amino acid sequence of a zinc-containing alcohol dehydrogenase
family protein of Synechocystis sp. PCC 6803, Identified by Genbank
Accession No. NP 443028.1.
[1585] FIG. 47D presents the amino acid sequence of a
zinc-containing alcohol dehydrogenase family protein of
Oceanobacter sp. RED65, identified by Genbank Accession No.
ZP.sub.--01306627.1.
[1586] FIG. 47E presents the amino acid sequence of an alcohol
dehydrogenase, zinc-binding protein of Limnobacter sp. MED105,
identified by Genbank Accession No. ZP.sub.--01914609.1.
[1587] FIG. 47F presents the amino acid sequence of an alcohol
dehydrogenase GroES-like protein of Psychrobacter cryohalolentis
K.sub.3, identified by Genbank Accession No. YP.sub.--581659.1.
[1588] FIG. 47G presents the amino acid sequence of an alcohol
dehydrogenase GroES-like domain family of Verrucomicrobiae
bacterium DG1235, identified by Genbank Accession No.
EDY84203.1.
[1589] FIG. 47H presents the amino acid sequence of a
zinc-containing alcohol dehydrogenase family protein of
Saccharophagus degradans 2-40, identified by Genbank Accession No.
YP.sub.--529423.1.
[1590] FIG. 47I presents the amino acid sequence of a
zinc-containing alcohol dehydrogenase family protein of Alteromonas
macleodii `Deep ecotype`, identified by Genbank Accession No.
YP.sub.--002126870.1.
[1591] The FIGS. 47J to 47S represent the Adh protein sequences of
sub-clade A of the above phylogenetic analysis.
[1592] In particular FIG. 47J presents the amino acid sequence of a
zinc-containing alcohol dehydrogenase family protein of
Acaryochloris marina MBIC11017, identified by Genbank Accession No.
YP.sub.--001519107.1.
[1593] FIG. 47K presents the amino acid sequence of an alcohol
dehydrogenase GroES domain protein of Cyanothece sp. PCC 7424,
identified by Genbank Accession No. YP.sub.--002380432.1.
[1594] FIG. 47L presents the amino acid sequence of an alcohol
dehydrogenase GroES domain protein of Cyanothece sp. PCC 7424,
Identified by Genbank Accession No. ZP.sub.--02976085.1.
[1595] FIG. 47M presents the amino acid sequence of an alcohol
dehydrogenase GroES domain protein of Cyanothece sp. PCC 7822,
identified by Genbank Accession No. ZP.sub.--03154326.1.
[1596] FIG. 47N presents the amino acid sequence of an alcohol
dehydrogenase GroES domain protein of Cyanothece sp. PCC 8801,
identified by Genbank Accession No. YP.sub.--002371662.1.
[1597] FIG. 47O presents the amino acid sequence of an alcohol
dehydrogense GroES domain protein of Cyanothece sp. PCC 8801,
identified by Genbank Accession No. ZP.sub.--02941996.1.
[1598] FIG. 47P presents the amino acid sequence of an alcohol
dehydrogenase GroES domain protein of Cyanothece sp. PCC 8802,
identified by Genbank Accession No. ZP.sub.--0143898.1.
[1599] FIG. 47Q presents the amino acid sequence of an alcohol
dehydrogenase GroES-like domain family of Microcoleus
chthonoplastes PCC 7420, identified by Genbank Accession No.
EDX77810.1.
[1600] FIG. 47R presents the amino acid sequence of an
uncharacterized zinc-type alcohol dehydrogenase-like protein of
Microcystis aeruginosa NIES-843, identified by Genbank Accession
No. YP.sub.--001659961.1.
[1601] FIG. 47S presents the amino acid sequence of an unnamed
protein product of Microcystis aeruginosa PCC 7806, identified by
Genbank Accession No. CA090817.1.
[1602] The FIGS. 47T to 47X show the amino acid sequences of the
Adh enzymes of the sub-clade C of the above phylogenetic
analysis.
[1603] In particular FIG. 47T presents the amino acid sequence of a
zinc-containing alcohol dehydrogenase superfamily protein of
Synechococcus sp. WH 5701, identified by Genbank Accession No.
ZP.sub.--01085101.1.
[1604] FIG. 47U presents the amino acid sequence of a
zinc-containing alcohol dehydrogenase superfamily protein of
Synechococcus sp. RS9917, identified by Genbank Accession No.
ZP.sub.--01079933.1.
[1605] FIG. 47V presents the amino acid sequence of a
zinc-containing alcohol dehydrogenase superfamily protein of
Synechococcus sp. WH 5701, identified by Genbank Accession No.
ZP.sub.--01085101.1.
[1606] FIG. 47W presents the amino acid sequence of a zn-dependent
alcohol dehydrogenase of Synechococcus sp. WH 7803, identified by
Genbank Accession No. YP.sub.--001224538.1
[1607] FIG. 47X presents the amino acid sequence of a
zinc-containing alcohol dehydrogenase superfamily protein of
Synechococcus sp. WH 7805, identified by Genbank Accession No.
ZP.sub.--01125148.1.
P.6 Ethanol Production Rates of Genetically Modified
Photoautotrophic Host Cells Containing Zymomonas mobilis Pdc as the
Only Second Genetic Modification
[1608] Almost ah organisms including photoautotrophic organisms
contain in their genomes genes coding for alcohol dehydrogenases
(Adh). Also the cyanobacterium Synechocystis PCC6803 exhibit Adh
activity in crude cell extracts and contains a corresponding adh
gene in the genome. However it is questionable whether this
endogenous Adh enzyme is active enough in order to ensure a high
level ethanol production in conjunction with an overexpressed Pdc
enzyme.
[1609] In order to test if this endogenous Adh enzyme is able to
convert efficiently the generated acetaldehyde produced by the
over-expressed Pdc enzyme, mutants were generated that express only
the Pdc enzyme without additional Adh enzyme. This mutant was
compared to an isogenic ethanol producing mutant of Synechocystis
that over-express Pdc enzyme together with an additional Adh enzyme
from Zymomonas mobilis.
Mutant Generation:
[1610] From a preexisting pVZ plasmid (pVZ321b-PisiA-Pdc/AdhII)
containing respective Pdc/Adh genes from Zymomonas mobilis the
coding region of AdhI was cut out by SacI/PstI digestion and
subsequent religation of the residual plasmid lead to
pVZ321b-PisiA-PDC (without AdhII). Mutants were selected on
streptomycin plates and grown in BG11 medium containing the
appropriate antibiotics (kanamycin 100 mg/l; streptomycin 10
mg/l).
Growth Conditions:
[1611] Mutant and Synechocystis wild-type strains were grown in
BG11 without iron, at 28.degree. C., under constant light (100
.mu.E m.sup.-2 s.sup.-1), aerated with CO.sub.2-enriched air (0.5%
CO.sub.2). The initial OD.sub.750 was 1.3 in a total culture volume
of 300 ml in a 500 ml Schott-flask.
[1612] The FIGS. 48A and 48B show the with as determined by
measurement of the OD.sub.250 and ethanol production of
Synechocystis wild type and mutants that express Pdc/Adh enzyme and
Pdc enzyme alone, respectively over the time course of 15 days.
Results and Conclusions:
[1613] Both ethanol producing mutants, the mutant overexpressing
Pdc enzyme alone and the mutant overexpressing Pdc/AdhII grow very
similar but show a reduced growth rate when compared to the wild
type.
[1614] Surprisingly, the mutant that expresses the Pdc enzyme alone
exhibit about the same ethanol production rate compared to the
mutant that co-expresses an additional Adh enzyme with the Pdc
enzyme. Thus, the endogenous Adh of Synechocystis is able to
convert efficiently the generated acetaldehyde produced by the
overexpressed Pdc enzyme into ethanol. Under the conditions tested
here it seems that no additional Adh enzyme is necessary to produce
ethanol in Synechocystis. These results further show that the
reaction catalyzed by the Pdc enzyme is the rate limiting step in
the ethanol production process. P.7 Comparison of Ethanol
Production Rates of Genetically Modified Photoautotrophic Host
Cells Containing Zymomonas mobilis Pdc as the Only Second Genetic
Modification with Photoautotrophic Host Cell Harboring Pdc Enzyme
in Conjunction with Various Adh Enzymes
[1615] Synechocystis PCC 6803 transformed with various plasmids
harboring either the Zymomonas mobilis Pdc enzyme alone or
combination with Zymomonas mobilis AdhII enzyme or the
Synechocystis Adh enzyme was cultivated under conditions of
CO.sub.2 limitation or with sufficient CO.sub.2 supply.
[1616] The condition of CO.sub.2 limitation was created by shaking
50 ml cyanobacterial cultures in 100 ml Erlenmeyer flasks at
28.degree. C. at a rate of 100 rpm. The light intensity was set to
40 .mu.E m.sup.-2 s.sup.-1.
[1617] The condition of sufficient CO.sub.2 supply was created by
cultivating cyanobacteria in aerated 200 ml flasks and subjecting
the cultures to a constant gas flow of 0.5% (v/v) of CO.sub.2 with
a rate of 10 ml/min. The temperature was at 28.degree. C. and the
light intensity was set at 100 .mu.m.sup.-2 s.sup.-1.
[1618] The graphical representations in the FIGS. 48C and 48D,
depict the time course of the ethanol concentration in % (v/v) as
determined with the enzymatic ethanol quantification methods as
described above for various Synechocystis cultures transformed with
the indicated plasmids and cultured under a condition of CO.sub.2
limitation.
[1619] These data show that under conditions of CO.sub.2 limitation
photoautotrophic cyanobacterial host cells transformed with Pdc
enzyme only exhibit about the same ethanol production rates as
photoautotrophic cells transformed with Pdc in combination with
Synechocystis Adh enzyme. In contrast to that, photoautotrophic
cells transformed with Pdc enzyme in conjunction with Zymomonas
mobilis AdhII enzyme showed lower ethanol production rates.
[1620] The graphical representations in the FIGS. 48E and 48F,
depict the time course of the ethanol concentration in % (v/v) as
determined with the enzymatic ethanol quantification methods as
described above for various Synechocystis cultures transformed with
the indicated plasmids and cultured under a condition of sufficient
CO.sub.2 supply.
[1621] These data suggest that at conditions of sufficient CO.sub.2
supply photoautotrophic cyanobacterial host cells harboring Pdc
only or harboring a combination of Pdc enzyme and Zymomonas mobilis
AdhII enzyme show comparable ethanol production rates, which are
lower than ethanol production rates for photoautotrophic host cells
with Pdc enzyme and Synechocystis Adh enzyme.
P.8 Ethanol Production Rates of Genetically Modified
Photoautotrophic Host Cells Containing Ethanologenic Enzymes Under
the Transcriptional Control of Various Inducible Promoters
[1622] In this section several natural occurring promoters from
Synechocystis were analyzed for their suitability to express the
Pdc enzyme in Synechocystis. In Tab.1 an overview of the chosen
promoters with their characteristics is shown. For all these
promoters corresponding mutants in Synechocystis PCC6803 were
already created and characterized. This section reports only a
summary of the best embodiments.
[1623] FIG. 49A shows a summary of the cyanobacterial promoters
used to express ethanologenic enzymes in Synechocystis 6803.
Characteristics were taken from the literature, mainly analyzed and
described for the cyanobacterium Synechocystis 6803.
Mutant Generation:
[1624] From a preexisting pVZ plasmid (pVZ321b-PisiA-PDC/ADHII)
containing Pdc/Adh genes from Zymomonas mobilis the respective
promoter fragment (PisiA) was cut out by SalI/EcoRI digestion and
subsequent ligation of a new promoter fragment into the residual
plasmid leading to a new pVZ321b-Pxxx-PDC/ADHII derivate with
exchanged promoter xxx. Mutants were selected on streptomycin
plates and grown in BG11 medium containing the appropriate
antibiotics (kanamycin 100 malt; streptomycin 10 mg/l).
Growth Conditions:
[1625] Cultures were grown in BG11 in continuous light (50-100 pE)
either on a shaker in 100 ml Erlenmeyer flasks (100 rpm) or in
bubbling flasks (200 ml) aerated with CO.sub.2-enriched air (0.5%).
Depending on the current promoter BG11 without iron or copper was
used as well as BG11 without nitrogen or supplemented with 5 mM
NH.sub.4Cl. Pre-cultures were harvested by centrifugation, the
supernatant discarded and the cell pellet resuspended in new medium
with or without the specific nutrient, needed for the regarding
promoter mutant. The growth of the cultures was monitored by
photodensitometrical measurements at 750 mm. The ethanol production
was determined in the culture supernatant by an optical enzymatic
test (Boehringer Mannheim).
Results and Conclusions:
[1626] Transconjugants with the isiA-promoter are well growing and
as pigmented in the same way as the wild type. Growth experiments
reveal that the ethanol formation in the culture strongly depends
on the availability of iron (FIG. 1). If iron is present the
ethanol production is lower and time-shifted compared to the
sub-culture without iron. As described in the literature iron
starvation leads to very strong induction of the isiA-promoter.
After transition of the cells to iron-free BG11 it needs usual 3-5
days until ethanol formation starts. Western blot analyses revealed
that Pdc accumulates within 48 hours past iron depletion (up to
50-fold), but it strongly depends on the growth phase and the iron
availability of the pre-culture. By supplementation the growth
medium with additional won (3.times. Fe) the ethanol production can
be disabled for long time and starts very late with a low rate as
depicted in FIG. 49C. FIG. 49B shows the growth of the same culture
monitored by determining the OD.sub.750. Thus, ethanol production
in Synechocystis is excellent adjustable by using the iron
depending isiA-promoter.
[1627] Until now best production rates were observed for the
isiA-promoter. In continuous light about 0.02% (v/v) ethanol and in
day/night cycle about 0.014% (v/v) ethanol was produced per day,
respectively (for at least 10 days). Since longer iron deficiency
limits the photosynthesis rate it is imaginable to use this
promoter in a biphasic manner in which after a production period
iron is added to regenerate the cells for the next production
period. Furthermore auto-induction by stationary growth is a
possibility for the application of the isiA-promoter, too.
[1628] Transconjugants with the nblA-promoter appear more slowly
growing compared to transconjugants with the isiA-promoter and are
also a bit more yellowish pigmented than the wild type. Growth
experiments reveal that the ethanol formation in the culture
depends on the availability of nitrogen as described in the
literature for the nblA-promoter. If nitrogen is absent the ethanol
production is significant higher compared to the control culture
with nitrogen (FIG. 49D). Western blot analyses revealed a fast and
strong induction of the Pdc expression after nitrogen starvation.
Within 48 hours the Pdc accumulates up to 25-fold compared to
control cells (with nitrogen). But the ethanol accumulation in the
culture stops after 5-6 days (see FIG. 49D) most likely due to the
nitrogen deficiency. Since Synechocystis is not able to fix
nitrogen from the atmosphere, nitrogen deprivation leads to a
reduction of photosynthesis because of the deficiency of amino acid
biosynthesis in the absence of an utilizable nitrogen source.
Within some days of nitrogen deprivation photosynthesis decreases
significantly. But by using of nitrogen-fixing cyanobacterial
species (e.g. Anabaena sp. PCC7120) the application of a
nitrogen-dependent promoter like the nblA-promoter might be well
suited.
[1629] FIG. 49D shows the ethanol production of Synechocystis 6803
pVZ321b-PnblA-PDC/ADH that express Pdc/Adh enzymes under the
control of the nitrogen dependent nblA-promoter. Cultures were
grown on a shaker in Erlenmeyer flasks in BG11 under continuous
light. A pre-culture was divided into 2 sub-cultures (start
OD.sub.750 nm=2), one with and the other without nitrate.
[1630] The next set of promoters consists of three promoters
inducible by the nutrient status. Two of them, PpetJ and PpetE are
inducible by the copper availability and the third one, PnirA,
depending from the nitrogen source, ammonia or nitrate.
[1631] According to the literature the nirA-promoter is repressed
if ammonia is present and turned on if nitrate is the sole nitrogen
source. Furthermore this promoter is described as tight regulated
and was already successful used for heterologous gene expression in
Synechocystis PCC6803. Transconjugants with the nirA-promoter
appear more yellowish compared to the wild type and grow very
slowly, If grown on usual BG11 plates. This phenotype is common for
strong ethanol producers and is not surprising since the sole
nitrogen source of BG11 is nitrate, which switches the
nirA-promoter on.
[1632] Growth experiments revealed that the ethanol accumulation
depends from the nitrogen source (FIGS. 49F and 49G) Without
supplementation of ammonia to the BG11, the culture grows more
slowly as shown in FIG. 49E and produces at the same time more
ethanol. If ammonia is present the ethanol production was
significant lower. At the 8th day new ammonia was added to the
culture to take care that enough ammonia is present for repression
of the nirA-promoter. Due to this elevated ammonia availability the
ethanol formation was transiently blocked whereas the reference
culture (BG11 without ammonia) continues accumulating ethanol with
a similar rate anymore. But already 5 days later most of the new
supplemented ammonia is consumed by the cells and the promoter
becomes activated and reaches ethanol production rates similar to
the reference culture. If the produced ethanol in each sub-culture
is normalized to the cell growth (optical density) a clear
difference in the ethanol productivity is visible (FIGS. 49F and
49G). The reference culture without ammonia produces at least two
times more ethanol per cell compared to the culture supplemented
with ammonia.
[1633] FIGS. 49E to 49G depict the growth, ethanol production and
productivity per growth of Synechocystis 6803 pVZ325PnirA-PDC.
Cultures were grown in Erlenmeyer flasks with BG11 medium in
continuous light A pre-culture was divided into two sub-cultures
(start OD.sub.750nm=3), one with and the other without ammonia
supplementation. At the 8th day new ammonia (again 5 mM) was added
to the subculture that already contained ammonia.
[1634] Thus, in general the nirA-promoter is applicable but in
contrast to the literature no tight repression seems to be
possible. If the leakiness of the nirA-promoter can be somehow
reduced, it is imaginable that in the up-scaling process ammonia
can be added to the BG11 to reach fast growth rates and reduced
activity of the nirA-promoter. By consuming the ammonia over the
time the culture induces itself, but can still grow by using the
second nitrogen source, the nitrate that will stimulate the ethanol
production. Thus, no medium exchange will be necessary.
[1635] Since copper is not essential for photosynthetic growth of
Synechocystis (in contrast to iron) promoters of copper-responsible
genes are very promising Well described in the literature are the
petJ- and the petE-promoter. The petJ-promoter is switched off if
copper is present whereas the petE promoter is switched on. Both
promoters have been already applied for heterologous expression in
cyanobacteria, the petJ mainly in Synechocystis, whereas the petE
was mainly used in Anabaena sp. PCC7120.
[1636] Transconjugants with the petJ-promoter show a reduced growth
rate compared to wild type and appear also a bit yellowish. This is
not surprising, since it is known that the limited copper
availability in BG11 medium (0.3 .mu.M) already activates the
pet-promoter to some extent Growth experiments revealed that the
ethanol formation in the culture with different concentrations of
copper strongly depends on the availability of copper (see FIGS.
49H to 49J). If copper is absent the ethanol production is
significant higher compared to the control cultures with 0.3 .mu.M
(1.times.) or 1.5 .mu.M copper (5.times.) but at the same time the
culture without copper grows more slowly.
[1637] Between 1.times. and 5.times. copper also a significant
difference in growth and ethanol accumulation is detectable. If
copper is added to the culture the growth rate is increased
depending on the amount. A control experiment with the wild type
was performed in which the growth was documented in dependence of
the copper availability. Neither growth improvement nor retardation
was detectable for the wild type by various copper concentrations
(data not shown). Therefore the faster growth of the mutant at
elevated copper concentration is not due to a growth stimulating
effect of copper, it is a consequence of the lower ethanol
production. The higher the ethanol production rate the lower the
growth rate of corresponding mutants. If the ethanol accumulation
is calculated per cell (ethanol per OD.sub.750nm) strong
differences in the productivity were obvious depending on the
copper availability (see FIG. 47-6C). Thus, it is possible to
adjust the ethanol production and the growth rate by copper
supplementation. The pet-promoter seems to be therefore well
suited. Till now best production rates for this promoter are 0.014%
(v/v) ethanol per day in continuous tight (for about 4 weeks) and
about 0.007% (v/v) ethanol in day/night cycles (for about 3
weeks).
[1638] FIGS. 49H to 49J show the growth, ethanol production and
productivity per growth of Synechocystis 6803
pVZ321b-PpetJ-PDC/ADH. Cultures were grown on a shaker in
Erlenmeyer flasks in BG11 in continuous light. A pre-culture
(1.times. copper) was divided into 3 sub-cultures (start
OD.sub.750nm=3) and different concentrations of copper were
added.
[1639] Since in contrast to Synechocystis PCC6803 for the
nitrogen-fixing cyanobacterium Anabaena PCC7120 it was shown that
the Anabaena petE-promoter responds to different copper
concentrations. Therefore, instead of the Synechocystis promoter
the petE-promoter from Anabaena PCC7120 was chosen for the
over-expression of Pdc/Adh in Synechocystis. Transconjugants with
the petE-promoter are well growing and as pigmented as the wild
type when grown on copper-free BG11-plates Growth experiments
reveal that the ethanol formation in the culture depends on the
availability of copper (FIG. 49L). If the copper concentration is
elevated (5K copper corresponds 1.5 .mu.M) the ethanol production
is significant higher and the culture grows more slowly at the same
time (compared to the reference culture in copper-free BG11). Thus,
the petE-promoter from Anabaena works well for the over-expression
of Pdc/Adh in Synechocystis
[1640] FIGS. 49K and 49L show the growth, ethanol production of
Synechocystis 6803 pVZ321b-PpetE-PDC/ADH. Cultures were grown on a
shaker in Erlenmeyer flasks with BG11 in continuous light A
pre-culture (1.times. copper) was divided into 2 sub-cultures
(start OD.sub.750nm=3) with different concentrations of copper
(without and 5.times. Cu).
[1641] The crhC-promoter (cold shock induced RNA helicase) was
amplified from the genome of Anabaena PCC7120, since the chrC-gene
from Synechocystis seems to be not regulated by the temperature or
alternatively exhibit no induction by cold-shock. The Pdc enzyme
expression level of the corresponding mutants is relatively low,
also when induced by cold-chock. But at least a 3-fold increase in
Pdc expression, verified by Western blot analysis, and also an
elevated ethanol formation was detectable if the culture was grown
at 20.degree. C. (compared to reference culture at 28.degree. C.).
Although the crhC-promoter works in general and seems to be
adjustable by temperature, this promoter allows only low expression
level of ethanologenic enzymes in Synechocystis. However for
Anabaena it was shown that the crhC-promoter works well. Therefore
it might be possible that the crhC-promoter works more efficient by
using other cyanobacterial species.
[1642] FIG. 49M shows the ethanol production of Synechocystis 6803
pVZ321b-PcrhC-PDC/ADH. Cultures were grown on a shaker in
Erlenmeyer flasks in BG11 under continuous light conditions at
20.degree. C. and 28.degree. C.
[1643] Further multi-stress responsible promoters, the
htpG-promoter (heat shock protein 90), the hspA-promoter (small
heat shock protein A), the clpB1-promoter (clp protease, HSP100)
and the hliB-promoter (high-light inducible protein B, BLIP) were
analyzed in order to test their suitability for over-expression of
ethanologenic ORFs in Synechocystis 6803.
[1644] All four mutants showed different degrees in growth
retardation and yellow pigmentation if grown on a plate. Strongest
yellow pigmentation and most slowly growth were observed for the
mutants with the hspA-promoter, followed by the htpG, the hliB and
the clpB1-promoter.
[1645] The growth experiment revealed that the mutant with the
hspA-promoter was most productive till the 10.sup.th day concerning
the ethanol formation, but grows more slowly compared to the three
other mutants (FIGS. 49N and 49O). But after 10 days of cultivation
the ethanol accumulation decreases compared to mutants with the
htpG- and the hliB-promoter which show a comparable ethanol
accumulation.
[1646] FIGS. 49N and 49O show the growth, ethanol production and
productivity per growth of Synechocystis 6803 pVZ321b-PhspA-PDC,
pVZ321b-PhtpG-PDC, pVZ321b-PhliB-PDC and pVZ321b-PclpB1-PDC.
Cultures were grown in a culture vessel in BG11 in continuous
light, bubbled with CO.sub.2 enriched air (0.5%).
[1647] If for these four mutants the ethanol production is
normalized to the culture growth the first observation or rather
the first assumption about the strength of each promoter (different
degree of yellow pigmentation and growth retardation indicates) can
be clearly confirmed. The hspA-promoter seems to be most active in
this set of multi-stress responsible promoters. The htpG- and the
hliB-promoter exhibit a quite similar expression level, but the
expression level of hliB-promoter can be additional elevated by
increasing the light intensity. The clpB1-promoter exhibit the
lowest expression in this selection of promoters, probably too low
for commercial application. Further tests are necessary to
elucidate the full performance of these kind of promoters, since no
stress conditions were tested which might increase the observed
expression level additionally, It is noteworthy that cultivation of
the mutant with the hspA-promoter revealed production rates of
about 0.015% (v/v) ethanol per day in continuous light and about
0.01% (v/v) ethanol in day/night cycles (both for about 2 weeks)
that is comparable to the maximal expression level of mutants with
the isiA- and petJ-promoter.
[1648] Multi-stress inducible promoters are especially of interest
because of their potential to respond to ethanol or side effects
the ethanol production (probably indirect). In this case some kind
of auto-induction or self-enhancement is imaginable, which might be
advantageous. e.g. In combination with other promoters.
[1649] It can be summarized that the genome of Synechocystis
contains several promoters useful for the ethanol production
process. Well working examples are the isiA-, petJ- and the
petE-promoter as well as the nirA-promoter, which are all
adjustable by the nutrient status. Furthermore the hspA and the
htpG as well as the HliB-promoter appear to be suited for the
production process.
P.9 Ethanol Production Rates of Genetically Modified
Photoautotrophic Host Cells Containing Ethanologenic Enzymes Under
Various Growth Conditions
Background:
[1650] In order to get an idea about the potential of generated
ethanologenic mutants, one ethanol producing mutant was cultivated
over a longer time scale. Three different culture conditions were
tested regarding the productivity and the duration of the ethanol
formation using the cyanobacterium Synechocystis sp. PCC6803 that
over-expresses the pyruvate decarboxylase from Zymomonas mobilis
and the endogenous alcohol dehydrogenase
(pVZ321b-PpetJ-PDC/SynADH).
Growth Conditions:
[1651] Synechocystis mutant was grown either at 28.degree. C., in
continuous light (150 .mu.E m.sup.-2 s.sup.-1) and aerated with
CO.sub.2-enriched air (0.5% CO.sub.2) or in day/night cycles (12
h/12 h) with a temperature cycle (25.degree. C. night/35.degree. C.
day) and aerated with 5% CO.sub.2. The initial OD.sub.750 was 3-5
in a total culture volume of either 200 ml (continuous light) or
600 ml (day/night cycle) in bubbled glass vessels. For comparison
of the ethanol production rates the mutant was cultivated in
freshwater BG11 or in seawater BG11 (without copper). After two
weeks of cultivation a nutrient-mix (100-fold BG11-concentrate) was
weekly added to assure sufficient supply of nutrients for optimal
growth conditions over longer periods of time.
TABLE-US-00059 Recipe for 11 artificial seawater (28 ppm): NaCl
28.05 g MgSO.sub.4 6.90 g MgCl.sub.2 5.49 g KCl 0.67 g CaCl.sub.2
1.47 g
Results and Conclusion:
[1652] Best ethanol production rates were observed for freshwater
BG11 medium and continuous light Cultivation of the mutant in
seawater BG11 (mutant was pre-adapted in seawater) leads to a
reduction of ethanol production of about 25%. This is probably due
to the fact that the energy- and carbon-consuming synthesis of
osmo-protectants (like glycosylglycerol), which allows the
freshwater strain Synechocystis sp. PCC6803 to overcome higher
salinities, decreases the availability of fixed carbon
(carbohydrates) for the ethanol formation.
[1653] When the mutant was cultivated under day/night cycles with a
temperature gradient that simulates the conditions of an outdoor
production facility, the ethanol production and the growth rate was
reduced compared to the continuous light conditions (FIGS. 49P, 49Q
and 49R). That is not surprising because carbon fixation, that is
necessary for growth and ethanol production occurs only during the
light phase. Thus both, ethanol production and biomass production
are reduced when cultivated in day/night cycles.
[1654] If the ethanol production is normalized to the optical
density (as an indicator for growth) the productivity for each of
the cultivation conditions appears relatively similar (FIG. 49R).
That means the fraction of fixed carbon that flows into the ethanol
branch is relatively constant despite the different growth
conditions (see Tab.1).
TABLE-US-00060 TABLE 1 Ethanol production rates of Synechocystis
sp. PCC6803 pV321b-PpetI-PDC/ SynADH at different growth
conditions. pVZ321b-PpetI- EtOH/OD.sub.750 nm * PDC/SynADH EtOH
EtOH/day EtOH/OD.sub.750 nm day after 38 days % (v/v) % (v/v) %
(v/v) % (v/v) freshwater, 0.46 0.0126 0.0479 0.00126 contin. light
seawater, contin. 0.41 0.0108 0.0413 0.00109 light seawater, 0.26
0.0068 0.0450 0.00118 day/night cycle
P.10 Ethanol Production Rates of Genetically Modified
Photoautotrophic Eukaryotic Host Cells Containing Ethanologenic
Enzymes as a Second Modification
[1655] Following the concept of direct ethanol production in
cyanobacteria, the aim of this project was to express Pdc and Adh
in the phototrophic microalga Chlamydomonas reinhardtii in order to
generate ethanol (EtOH) in a eukaryotic system. C. reinhardtii
(hereafter Chlamydomonas) was chosen, because this unicellular
green alga is easy to cultivate up to high cell densities and well
established for transformation. In general, however, the concept of
EtOH production is applicable to all eukaryotic phototrophic algae
as long as stable transformants can be generated. As gene source
for PDC and Adh we chose Saccharomyces cerevisiae (Sc). This yeast
has a very high fermentative activity; its genome is completely
sequenced and well annotated (available on the world wide web at
yeastgenome.org).
[1656] After cloning of ScPdc and ScAdh into eukaryotic expression
vectors, Chlamydomonas was transformed [Kindle (1990) Proc Natl
Acad Sci USA 87:1228]. After selection, transformation was
confirmed via PCR. The expression of heterologous proteins was
confirmed by immune staining (Western blotting). The production of
EtOH was assayed via a coupled enzymatic test (as previously
described for cyanobacteria).
Chlamydomonas Strains and Growth Conditions
[1657] Wild type Chlamydomonas strains (CC-1960, CC-620 and CC-621)
were obtained from the Chlamydomonas Culture Collection at Duke
University (available on the world wide web at chlamy.org). The
cell wall deficient, arginine requiring strain (cw15 arg-) is a
gift from Dr. Daniel Karcher, MPI-MP (Golm). Cells were grown at
25.degree. C. in Tris-acetate-phosphate (TAP) medium [Harris (1989)
The Chlamydomonas sourcebook a comprehensive guide to biology and
laboratory use. Academic Press, San Diego] on a rotary shaker (110
rpm) in continuous light (100 .mu.E m.sup.-2 s.sup.-1). Arginine
was added at 200 mg l.sup.-1 (TAP+R) when required. For solid
media, 15% agar was used.
Pdc and Adh Genes
[1658] S. cerevisiae encodes three structural genes for Pdc of
which Pdc1 is the most active [Hohmann and Cederberg (1990) Eur. J.
Biochem. 188:615; Hohmann (1991). Bacteriol. 173:7963]. For Adh,
there are four structural genes [Johnston and Carlson (1992) In:
The molecular cell biology of the yeast Saccharomyces. Vol 2 pp.
193] of which AdhI appears best suited for our purpose. It is
Zn-dependent and catalyzes the forward reaction from acetaldehyde
to EtOH with highest efficiency. Therefore, ScPDC1 and ScADH1 were
chosen for expression in Chlamydomonas.
[1659] The nucleotide sequence of ScPDC1 is shown in FIG. 50A and
the corresponding protein sequence in FIG. 50B. The nucleotide
sequence of ScADH1 is depicted in FIG. 50C and the corresponding
protein sequence in FIG. 50D.
Eukaryotic Promoter Systems (CYC6 and FEA1)
[1660] As eukaryotic promoters, inducible promoters were chosen in
order to be able to control EtOH production and restrict production
to specific growth phases.
Nucleotide Sequence of Pcyc6
[1661] The CYC6 gene of Chlamydomonas encodes cytochrome c6 (cyt
c6). Gene expression is regulated by Pcyc6 (located upstream [-127
to -7] of the transcription start) and induced by copper starvation
[Quinn and Merchant (1995) Plant Cell 7:623]. Pcyc6 (nucleotide
sequence is shown in FIG. 50E) was obtained from the plasmid pXX311
(a gift from Prof. Peter Hegemann, Humboldt University Berlin).
Nucleotide Sequence of Pfea1
[1662] FEA1 and FEA2 encode two proteins which are secreted as an
answer to iron deficiency by Chlamydomonas. They subsequently
facilitate high affinity iron uptake [Merchant et al (2006) Biochin
Biophys Acta 1763:578; Allen et al. (2007) Eukaryot Cell 6:184].
The iron-responsive element, Pfea1 was obtained from the plasmid
p5'FEA1-ARS2 [Allen et al. (2007) Eukaryot Cell 6:184], purchased
from the Chlamydomonas Center (available on the world wide web at
chlamy.org). The nucleotide sequence of FEA1 is shown in FIG.
50F.
Selectable Markers (ble and ARG7)
[1663] As selectable markers the introduction of antibiotic
resistance as well as the recovery of auxotrophy for essential
nutrients in metabolic mutants were used.
Nucleotide Sequence of the ble Marker
[1664] For selection on antibiotics the synthetic ble gene was
used, which confers resistance against the antibiotics
bleomycin/zeocin. (TAP agar supplemented with 5, 10, 15 or 20 .mu.g
zeocin ml.sup.-1 were used.) The marker gene ble was obtained from
the plasmid pSP124S [Lumbreras et al. (1998) Plant J 14:441],
purchased from the Chlamydomonas Center (available on the world
wide web at chlamy.org). Capital letters in FIG. 50G represent the
coding sequence.
Nucleotide Sequence of the ARG7 Marker
[1665] The ARG7 gene of Chlamydomonas encodes argininosuccinate
lyase, the last enzyme in arginine biosynthetic pathway. For
selection on nutrient-deficient plates, we used arg7' mutants that
require exogenous arginine (gifts from Dr. Daniel Karcher, MPI-MP).
Prior to transformation, cells were grown in TAP medium
supplemented with arginine (TAP+R). The cells were transformed with
a plasmid carrying the ARG7 gene and selected on TAP plates lacking
exogenous arginine (for preparation of plates Sigma agar was used,
because Bacto agar may contain traces of arginine). The ARG7 marker
gene was obtained from the plasmid pXX311 (a gift from Prof. Peter
Hegemann, Humboldt University Berlin). ARG7 was subcloned into the
NotI/XbaI site of pBluescript II KS+ (pKS) to give ARG7_pKS, which
was subsequently used for expression (see below). Capital letters
in FIG. 50H represent the coding sequence.
Expression Plasmids (pSP124, pXX311 and ARG7_pKS) pSP124S
[1666] The plasmid pSP124S was purchased from the Chlamydomonas
Center (available on the world wide web at chlamy.org). It contains
the Amp.sup.R marker (bla) for selection in E. coli and the ble
gene (see 1.5.1.) for selection in Chlamydomonas. The Chlamydomonas
RbcS2 promoter and 3' UTR ("untranslated region") sequence were
used as promoter and 3'UTR for BLE expression (shaded in grey in
the nucleotide sequence below).
[1667] pSP124S was used for expression of ScPDC1 and ScADH1 and is
schematically shown in FIG. 50I. The nucleotide sequence of pSP124S
is depicted in FIG. 50-58.
pXX311
[1668] The plasmid pXX311 was a gift from Prof. Peter Hegemann
(Humboldt University Berlin). It contains an Amp.sup.R (bla) and a
Km.sup.R marker for selection in E. coli and the ARG7 gene for
selection in Chlamydomonas (arg mutants). The coding sequence of
ARG7 is given above, 5' regulatory (incl. promoter) and 3' UTR
sequences are shaded in grey in the pXX311 nucleotide sequence
shown in FIG. 50L.
[1669] The original pXX311 plasmid contains luciferase as a
reporter gene. This gene was deleted and ScPDC1 and ScADH1 were
cloned behind the CYC6 promoter. A graphical representation of
pXX311 is shown in FIG. 50K.
ARG17pKS
[1670] The commercial cloning vector pBluescript II contains the
Amp.sup.R marker (bla) for selection in E. coli. For selection in
Chlamydomonas, the ARG7 marker (derived from pXX311) was inserted
between NotI and XbaI sites. Subsequently. ARG7_pKS was used for
cloning of the double expression cassette containing ScPDC1 and
ScADH1 A graphical representation of ARG7_pKS is shown in FIG.
50M.
Cloning Strategy
[1671] ScPDC1 and ScADH1 genes were PCR-amplified from yeast
genomic DNA. For subsequent cloning steps, the forward primer
carried a restriction site for XhoI, the reverse primer for
BamHI.
TABLE-US-00061 (SEQ ID NO: 225) ScPDC1-XhoI-F
catgctcgagATGTCTGAAATTACTTTGGGTAA (SEQ ID NO: 226) ScPDC1-BamHI-R
catgggatccTTATTGCTTAGCGTTGGTAG (SEQ ID NO: 227) ScADH1-XhoI-F
catgctcgagATGTCTATCCCAGAAACTCAAA (SEQ ID NO: 228) ScADH1-BamHI-R
catgggatccTTATTTAGAAGTGTCAACAACGT
[1672] The promoters Pcyc6 and Pfea1 were PCR-amplified from the
plasmids pXX311 and p5'FEA1-ARS2, respectively. For subsequent
cloning steps, two PCRs were run for each construct in the primary
PCR, the forward primers carried a NotI restriction site. In the
second PCR, the forward primers carried an additional SpeI
restriction site. In both the first and second PCR, the same
reverse primers, which carried an (endogenous) XhoI site, were
used.
TABLE-US-00062 (SEQ ID NO: 229) Pcyc6-NotI-F
gcggccgcCACTGAAGACTGGGATGAGC (SEQ ID NO: 230) Pcyc6-NotI-SpeI-F
gcggccgcactagtCACTGAAGACTGGGATG AGC (SEQ ID NO: 231) Pcyc6-XhoI-R
CTCGAGCATGTTTATGGAGTAGG (SEQ ID NO: 232) Pfea1-NotI-F
gcggccgcAGGACAGAGTGCGTGTGG (SEQ ID NO: 233) Pfea1-NotI-SpeI-F
gcggccgcactagtAGGACAGAGTGCGTGTGG (SEQ ID NO: 234) Pfea1-XhoI-R
CTCGAGCATGGTTAACTGTG
[1673] The 3'UTR sequence (required for correct translation and
protein assembly in eukaryotes) was PCR-amplified from the pXX311
plasmid. For subsequent cloning steps, the forward primer carried
an (endogenous) BamHI restriction site. The reverse primer carried
two restriction sites in tandem: XbaI and Kpnl.
TABLE-US-00063 (SEQ ID NO: 255) 3'UTR-BamHI-F catgGGATCC
CCGCTCCGTGTA (SEQ ID NO: 236) 3'UTR-XbaI-KpnI-R
catgggtacctctagaCGCTTCAAATACGCCCAG
[1674] For intermediate cloning, the 3'UTR sequence was cloned into
pBluescript II SK+ (pSK; BamHI/Kpnl). All other PCR products were
cloned into pJET1.2/blunt (Fermentas).
[1675] After cloning of ScPDC1 and ScADH1, they were subcloned in
front of the 3'UTR sequence in pSK via NotI/BamHI (Not sites
derived from multi cloning sites of cloning vectors). For the sake
of briefness, only one construct is illustrated in FIG. 50N. Other
constructs were generated accordingly.
[1676] Afterwards, the respective promoter (with NotI/XhoI sites)
was connected to the ScPDC1.sub.--3'UTR construct via NotI/XhoI
restriction as shown in FIG. 50O. Similarly, the respective
promoter (with Not, SpeI/XhoI sites) was connected to the
ScADH1.sub.--3'UTR construct. [Note: The gene of interest (ScPDC1)
which will later be the first of two in a double expression
cassette has to be linked with a promoter carrying NotI/XhoI sites,
while the second (ScADH1) has to be linked with a promoter carrying
NotI, SpeI/XhoI sites. The Internal SpeI site will be lost during
ligation of the two constructs.]
[1677] In order to have a double expression-construct for Pdc and
Adh, the promoter-ScADH1-3'UTR cassette was excised via SpeI/XbaI
and ligated into the XbaI site of the promoter-ScPDC-3'UTR
construct as shown in FIG. 50P. SpeI and XbaI generate compatible
ends, and, after ligation, both the SpeI and XbaI sites are lost.
This way, the double expression-cassette could be excised by
NotI/XbaI for the final cloning step. The correct orientation of
the double expression cassette was verified by sequencing.
[1678] For the final cloning step, the double expression cassette
was excised by NotI/XbaI and ligated into the NotI/SpeI site of the
expression plasmid pSP124S (containing the ble gene) or into the
NotI/SpeI site of ARG7_pKS.
[1679] In addition to the PDC-ADH double expression constructs the
Pcyc6 ScPDC1 single construct was also cloned into pXX311. This was
done to examine the effect of heterologous ScPDC in concert with
endogenous CrADH. As described herein, results with cyanobacteria
have shown that cells expressing only a foreign PDC and relying on
their own ADH activity can generate significant amounts of
EtOH.
[1680] The resulting expression plasmids are depicted in FIGS. 50Q,
50R, 50S, 50T and 50U respectively.
Transformation of Chlamydomonas
[1681] For transformation of Chlamydomonas, the glass bead method
was used [Kindle (1990) Proc Natl Acad Sci USA 87:1228]. This
method can only be applied to cells with a degenerated cell wall.
This can either be achieved by a mutation (we used the cw.sup.13
mutants) or by treatment of wild type cells with autolysin. Prior
to gene transfer, expression plasmids were linearized (XmnI).
Protocol for Transformation
[1682] 1. Transformation of cw15 arg-cells with ARG7
[1683] 1) Inoculate 25 ml TAP+R with a loopful of cells and grow
for 3 days
[1684] 2) Transfer 2 ml of the preculture to 150 ml fresh TAP+R,
and grow the cells for 2 days (OD.sub.750=0.3 to 0.5)
[1685] 3) Collect cells by centrifugation
[1686] 4) Wash and resuspend cells in TAP, incubate for 2 h with
gentle shaking.
[1687] 5) Collect the cells by centrifugation
[1688] 6) Resuspend the cells in 3 ml of TAP
[1689] 7) Glass beads transformation:
[1690] i. In a 1.5 ml tube that contains 4 ug of linearized DNA,
add 300 ul of the cell suspension and 100 ul of 20% PEG8000
[1691] ii. Transfer the mixture into a glass tube that contains
sterile 300 mg glass beads (0.5 um) [1653] iii. Vortex at the top
speed for 15 s
[1692] 8) Spread the cell suspension on 2 plates of TAP agar (1.5%
sigma agar)
[1693] 2. Transformation of cw15 arg-cells with ble
[1694] Steps 1)-3): same as 1
[1695] 4) Wash and resuspend cells in 3 ml of TAP+R
[1696] 5) Glass beads transformation
[1697] 6) Transfer the cells in a flask, add 10 ml of TAP+R, and
shake for 1 day under the growth conditions
[1698] 7) Collect the cells by centrifugation
[1699] 8) Resuspend the cells in 1 ml of TAP+R
[1700] 9) Spread the cells on 4 plates of TAP+R agar (1.5%
Bacto-Agar) that contain 5-20 .mu.g/ml zeocin
[1701] 3. Transformation of CC-1960 cells with ble
[1702] 1) Inoculate 25 ml TAP with a loopful of cells and grow for
3 days
[1703] 2) Transfer 2 ml of the preculture to 150 ml fresh TAP, and
grow the cells for 2 days [1666]3) Collect the cells by
centrifugation
[1704] 4) Wash and resuspend the cells in 25 ml of autolysin
preparation (see below). Incubate for 1 h with gentle shaking.
[1705] 5) Wash and resuspend the cells in 3 ml TAP
[1706] 6) Glass beads transformation
[1707] 7) Transfer the cells in a flask, add 10 ml of TAP, and
shake for 1 day under the growth conditions
[1708] 8) Centrifuge and resuspend the cells in 1 ml of TAP
[1709] 9) Spread the cells on 4 plates of TAP agar (1.5%
Bacto-Agar) that contain 5-20 .mu.g/ml zeocin
[1710] 4. Preparation of Autolysin
[1711] 1) Cultivate the two different mating types of Chlamydomonas
(CC-620 & CC-621) into early exponential phase
(3.times.10.sup.5 cells ml.sup.-1) (use 250 ml TAP medium in a 1 L
flask)
[1712] 2) Collect cells and resuspend in TAP-N(NH.sub.4Cl was
replaced with the same concentration of KCl) (use 1 L TAP-N in a 2
L flask)
[1713] 3) Shake gently under light for 24 h (induction of gamete
formation)
[1714] 4) Harvest cells and resuspend each culture in 200 ml
TAP-N
[1715] 5) Mix both cultures in a 2 L flask
[1716] 6) Keep the flask in the light without shaking for 1-2 h
(mating)
[1717] 7) Remove the cells by centrifugation
[1718] 8) Freeze the supernatant (clued extract of autolysin) and
store at -80.degree. C.
[1719] Transformation of the Chlamydomonas wild type (strain
CC-1960) and mutant strain cw.sup.15 arg.sup.- (defective in cell
wall and arginine biosynthesis) was carried out with the expression
constructs listed below.
TABLE-US-00064 Expressed Selectable C. reinhardrii gene(s) Promoter
marker Plasmid strain(s) ScPDC1 CYC6 ARG pKS cw.sup.15arg.sup.-
scADH1 (pXX311) ScPDC1 FEA1 ARG pKS cw.sup.15arg.sup.- scADH1
(pXX311) ScPDC1 CYC6 BLE pSP124S CC-1960 & scADH1
cw.sup.15arg.sup.- ScPDC1 FEA1 BLE pSP124S CC-1960 & scADH1
cw.sup.15arg.sup.- ScPDC1 CYC6 ARG pXX311 cw.sup.15arg.sup.-
[1720] For all transformations, PCR positive colonies were
obtained. The rate of positives, however, was significantly higher
for the ARG marker (90% positives) than for the BLE marker (10%
positives).
EtOH Production
[1721] EtOH production was assayed by an optic enzymatic test (as
described herein). Cells were grown in TAP medium at 25.degree. C.
on a rotary shaker in continuous light. For transformants carrying
the synthetic ble gene as a marker, zeocine (3 .mu.g ml.sup.-1) was
added to the medium. EtOH production was triggered by a transfer of
cultures to TAP-CU (for transformants carrying the CYC6 promoter)
and TAP-Fe (for transformants carrying the FEA1 promoter),
respectively.
[1722] The following table gives representative values for EtOH
production in Chlamydomonas. These data are also depicted in the
graph below. Non-induced transformants as well as non-transformed
cells were run as control. A graphical representation of these data
is given in FIG. 50V.
TABLE-US-00065 EtOH content [.mu.M] of the cell-free medium 0 6 13
20 24 Time (d) Non-transformed wild type (CC1960) 0 30 10 25 20
[mean of 6 independent cultures] Non-transforrned background strain
0 20 45 45 43 (cw.sup.15arg.sup.-) [mean of 4 independent cultures]
Non-induced transformant (cw.sup.15arg.sup.-) 0 20 30 45 90 [pKS
ARG Pcyc6 PDC ADH] [mean of 5 independent cultures] Time(d) after
induction Induced transformant (cw.sup.15arg.sup.-) 0 40 80 150 225
[pKS ARG Pcyc6 PDC ADH] [mean of 6 independent cultures] Induced
transformant (cw.sup.15arg.sup.-) 0 20 110 170 240 [pKS ARG Pfeal
PDC ADH] [mean of 4 independent cultures]
[1723] The Chlamydomonas transformants pKS_ARG_Pcyc6_PDC_ADH and
pKS_ARG_Pfea1_PDC_ADH, both generated in the cw.sup.15 arg.sup.-
background, produced significant amounts of extracellular ethanol
after induction (i.e. copper depletion for pKS_ARG_Pcyc6_PDC_ADH
transformants and iron depletion for pKS_ARG_Pfea1_PDC_ADH
transformants). After 24 d, final concentrations of 225 and 240
.mu.M ethanol were reached in the medium. The non-transformed
control strains (wild type strain CC1960 as well as background
strain cw.sup.15 arg.sup.-) did not produce significant amounts of
extracellular ethanol during the same time span. The level of
extracellular ethanol in non-induced transformants remained on a
baseline level for about 20 d, but started to increase after that.
This is most likely due to self-induction of the culture after the
onset of copper-/iron-depletion.
[1724] Compared to ethanol production in cyanobacteria, ethanol
levels reached with Chlamydomonas transformants were rather low.
This is most likely due to differences in the codon-usage of
Chlamydomonas and Saccharomyces cerevisiae (donor organism for Pdc
and Adh genes), resulting in a low expression of ScPDC and ScADH.
While the green alga has a strong G/C-bias [Goldschmidt-Clermont
(1991) Nucleic Acids Res 19: 4083-4089; Kindle and Sodeinde (1994)
J. Appl. Phycol 6:231-238] the yeast genes exhibit only an average
G/C-content. This would clearly impair expression of heterologous
proteins in Chlamydomonas as also reported in other instances
[Fuhrmann et al (1999) Plant J. 19: 353-361; Fuhrmann et al (2004)
Plant Mol Biol 55: 869-881]. However, the use of endogenous
Chlamydomonas promoters (CYC6 and FEA1) apparently supported
protein expression to such a degree that ethanol production in
transformants was clearly detectable. In the future, a focus will
be on codon optimization of Pdc and Adh in order to promote protein
expression in Chlamydomonas and thereby reach higher ethanol
production in the green alga.
[1725] Detailed Description of Various Embodiments for Testing a
Photoautotrophic Strain for a Desired Growth Property
[1726] In the following various detailed protocols for different
tests to Identify a photoautotrophic strain with a desired growth
property we be explained:
Initial Ethanol Tolerance Test (Also Called Short Term Ethanol
Tolerance Test)
Method
[1727] testing of all strains for tolerance against ethanol by
stepwise increasing of ethanol concentration in 5% steps up to an
concentration of 20% [1728] measurement of optical densities at
certain points as well as microscopic analyses using a
light/fluorescence microscope containing percentage estimation of
ratio of living to death cells (using the red auto fluorescence of
chlorophyll and actual conditions of cells like e.g. green colored
or bleached). A photoautotrophic strain has passed this test if
less than 50% of the cells were found to be bleached or lysed.
[1729] end concentrations of ethanol in the test can vary from 0.5
up to 20 percent (v/v), time of experiment can vary from 1 day up
to 2 weeks
Protocol
1st Day:
[1729] [1730] 20 ml of culture are transferred into 100 ml
Erlenmeyer flask [1731] taking 1 ml culture for measuring start--OD
(photometer by 750 nm) [1732] adding 1 ml ethanol up to an end
concentration of 5% ethanol in the culture [1733] after 10 minutes
at 5% ethanol end concentration taking 1 ml culture for measuring
of OD [1734] macroscopic observation of the culture by eyes as well
as microscopic analysis (as described above) [1735] adding 1 ml
ethanol up to an end concentration of 10% ethanol in the
culture.
2nd Day:
[1735] [1736] after 24 hours at 10% ethanol taking 1 ml culture for
measuring of OD and microscopic analysis [1737] adding 1 ml ethanol
up to an end concentration of 15% ethanol in the culture
3rd Day:
[1737] [1738] after another 24 hours at 15% ethanol taking 1 ml
culture for measuring of OD and microscopic analysis [1739] adding
1 ml ethanol up to an end concentration of 20% ethanol in the
culture [1740] after 2 hours at 20% ethanol taking 1 ml culture for
measuring of OD and microscopic analysis
[1741] If the OD.sub.750 was reduced>50% or if >50% of cells
bleached or lysed (LM-microscope) at a certain ethanol
concentration, the culture has failed the respective ethanol
concentration. The result is given as the highest EtOH
concentration that was passed by the strain.
Recultivation:
[1742] 20% ethanol cultures are transferred into a 50 ml
Falcon-tube and harvested by centrifugation for 10 minutes at 3.000
rpm (about 3.000 to 4.000 g) [1743] if strains are
self-sedimenting, ethanol containing media is removed after
self-sedimentation of cells [1744] cell pellets are resuspended in
20 ml fresh media and transferred into 100 ml flasks [1745] 1 ml is
taken for measuring OD [1746] cultures are cultivated for 72 hours
and OD was measured again after 24, 48 and 72 hours
respectively.
[1747] A photoautotrophic strains was found to be recultivable in
the case that the optical density is rising in the 72 h after the
cells were resuspended in fresh medium without ethanol after the
short term ethanol test.
Exact Ethanol Tolerance Test
Method
[1748] testing of all strains for tolerance against ethanol by
continuous and fast Increasing of ethanol concentration up to a
concentration of 20% (v/v) [1749] measurement of optical densities
at certain points as well as microscopic analyses using a
light/fluoresce microscope containing percentage estimation of
ratio of living to death cells (using the red auto fluorescence of
chlorophyll and actual conditions of cells like e.g. green colored
or bleached) [1750] end concentrations of ethanol in the test can
vary from 2 up to 20 percent, time of experiment can vary from 6
hours up to 2 days Mic=abbreviation for microscopic analysis
1st Day:
[1750] [1751] 650 ml of culture were transferred into a 2 l
Erlenmeyer flask [1752] taking 1 ml culture for measuring of
start--OD (photometer at 750 nm) and Mic as well as 50 ml for
pyruvate determination and 50 ml for recultivation at the end of
experiment [1753] start of adding ethanol with MS-pumps to an end
concentration of 10% ethanol in the culture after 18 h
2nd Day:
[1754] after 18 h when 10% were reached, taking 1 ml culture for
measuring of OD and Mic, 50 ml for recultivation after 2 h and 50
ml for recultivation at the end of experiment
[1755] after 20 h recultivation of the 50 ml sample taken at 10%
EtOH concentration
[1756] after 205 h when 15% were reached, taking 1 ml culture for
measuring of OD and Mic, 50 ml for recultivation after 2 h and 50
ml for recultivation at the end of experiment
[1757] after 22.5 h recultivation of the 50 ml sample taken at 15%
EtOH concentration
[1758] after 24 h when 20% are reached, taking 1 ml culture for
measuring of OD and Mic, 50 ml for recultivation after 2 h and 50
ml for pyruvate determination
[1759] after 26 h recultivation of the 50 ml sample taken at 20%
EtOH concentration
Long Term Ethanol Tolerance Test
Method
[1760] testing of all strains for tolerance against ethanol in a
long term test whereas the ethanol concentration is 0.2%, 0.5%, 1%
or 5%
[1761] measurement of optical densities at certain points as well
as microscopic analyses using a light/fluorescence microscope
containing percentage estimation of ratio of living to death cells
(using the red auto fluorescence of chlorophyll and actual
conditions of cells like e.g. green colored or bleached).
Protocol
[1762] 20 ml of culture are transferred into two 100 ml Erlenmeyer
flasks, [1763] taking 1 ml culture for measuring start--OD
(Spectrophotometer at 750 nm) [1764] adding ethanol up to an end
concentration of e.g. 1% and 5% ethanol in the culture [1765] daily
taking 1 ml culture for measuring of OD and analysis of the cells
under fluorescence microscope [1766] to keep the ethanol
concentration constant, twice a week the ethanol concentration is
analyzed and evaporation of ethanol is compensated by adding
ethanol in appropriate volume [1767] the evaporation of water is
compensated by adding corresponding amounts of sterile water
whenever necessary [1768] The experiment is running as long as the
culture is alive or growing and the result is documented in a
growth curve (optical density versus time). Microscopic
observations are noted. [1769] A growth rate as well as the highest
possible cell density can be determined also via determination of
dry cell mass, determination of biovolume, counting of cell numbers
beside the determination of optical density.
[1770] The long term ethanol tolerance experiment is ended when
more than 50% of the cells as determined by light microscopy are
bleached or lysed. A particular photoautotrophic strain is
considered to have passed the long term ethanol tolerance test if
it survived at least for 5 weeks with an ethanol concentration of
1% (v/v) in the growth medium.
[1771] Thermo Tolerance and Mechanical Stress Tolerance Test
Method
[1772] testing of all strains against higher temperature and
mechanical stress tolerance
Protocol
[1773] 40 ml of culture are transferred into 100 ml Erlenmeyer
flasks
[1774] for every culture 3 parallels at the same light conditions
e.g. of 40 .mu.E/m2*s are observed [1775] Blind culture: 28.degree.
C. on shaker [1776] Thermo stress: 45.degree. C. on shaker [1777]
Mechanical stress: magnetic stirrer in culture flask under highest
rotations (5.000 rpm; max. speed)
[1778] 1 ml sample is taken after 48 and 96 hours for measuring
OD
[1779] these samples are also microscopically checked and
observations are noticed
[1780] at the end of experiment macroscopic photos were taken
[1781] in case of non-unicellular cultures also microscopic photos
were taken take samples after 48 and 96 h for OD.sub.750 nm and
LM-microscopic analysis
[1782] compare growth of control culture to that of stressed
cultures and evaluate the results as follows:
[1783] positive=same or faster growth of the stressed culture than
growth of the control culture;
[1784] positive/negative results or indefinite results=slower
growth of the stressed culture than control
[1785] negative result=death of the stressed culture (within these
4 days).
Test for Growth in Salty Medium
[1786] Freshwater strains are investigated for their ability to
grow in marine medium.
[1787] dilute 25 ml BG11-grown cultures with 25 ml salty medium,
resulting in a 0.5 (salty medium for an initial adaptation of cells
to increased salt levels [1788] grow cells in 0.5.times. salty
medium for one week [1789] wash and cultivate cells in 1.times.
salty medium (start--OD.sub.750 nm e.g. Synechocystis 1, 5-2)
[1790] parallel growth of the same culture in freshwater medium
(same start--OD.sub.750 nm) for control [1791] cultivate cells for
4 weeks; sampling two times a week OD.sub.750 nm and chlorophyll
content of cells [1792] comparison of both growth curves of the
stressed culture in salty medium and the control culture for
analysis compare growth of control culture to that of stressed
cultures and evaluate the results as follows: [1793] positive=same
or faster growth of the stressed culture than growth of the control
culture; [1794] positive/negative results or indefinite
results-slower growth of the stressed culture than control [1795]
negative result-death of the stressed culture.
[1796] Salty medium can be prepared by mixing half of the
ingredients of the BG-11 medium (see above) with 1 liter of
artificial seawater and adding the trace element mix for BG-11 (see
above).
[1797] Recipe for 1 l of artificial seawater:
[1798] Recipe for 1 l artificial seawater (28 ppm):
TABLE-US-00066 NaCl 28.05 g MgSO.sub.4 6.90 g MgCl.sub.2 5.40 g KCl
0.67 g CaCl.sub.2 1.47 g
HPLC Analysis for Natural Product Content
Protocol for Natural Product Extraction and Sample Preparation for
HPLC/MS Analysis
[1799] 50 ml of cell culture (optical density around 1) is
centrifuged, supernatant is discarded
[1800] cell pellet is resuspended in 2 ml 50% methanol and cells
are broken by ultrasonic bar (ultrasonic treatment for 30 seconds
at max. intensity, three times repeated)
[1801] extract is centrifuged (6000 rpm=about 6.000 g, 10 min,
4.degree. C.), supernatant is transferred into a new tube
[1802] cell pellet is resuspended in 2 ml 50% methanol and cells
are broken by ultrasonic bar as before
[1803] extract is centrifuged (6000 rpm, 10 min, 4.degree. C.),
supernatant is united with the first one
[1804] cell pellet is resuspended in 2 ml 80% methanol and cells
are broken by ultrasonic bar as before
[1805] extract is centrifuged (6000 rpm, 10 min, 4.degree. C.),
supernatant united with the other ones
[1806] cell pellet is resuspended in 2 ml 80% methanol and cells
are broken by ultrasonic bar as before
[1807] extract is centrifuged (6000 rpm, 10 min, 4.degree. C.),
supernatant united with the other ones
[1808] drying the supernatant in a vacuum rotator until pellet is
dry
[1809] the pellet is resuspend in 1.6 ml 20% methanol, centrifuged
at 4.degree. C., 13000 rpm (about 15.000 g) and filtered (0.45
.mu.m CA membrane)
[1810] HPLC/MS analysis (Detector ELSD, PDA, MS)
Exact Growth Test
Method
[1811] determination of growth speed and max. optical density
[1812] stepwise increase of light intensity and CO.sub.2
supplementation
[1813] samples are taken for analysis of metabolites at certain
growth phases
Protocol
[1814] 500 ml of culture in 11 culture vessel:
TABLE-US-00067 height about 9 cm diameter about 11 cm volume of
vessel 1 l used volume 500 ml
[1815] light conditions starts with 40 pE/m.sup.2*s; culture
conditions of 30.degree. C. or 21.degree. C. respectively. CO.sub.2
concentration starts with 2%
[1816] when growth becomes stationary light conditions are
increased in 2 steps (120 pE/m.sup.2*s and 220 pE/m.sup.2*s)
[1817] daily taking samples of 1 ml for OD and microscopic
observations, the evaporation is compensated by adding sterile
water whenever necessary
[1818] a growth curve is drawn and the growth rate can be
calculated (optical density versus time)
[1819] start optical density is about 0.2.
[1820] a growth rate as well as the highest possible cell density
can be determined also via determination of dry cell mass,
determination of biovolume, counting of cell numbers beside the
determination of optical density.
Initial Growth Test
Method/Protocol
[1821] Testing of all strains for growth in microtiter plates on a
rotary shaker (between 6 or 96 well plates, preferred are 6 to 24
well plates due to the larger volume)
[1822] Measurement of optical density using a photometer plate
reader at 750 nm.
[1823] growth rate can be determined also via determination of dry
cell mass (only for 6 and 12 well plates), determination of
biovolume, counting of cell numbers beside the determination of
optical density.
[1824] strains which need more than 48 hours for doubling fall,
others go to the next test
Test for Photosynthetic Activity
Measurement of Oxygen Generation of Strains in Different Growth
Phases (Lag Phase, Log Phase, Stationary Phase) Using an Oxygen
Electrode (Clark-Type Electrode):
[1825] Measurement of chlorophyll content of the cyanobacterial
culture according to N. Tandeau De Marsac and J. Houmard (in:
Methods in Enzymology, Vol. 169, 318-328. L. Packer, ed., Academic
Press, 1988)
[1826] Centrifugation of the culture and resuspension of the cells
in fresh BG11-Medium adjusting a chlorophyll concentration of about
10 .mu.g/m
[1827] Addition of 25 mM NaHCO.sub.3 as carbon source
[1828] Cultures are then filed into a 2.5 ml cuvette with an
integrated Clark-electrode (oxygen electrode).
[1829] Measurement of oxygen generation using light saturated
conditions (about 500 .mu.E/m.sup.2.times.s) at 25.degree. C. over
the time and record of data using a chart recorder.
[1830] Calculation of oxygen generation using the following
formula: oxygen rate in .mu.mol O.sub.2 per h and .mu.g
chlorophyll=.DELTA..units of measurement.times.0.253 .mu.mol per
ml.times.60/chlorophyll concentration in .mu.g per
ml.times..DELTA..units of calibration.DELTA.t of measurement in
min.
[1831] The .DELTA.units are recorded by the chart recorder. The
.DELTA.units of calibration are determined by measuring the
amplitude of difference of a O.sub.2 saturated water solution and a
water solution without any O.sub.2 after adding of Sodium
dithionite (zero point). That means .DELTA.units of calibration
correspond directly to the oxygen concentration of air-saturated
water at 25.degree. C. of 0.253 .mu.mol per ml.
[1832] A photoautotrophic strain passes this test if a
photosynthetic oxygen evolution of at least 150 .mu.mol
O.sub.2/h*mg chl can be detected.
Photometric Quantification of Chlorophyll in Cyanobacterial
Cultures
Chemicals and Solutions:
[1833] 100% methanol (4.degree. C.)
Principle of the Method:
[1834] Cyanobacterial cells are extracted with methanol (90% v/v).
The chlorophyll content in the extract is measured
spectrophotometrically.
Method:
[1835] Batches of cyanobacteria cultures are centrifuged. The
pellets are resuspended in 90% methanol, for example by leaving 100
.mu.l of the supernatant and addition of 900 .mu.l of 100%
methanol. After resuspension and incubation (at 4.degree. C., dim
light, at least 1 hour) the sample is centrifuged and the
absorbance of the supernatant is measured at 665 nm against
methanol. The chlorophyll content of the methanol extract is
calculated using equation:
A.sub.665.times.13.9=chlorophyll [.mu.g/ml]
[1836] For the calculation of the chlorophyll content of the
cyanobacteria culture the dilution factor has to be considered.
[1837] Using the above mentioned methods for testing the
photoautotrophic strains, inter alia the following strains from the
public databases Pasteur culture collection (PCC) or the Gottinger
Algensmmlung (SAG) have been identified, which are prime candidates
for genetic modification due to their positive behavior during the
above screening procedures:
[1838] SAG 37.79, PCC 7715, Calothrix thermalis
[1839] PCC 8937, Lyngbya sp.
[1840] SAG 12.89, Phormidium africanum
[1841] PCC 7321, Pkeurocapsa sp.
[1842] PCC 6715, Synechococcus sp.
[1843] In particular these strains performed during the screening
procedures as indicated in the below table:
TABLE-US-00068 Initial Exact Mechanical ethanol ethanol- Thermo
stress tolerance tolerance tolerance tolerance Strain test test
Recultivation test test SAG 12.89 up to up to up to 15% pos. pos.
20% 20% PCC 8937 up to up to pos. pos. 20% 20% PCC 7321 up to up to
up to 10% pos./neg. pos. 20% 20% (2 h) SAG 37.79 up to neg. pos.
20% PCC 6715 up to up to up to 0% pos. pos. 20% 20%
[1844] Further examples of photoautotrophic strains which passed
and failed the screening test are shown in FIG. 50-13.
[1845] Detailed description of embodiments related to adding a
substrate to the growth medium of a growing culture, which is used
by the at least one overexpressed enzyme for ethanol formation to
produce ethanol:
Effect of Acetaldehyde on Ethanol Production by Cyanobacteria
[1846] Background: The bottle neck of the ethanol formation in the
metabolism of our transgenic cyanobacteria has not been detected.
Addition of pyruvate and 3-PGA to cyanobacteria expressing Pdc and
Adh did not result in an increased ethanol production, but
according to our experiments this metabolites of glycolysis were
not absorbed by the cells. We now performed feeding experiments
with acetaldehyde. The goal was to elucidate whether the ethanol
production is limited solely by this immediate ethanol precursor,
or by other factors, i.e. the availability of reduced co-substrates
(NADH and/or NADPH).
[1847] Methods: Synechocystis PCC 6803 wild type and the transgenic
strain "6803pVZ-PisiA", corresponding to the above described
Synechocystis pVZ-PisiA-Pdc-AdhII, were washed twice with BG11
(centrifugation 15 min, 4500 rpm, 4.degree. C.; Rotina 420R,
Hettich) and re-dissolved in BG11. Aliquots of 2 ml were spiked
with acetaldehyde. The assays were incubated at room temperature
under Illumination. Samples of 250 .mu.l were removed in defined
time intervals (5 min or 10 min) and centrifuged (3 min, 14000 rpm,
room temperature, Micro 200R, Hettich). The supernatants were
stored at -70.degree. C., subsequently the ethanol content was
measured.
[1848] Ethanol was quantified with a described protocol. The method
is based on oxidation of ethanol catalyzed by alcohol dehydrogenese
(Sigma, Adh of S. cerevisiae). NADH formed in this reaction, reacts
with the PMS/MTT reagent to a dye. Its absorption (measured at 580
nm) is proportionate to the ethanol content of a sample.
Principle of Ethanol Quantification:
[1849] Ethanol is oxidized by nicotinamide-adenine dinucleotide
(NAD.sup.+) to acetaldehyde in a reaction, which is catalyzed by
the enzyme alcohol dehydrogenase (ADH) (reaction 1). The
acetaldehyde, which is formed in the reaction, is quantitatively
oxidized to acetic acid by the enzyme aldehyde dehydrogenase
(AI-DH) (reaction 2).
##STR00003##
[1850] In reactions (1) and (2) reduced nicotinamide-adenine
dinucleotide (NADH) is formed. The amount of NADH formed is
proportionate to the amount of ethanol in the sample. NADH is
easily quantified by means of its light absorbance. The absorbance
is usually measured at 340 nm, Hg 365 nm or Hg 334 nm.
Procedure:
[1851] Preparation of solutions: Solution 1: 1.3 mg/ml NAD and 0.27
U aldehyde dehydrogenase in potassium diphosphate buffer, pH 9.0.
Solution 2: Suspension of alcohol dehydrogenase (ADH) with approx.
4000 U/mind. Alternatively, the chemicals and solutions of the
ethanol determination kit of Boehringer Mannheim/R-Biopharm (Cat.
No. 10 176 290 035) can be used. Sample and solution 1 are mixed in
a ratio of 3 ml solution 1 and 0.1 ml sample (If necessary the
sample is diluted with water). After approx. 3 min the absorbance
is measured (A.sub.1). The reaction is then started by the addition
of ADH suspension (solution 2, 0.050 ml for 3 ml solution 1 and 0.1
ml sample). After completion of the reaction (approx. 5 to 10 min)
the absorbance is measured again (A.sub.1). The absorption
measurements can be performed using a photometer or a microplate
reader. For plate reader measurements all volumes are
downscaled.
[1852] From the measured absorbance difference
.DELTA.A=(A.sub.2-A.sub.1) the ethanol concentration in the sample
is calculated with the equation:
c = V .times. MG .times. d .times. v .times. 2 .times. 1000 .times.
.DELTA. A ##EQU00002##
c, ethanol concentration [g/L]; V, total volume [mL]; MG, molecular
weight of ethanol (46.07 g/mol); e, extinction coefficient (6.3
L.times.mmol.sup.-1.times.cm.sup.-1 at 340 nm); d, light path [cm];
v, sample volume [mL]
Literature:
[1853] Protocol of the kit Ethanol, UV method for the determination
of ethanol in foodstuff and other materials, Cat. No. 10176290035,
R-Biopharm AG, Darmstadt, Germany.
[1854] H.-O. Beutler (1984) in: Methods in Enzymatic Analysis
(Bergmeyer, H. U. ed.) 3.sup.rd ed. Vol. VI, pp. 598-606, Verlag
Chemie, Weinheim, Germany.
[1855] Acetaldehyde was quantified by a modification of the
protocol of a kit for ethanol quantification (Ethanol kit.
R-Biopharm AG). Acetaldehyde is converted by aldehyde dehydrogenase
under formation of NADH, which is quantified by its absorption at
340 nm. The amount is proportionate to the acetaldehyde content of
the sample.
[1856] For preparation of crude extracts, cells were harvested,
washed with 40 mM MES/Tris (pH 6.5), 1 mM DTT and broken
(beadbeater, 2.times.10 min). The supernatant of a centrifugation
(15 min, 14000 rpm, 4.degree. C., Micro 200R, Hettich) was used for
the determination of Adh activity in cells.
[1857] Assays for measurement of the Adh activity in the direction
of ethanol formation contained in a total volume of 800 .mu.l 40 mM
MES adjusted with Tris base to pH 6.5, 1 mM DTT, different
concentrations of acetaldehyde, 50 .mu.l crude extract and 0.3 M
NADH. The initial velocity was calculated from the dE/min at 340
nm.
[1858] Results: Addition of acetaldehyde to final concentrations in
the range of 6.6 .mu.M to 200 .mu.M resulted in an increase of
ethanol in the medium of cultures of the transgenic strain
6803-pVZ-PisiA. The rates of ethanol production per minute were
linear at the beginning of the experiment (for at least 30 min),
but finally decelerated, obviously because of the expiration of the
supply of acetaldehyde (FIG. 51A).
[1859] In FIG. 51A, ethanol production is measured after addition
of acetaldehyde Different concentrations were added to a culture of
strain 6803pVZPisiA and the ethanol content in the medium was
measured for 60 minutes.
[1860] A plot of the initial velocity of the ethanol production
versus the substrate concentration resulted in a graph similar to
the substrate saturation curves of enzymes with Michaelis-Menten
kinetics (FIG. 51B). K.sub.m and V.sub.max were calculated from a
"Lineweaver-Burk" plot (1/v versus 1/[S] FIG. 6) with K.sub.m for
acetaldehyde-18 .mu.M and Vmax-3.2 .mu.Mol L.sup.-1 min.sup.-1.
OD.sub.750 of the culture was 0.56.
[1861] FIG. 51B presents a correlation of ethanol production rate
and acetaldehyde concentration. Given are the initial ethanol rates
(calculated with FIG. 4) in correlation to the initial acetaldehyde
concentrations.
[1862] FIG. 51C presents a Lineweaver-Burk-Plot. Reciprocal of the
initial velocity versus the reciprocal of the acetaldehyde
concentration. Intact cells were used.
[1863] This experiment was repeated with a different culture of
strain 6803-pVZ-PisiA-PDC/ADHII of OD.sub.750 of 1.353 and a
chlorophyll concentration of 4.6 .mu.g/ml. Similar results were
obtained. The K.sub.m for acetaldehyde was calculated with 25 .mu.M
(FIG. 51D). V.sub.max was 4.35 .mu.Mol L.sup.-1 min.sup.-1, or 0.95
.mu.Mol L.sup.-1 mg.sup.-1 using chlorophyll as reference.
[1864] FIG. 51D presents a Lineweaver-Burk-Plot in which the
reciprocal of the initial velocity versus the reciprocal of the
acetaldehyde concentration. The results shown are from a repeat of
the experiment with intact cells summarized in FIGS. 51A to
51C.
[1865] In order to compare the dates acquired with intact cells,
the kinetic constants of alcohol dehydrogenase in crude extracts of
strain 6803-pVZ-PisiA-PDC/ADHII were measured. The measurements
were carried out in the direction of ethanol formation at pH 6.5,
following a protocol in the literature. A graphical representation
of the results obtained is given in form of a "Lineweaver-Burk"
plot (FIG. 8). The K.sub.m for acetaldehyde was calculated with 45
.mu.M and V.sub.max was 7.2 .mu.Mol L.sup.-1 mg.sup.-1
chlorophyll.
[1866] In a second experiment the Adh activity was measured at pH
7.5. NADH and NADPH were used as co-substrates. Activity was not
significantly different for NADH and NADPH in the concentrations
used (NADH 0.25 M, NADPH 0.21 M final concentration). The V.sub.max
was calculated with 0.89 .mu.Mol L.sup.-1 mg.sup.-1 chlorophyll,
the K.sub.m for acetaldehyde was determined in this experiment with
100 .mu.M (FIG. 9).
[1867] FIG. 51E presents a Lineweaver-Burk-Plot in which Adh
activities of a crude extract of strain 6803pVZ-PisiA-PDC/ADHII
were measured in presence of different concentration of
acetaldehyde. In contrast to the experiments with intact cells in
this experiment NADH was added in excess. Shown is the reciprocal
of the initial velocity versus reciprocal of the concentration of
acetaldehyde.
[1868] FIG. 51F is a Lineweaver-Burk-Plot Similar to the experiment
summarized in FIG. 51E Adh activities of a crude extract of strain
6803PVZPisiA were measured in the presence of different
concentrations of acetaldehyde. The assays contained an over excess
either of NADH or of NADPH. Substantial differences between NADH
(squares) and NADPH (diamonds) were not observed.
[1869] Summary: Acetaldehyde added to the medium is absorbed and
converted into ethanol by intact cells. The K.sub.m for
acetaldehyde of the entire process of uptake and ethanol formation
was determined with approx. 20 to 25 .mu.M. This value is similar
to the K.sub.m for acetaldehyde of the purified AHDII of Z.
mobilis, measured at pH 6.5. The correlation of the rate of ethanol
formation and the acetaldehyde concentration clearly shows that the
ethanol formation is to a larger extent limited by the availability
acetaldehyde. Maximum ethanol formation rates were obtained with
200 .mu.M acetaldehyde. When acetaldehyde was added in significant
higher concentration, we tested the range of 1 mM to 10 mM, a
decrease of ethanol formation was observed. It is assumed, that the
acetaldehyde, which is very reactive, is in higher concentrations
rapidly poisoning the cells.
[1870] The scope of protection of the invention is not limited to
the examples given hereinabove. The invention is embodied in each
novel characteristic and each combination of characteristics, which
particularly includes every combination of any features which are
stated in the claims, even if this feature or this combination of
features is not explicitly stated in the claims or in the examples.
Sequence CWU 1
1
2361477PRTSynechocystis sp. PCC 6803 1Met Lys Ile Leu Phe Val Ala
Ala Glu Val Ser Pro Leu Ala Lys Val 1 5 10 15 Gly Gly Met Gly Asp
Val Val Gly Ser Leu Pro Lys Val Leu His Gln 20 25 30 Leu Gly His
Asp Val Arg Val Phe Met Pro Tyr Tyr Gly Phe Ile Gly 35 40 45 Asp
Lys Ile Asp Val Pro Lys Glu Pro Val Trp Lys Gly Glu Ala Met 50 55
60 Phe Gln Gln Phe Ala Val Tyr Gln Ser Tyr Leu Pro Asp Thr Lys Ile
65 70 75 80 Pro Leu Tyr Leu Phe Gly His Pro Ala Phe Asp Ser Arg Arg
Ile Tyr 85 90 95 Gly Gly Asp Asp Glu Ala Trp Arg Phe Thr Phe Phe
Ser Asn Gly Ala 100 105 110 Ala Glu Phe Ala Trp Asn His Trp Lys Pro
Glu Ile Ile His Cys His 115 120 125 Asp Trp His Thr Gly Met Ile Pro
Val Trp Met His Gln Ser Pro Asp 130 135 140 Ile Ala Thr Val Phe Thr
Ile His Asn Leu Ala Tyr Gln Gly Pro Trp 145 150 155 160 Arg Gly Leu
Leu Glu Thr Met Thr Trp Cys Pro Trp Tyr Met Gln Gly 165 170 175 Asp
Asn Val Met Ala Ala Ala Ile Gln Phe Ala Asn Arg Val Thr Thr 180 185
190 Val Ser Pro Thr Tyr Ala Gln Gln Ile Gln Thr Pro Ala Tyr Gly Glu
195 200 205 Lys Leu Glu Gly Leu Leu Ser Tyr Leu Ser Gly Asn Leu Val
Gly Ile 210 215 220 Leu Asn Gly Ile Asp Thr Glu Ile Tyr Asn Pro Ala
Glu Asp Arg Phe 225 230 235 240 Ile Ser Asn Val Phe Asp Ala Asp Ser
Leu Asp Lys Arg Val Lys Asn 245 250 255 Lys Ile Ala Ile Gln Glu Glu
Thr Gly Leu Glu Ile Asn Arg Asn Ala 260 265 270 Met Val Val Gly Ile
Val Ala Arg Leu Val Glu Gln Lys Gly Ile Asp 275 280 285 Leu Val Ile
Gln Ile Leu Asp Arg Phe Met Ser Tyr Thr Asp Ser Gln 290 295 300 Leu
Ile Ile Leu Gly Thr Gly Asp Arg His Tyr Glu Thr Gln Leu Trp 305 310
315 320 Gln Met Ala Ser Arg Phe Pro Gly Arg Met Ala Val Gln Leu Leu
His 325 330 335 Asn Asp Ala Leu Ser Arg Arg Val Tyr Ala Gly Ala Asp
Val Phe Leu 340 345 350 Met Pro Ser Arg Phe Glu Pro Cys Gly Leu Ser
Gln Leu Met Ala Met 355 360 365 Arg Tyr Gly Cys Ile Pro Ile Val Arg
Arg Thr Gly Gly Leu Val Asp 370 375 380 Thr Val Ser Phe Tyr Asp Pro
Ile Asn Glu Ala Gly Thr Gly Tyr Cys 385 390 395 400 Phe Asp Arg Tyr
Glu Pro Leu Asp Cys Phe Thr Ala Met Val Arg Ala 405 410 415 Trp Glu
Gly Phe Arg Phe Lys Ala Asp Trp Gln Lys Leu Gln Gln Arg 420 425 430
Ala Met Arg Ala Asp Phe Ser Trp Tyr Arg Ser Ala Gly Glu Tyr Ile 435
440 445 Lys Val Tyr Lys Gly Val Val Gly Lys Pro Glu Glu Leu Ser Pro
Met 450 455 460 Glu Glu Glu Lys Ile Ala Glu Leu Thr Ala Ser Tyr Arg
465 470 475 2491PRTSynechocystis sp. PCC 6803 2Met Tyr Ile Val Gln
Ile Ala Ser Glu Cys Ala Pro Val Ile Lys Ala 1 5 10 15 Gly Gly Leu
Gly Asp Val Ile Tyr Gly Leu Ser Arg Glu Leu Glu Leu 20 25 30 Arg
Gly His Cys Val Glu Leu Ile Leu Pro Met Tyr Asp Cys Met Arg 35 40
45 Tyr Asp His Ile Trp Gly Leu His Asp Ala Tyr Arg Asn Leu Glu Val
50 55 60 Pro Trp Tyr Gly Ser Ser Ile Phe Cys Asp Val Phe Cys Gly
Trp Val 65 70 75 80 His Gly Arg Leu Cys Phe Phe Ile Gln Pro Lys Ser
Ser Asp Asn Phe 85 90 95 Phe Asn Arg Gly His Tyr Tyr Gly Ala Leu
Asp Asp His Met Arg Phe 100 105 110 Ala Phe Phe Ser Lys Ala Ala Met
Glu Phe Leu Leu Arg Ser Asn Lys 115 120 125 Arg Pro Asp Ile Ile His
Cys His Asp Trp Gln Thr Gly Leu Val Pro 130 135 140 Val Leu Leu Tyr
Glu Ile Tyr Arg Phe His Gly Met Asp His Gln Arg 145 150 155 160 Val
Cys Tyr Thr Ile His Asn Phe Lys His Gln Gly Ile Ala Gly Ala 165 170
175 Asn Ile Leu His Ala Thr Gly Leu Asn Asn Asp Ser Tyr Tyr Phe Ser
180 185 190 Tyr Asp Arg Leu Gln Asp Asn Phe Asn Pro Asn Ala Ile Asn
Phe Met 195 200 205 Lys Gly Gly Ile Val Tyr Ser Asn Tyr Val Asn Thr
Val Ser Pro His 210 215 220 His Ala Trp Glu Ala Arg Phe Ser Asp Ile
Ser Cys Gly Leu Gly His 225 230 235 240 Thr Leu Glu Ile His Gln Gln
Lys Phe Gly Gly Ile Leu Asn Gly Leu 245 250 255 Asp Tyr Glu Val Trp
Asn Pro Glu Ile Asp Pro Leu Leu Ala Ser Asn 260 265 270 Phe Ser Val
Lys Thr Phe Gly Asp Lys Ala Lys Asn Lys Gln Ala Leu 275 280 285 Arg
Glu Arg Leu Leu Leu Glu Thr Asp Asp Lys Lys Pro Met Leu Cys 290 295
300 Phe Ile Gly Arg Leu Asp Gly Gln Lys Gly Val His Leu Val His His
305 310 315 320 Ser Ile Tyr Tyr Ala Leu Ser Gln Gly Ala Gln Phe Val
Leu Leu Gly 325 330 335 Ser Ala Thr Glu Pro Asn Leu Ser Lys Trp Phe
Trp His Glu Lys Gln 340 345 350 His Leu Asn Asp Asn Pro Asn Val His
Leu Glu Leu Gly Phe Asp Glu 355 360 365 Glu Leu Ala His Leu Ile Tyr
Gly Ala Ala Asp Ile Ile Val Val Pro 370 375 380 Ser Asn Tyr Glu Pro
Cys Gly Leu Thr Gln Met Ile Gly Leu Arg Tyr 385 390 395 400 Gly Ala
Val Pro Val Val Arg Gly Val Gly Gly Leu Val Asn Thr Val 405 410 415
Phe Asp Arg Asp Tyr Asp Gln Asn His Pro Pro Glu Lys Arg Asn Gly 420
425 430 Phe Val Phe Tyr Gln Pro Asp Glu Tyr Ala Leu Glu Thr Ala Leu
Ser 435 440 445 Arg Ala Ile Ala Leu Tyr Lys Asp Asp Pro Val Ala Phe
Lys Thr Leu 450 455 460 Ala Leu Gln Gly Met Ala Tyr Asp Tyr Ser Trp
Asn Lys Pro Gly Leu 465 470 475 480 Gln Tyr Val Glu Ala Tyr Glu Tyr
Ile Arg Ala 485 490 35069DNAartificialconstruct pUC 19-glgA1-Cm
3tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
60cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
120ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta
ctgagagtgc 180accatatgcg gtgtgaaata ccgcacagat gcgtaaggag
aaaataccgc atcaggcgcc 240attcgccatt caggctgcgc aactgttggg
aagggcgatc ggtgcgggcc tcttcgctat 300tacgccagct ggcgaaaggg
ggatgtgctg caaggcgatt aagttgggta acgccagggt 360tttcccagtc
acgacgttgt aaaacgacgg ccagtgaatt cgagctcggt accaactaaa
420gtctgcccgc atggcccgtt gctgtaattt ttgccaatct gccttgaaac
ggaaaccctc 480ccaggcccgc accatggccg taaagcaatc caggggttca
taacggtcaa agcaatagcc 540ggtgccggct tcattgatag gatcgtagaa
ggataccgta tccaccaaac cccctgtccg 600ccgcacaatg gggatacagc
cataacgcat ggccatcaat tgactcagcc cacagggctc 660aaagcgagaa
ggcattaaaa acacatccgc cccggcatag actcgacggg aaagggcatc
720gttgtggagt aattgcaccg ccatccgccc aggaaatcgg gaagccatct
gccaaagttg 780ggtttcgtaa tggcgatcgc cagtgccgag gataattaac
tgggaatcgg tgtaggacat 840gaagcggtca aggatctgaa tcaccaaatc
aatccccttt tgttccacca agcgagccac 900tatacccacg taagaggttc
caactttcac cataatgaaa taagatcact accgggcgta 960ttttttgagt
tatcgagatt ttcaggagct aaggaagcta aaatggagaa aaaaatcact
1020ggatatacca ccgttgatat atcccaatgg catcgtaaag aacattttga
ggcatttcag 1080tcagttgctc aatgtaccta taaccagacc gttcagctgg
atattacggc ctttttaaag 1140accgtaaaga aaaataagca caagttttat
ccggccttta ttcacattct tgcccgcctg 1200atgaatgctc atccggaatt
ccgtatggca atgaaagacg gtgagctggt gatatgggat 1260agtgttcacc
cttgttacac cgttttccat gagcaaactg aaacgttttc atcgctctgg
1320agtgaatacc acgacgattt ccggcagttt ctacacatat attcgcaaga
tgtggcgtgt 1380tacggtgaaa acctggccta tttccctaaa gggtttattg
agaatatgtt tttcgtctca 1440gccaatccct gggtgagttt caccagtttt
gatttaaacg tggccaatat ggacaacttc 1500ttcgcccccg ttttcaccat
gggcaaatat tatacgcaag gcgacaaggt gctgatgccg 1560ctggcgattc
aggttcatca tgccgtctgt gatggcttcc atgtcggcag aatgcttaat
1620gaattacaac agtactgcga tgagtggcag ggcggggcgt aattttttta
aggcagttat 1680tggtgccctt aaacgcctgg tgctacgcct gaataagtga
taataagcgg atgaatggca 1740gaaattcgaa agcaaattcg acccggtcgt
cggttcaggg cagggtcgtt aaatagccgc 1800ttatgtctat tgctggttta
ccggtttatt gactaccgga agcagtgtga ccgtgtgctt 1860ctcaaatgcc
tgaggccagt ttgctcaggc tctccccgtg gaggtaataa ttgacgatat
1920gatcatttat tctgcctccc agagcctgat aaaaacggtt agcgcttcgt
taatacagat 1980gtaggtgttc cacagggtag ccagcagcat cctgcgatgc
atggcattac gattaatttc 2040taaccccgtt tcctcctgga tggcaatttt
atttttcacc cgcttgtcca aactgtccgc 2100atcgaaaaca ttgctgataa
agcggtcttc cgccgggttg taaatctccg tatcaatacc 2160gttgagaata
ccgactaaat taccactcag gtaggacaat aacccttcca gcttttcccc
2220ataggccggg gtttggatct gttgggcata ggtgggagaa acggtagtca
cccgattggc 2280aaattgaatc gccgccgcca tcacattgtc tccctgcatg
taccaaggac accaagtcat 2340agtttcaagc aagccccgcc agggcccttg
gtaagcaaga ttatggatgg tgaaaacggt 2400ggcgatgtct ggggactgat
gcatccaaac agggatcatg ccagtgtgcc aatcatggca 2460atggataatt
tccggcttcc aatggttcca ggcaaattca gctgccccgt tagaaaaaaa
2520agtgaaccgc cacgcctcgt catctccgcc atagatcctt cgggagtcga
aagctggatg 2580gccgaacaag tagagaggaa ttttggtgtc cggtagatag
gactggtaaa cagcaaactg 2640ctggaacatg gcttcccctt tccagaccgg
ctccttgggc acatcaatct tgtcgccgat 2700gaaaccgtag tagggcatga
agacacggac atcatggccc aactgatgca gaactttagg 2760cagggaaccc
accacatccc ccatgccacc tacctttgct aggggggata cttccgccgc
2820cacaaataaa agcttggcgt aatcatggtc atagctgttt cctgtgtgaa
attgttatcc 2880gctcacaatt ccacacaaca tacgagccgg aagcataaag
tgtaaagcct ggggtgccta 2940atgagtgagc taactcacat taattgcgtt
gcgctcactg cccgctttcc agtcgggaaa 3000cctgtcgtgc cagctgcatt
aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat 3060tgggcgctct
tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
3120agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
gggataacgc 3180aggaaagaac atgtgagcaa aaggccagca aaaggccagg
aaccgtaaaa aggccgcgtt 3240gctggcgttt ttccataggc tccgcccccc
tgacgagcat cacaaaaatc gacgctcaag 3300tcagaggtgg cgaaacccga
caggactata aagataccag gcgtttcccc ctggaagctc 3360cctcgtgcgc
tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
3420ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
cggtgtaggt 3480cgttcgctcc aagctgggct gtgtgcacga accccccgtt
cagcccgacc gctgcgcctt 3540atccggtaac tatcgtcttg agtccaaccc
ggtaagacac gacttatcgc cactggcagc 3600agccactggt aacaggatta
gcagagcgag gtatgtaggc ggtgctacag agttcttgaa 3660gtggtggcct
aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa
3720gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
ccaccgctgg 3780tagcggtggt ttttttgttt gcaagcagca gattacgcgc
agaaaaaaag gatctcaaga 3840agatcctttg atcttttcta cggggtctga
cgctcagtgg aacgaaaact cacgttaagg 3900gattttggtc atgagattat
caaaaaggat cttcacctag atccttttaa attaaaaatg 3960aagttttaaa
tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
4020aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
ttgcctgact 4080ccccgtcgtg tagataacta cgatacggga gggcttacca
tctggcccca gtgctgcaat 4140gataccgcga gacccacgct caccggctcc
agatttatca gcaataaacc agccagccgg 4200aagggccgag cgcagaagtg
gtcctgcaac tttatccgcc tccatccagt ctattaattg 4260ttgccgggaa
gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
4320tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
gctccggttc 4380ccaacgatca aggcgagtta catgatcccc catgttgtgc
aaaaaagcgg ttagctcctt 4440cggtcctccg atcgttgtca gaagtaagtt
ggccgcagtg ttatcactca tggttatggc 4500agcactgcat aattctctta
ctgtcatgcc atccgtaaga tgcttttctg tgactggtga 4560gtactcaacc
aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
4620gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
tcattggaaa 4680acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
ttgagatcca gttcgatgta 4740acccactcgt gcacccaact gatcttcagc
atcttttact ttcaccagcg tttctgggtg 4800agcaaaaaca ggaaggcaaa
atgccgcaaa aaagggaata agggcgacac ggaaatgttg 4860aatactcata
ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
4920gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
cgcgcacatt 4980tccccgaaaa gtgccacctg acgtctaaga aaccattatt
atcatgacat taacctataa 5040aaataggcgt atcacgaggc cctttcgtc
506945533DNAartificialconstruct pUC 19-glgA2-Kan 4tcgcgcgttt
cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60cagcttgtct
gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
120ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta
ctgagagtgc 180accatatgcg gtgtgaaata ccgcacagat gcgtaaggag
aaaataccgc atcaggcgcc 240attcgccatt caggctgcgc aactgttggg
aagggcgatc ggtgcgggcc tcttcgctat 300tacgccagct ggcgaaaggg
ggatgtgctg caaggcgatt aagttgggta acgccagggt 360tttcccagtc
acgacgttgt aaaacgacgg ccagtgaatt ctcctccagt gcttgcaagg
420gaggggcaat ataggaaaat acaatcaact cgatcgccgt cgagccgaag
tcgagtaaaa 480accgctatca ggagcctcta tgtacatcgt tcaaattgcc
tcagaatgcg cccccgtcat 540taaggctggg ggattggggg atgttattta
cggcctaagc cgtgaattgg aactgcgggg 600ccattgcgtc gagctaatcc
tacccatgta cgattgcatg cgctatgacc acatctgggg 660tttacacgat
gcttaccgca acctagaggt gccctggtat ggaagctcaa tcttctgtga
720tgttttttgt ggctgggttc acggtaggct ctgcttcttc attcagccca
aatcttctga 780taactttttc aatcggggtc attattatgg cgctctagac
gaccatatgc gctttgcctt 840tttctccaag gcggccatgg agtttttgct
acgcagtaac aaacgcccag acattatcca 900ctgccacgat tggcaaacgg
gactggtgcc ggtgttgttg tatgaaattt accgtttcca 960tggcatggac
catcaacggg tttgttacac catccacaat ttcaaacacc agggtattgc
1020tggagccaat attctccacg ccactgggct caataatgac agttattatt
tcagctacga 1080tcgcctgcag gataatttca atcccaatgc gattaacttc
atgaaggggg gcattgtcga 1140cctgcagggg ggggggggaa agccacgttg
tgtctcaaaa tctctgatgt tacattgcac 1200aagataaaaa tatatcatca
tgaacaataa aactgtctgc ttacataaac agtaatacaa 1260ggggtgttat
gagccatatt caacgggaaa cgtcttgctc gaggccgcga ttaaattcca
1320acatggatgc tgatttatat gggtataaat gggctcgcga taatgtcggg
caatcaggtg 1380cgacaatcta tcgattgtat gggaagcccg atgcgccaga
gttgtttctg aaacatggca 1440aaggtagcgt tgccaatgat gttacagatg
agatggtcag actaaactgg ctgacggaat 1500ttatgcctct tccgaccatc
aagcatttta tccgtactcc tgatgatgca tggttactca 1560ccactgcgat
ccccgggaaa acagcattcc aggtattaga agaatatcct gattcaggtg
1620aaaatattgt tgatgcgctg gcagtgttcc tgcgccggtt gcattcgatt
cctgtttgta 1680attgtccttt taacagcgat cgcgtatttc gtctcgctca
ggcgcaatca cgaatgaata 1740acggtttggt tgatgcgagt gattttgatg
acgagcgtaa tggctggcct gttgaacaag 1800tctggaaaga aatgcataag
cttttgccat tctcaccgga ttcagtcgtc actcatggtg 1860atttctcact
tgataacctt atttttgacg aggggaaatt aataggttgt attgatgttg
1920gacgagtcgg aatcgcagac cgataccagg atcttgccat cctatggaac
tgcctcggtg 1980agttttctcc ttcattacag aaacggcttt ttcaaaaata
tggtattgat aatcctgata 2040tgaataaatt gcagtttcat ttgatgctcg
atgagttttt ctaatcagaa ttggttaatt 2100ggttgtaaca ctggcagagc
attacgctga cttgacggga cggcggcttt gttgaataaa 2160tcgaactttt
gctgagttga aggatcagat cacgcatctt cccgacaacg cagaccgttc
2220cgtggcaaag caaaagttca aaatcaccaa ctggtccacc tacaacaaag
ctctcatcaa 2280ccgtggctcc ctcactttct ggctggatga tggggcgatt
caggcctggt atgagtcagc 2340aacaccttct tcacgaggca gacctcagcg
cccccccccc cctgcaggtc tactccaact 2400atgtcaacac cgtttccccc
caccatgctt gggaagcccg tttttccgat atttcctgtg 2460gcttgggcca
taccctggaa atccatcagc aaaaattcgg cggtattttg aacggtttgg
2520attacgaagt gtggaaccca gaaattgatc ctttactggc gagtaacttc
agtgtcaaaa 2580cctttggcga taaggcaaaa aataagcaag cgttacggga
aagattactg ttagaaacgg 2640atgataaaaa acccatgctc tgctttattg
gccgcttgga tggacaaaaa ggtgtgcact 2700tggtgcatca ctccatctac
tacgccctca gccagggagc gcaatttgtc ctgctcggct 2760ccgccaccga
acccaatctg agcaaatggt tctggcatga aaaacaacat ctcaacgata
2820accccaatgt ccatctagag ttgggctttg acgaggagct ggcccactta
atttacggag 2880cggcggacat tattgtggtg cccagtaact acgaaccctg
tggtttgacc caaatgattg 2940gtctgcgtta tggggccgtt ccggtggtgc
ggggagtagg cggtttggta aatactgttt 3000tcgaccggga ttatgaccag
aaccatcccc cggagaaacg taatggtttt gttttctatc 3060aaccggatga
gtatgccctg gaaacggccc tcagtcgggc gatcgccttg tataaggatg
3120atcccgtggc ttttaaaacc ttggccttgc agggcatggc ctacgactac
tcttggaata 3180aaccagggct ccaatatgtg gaagcctacg aatacatccg
ggcttaacac ctcgggtttg 3240taacagtttc gttacactag ttcagaggcg
gagtcaaagc agttggtcag aataagcttg 3300gcgtaatcat ggtcatagct
gtttcctgtg tgaaattgtt atccgctcac aattccacac 3360aacatacgag
ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc
3420acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
gtgccagctg 3480cattaatgaa tcggccaacg
cgcggggaga ggcggtttgc gtattgggcg ctcttccgct 3540tcctcgctca
ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
3600tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
gaacatgtga 3660gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
cgttgctggc gtttttccat 3720aggctccgcc cccctgacga gcatcacaaa
aatcgacgct caagtcagag gtggcgaaac 3780ccgacaggac tataaagata
ccaggcgttt ccccctggaa gctccctcgt gcgctctcct 3840gttccgaccc
tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
3900ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
ctccaagctg 3960ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
ccttatccgg taactatcgt 4020cttgagtcca acccggtaag acacgactta
tcgccactgg cagcagccac tggtaacagg 4080attagcagag cgaggtatgt
aggcggtgct acagagttct tgaagtggtg gcctaactac 4140ggctacacta
gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
4200aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
tggttttttt 4260gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
aagaagatcc tttgatcttt 4320tctacggggt ctgacgctca gtggaacgaa
aactcacgtt aagggatttt ggtcatgaga 4380ttatcaaaaa ggatcttcac
ctagatcctt ttaaattaaa aatgaagttt taaatcaatc 4440taaagtatat
atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
4500atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
cgtgtagata 4560actacgatac gggagggctt accatctggc cccagtgctg
caatgatacc gcgagaccca 4620cgctcaccgg ctccagattt atcagcaata
aaccagccag ccggaagggc cgagcgcaga 4680agtggtcctg caactttatc
cgcctccatc cagtctatta attgttgccg ggaagctaga 4740gtaagtagtt
cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
4800gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
atcaaggcga 4860gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
ccttcggtcc tccgatcgtt 4920gtcagaagta agttggccgc agtgttatca
ctcatggtta tggcagcact gcataattct 4980cttactgtca tgccatccgt
aagatgcttt tctgtgactg gtgagtactc aaccaagtca 5040ttctgagaat
agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
5100accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
ttcggggcga 5160aaactctcaa ggatcttacc gctgttgaga tccagttcga
tgtaacccac tcgtgcaccc 5220aactgatctt cagcatcttt tactttcacc
agcgtttctg ggtgagcaaa aacaggaagg 5280caaaatgccg caaaaaaggg
aataagggcg acacggaaat gttgaatact catactcttc 5340ctttttcaat
attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
5400gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
aaaagtgcca 5460cctgacgtct aagaaaccat tattatcatg acattaacct
ataaaaatag gcgtatcacg 5520aggccctttc gtc 55335360PRTSynechocystis
sp. strain PCC6803 5Met Glu Ile Gly Val Pro Lys Glu Ile Lys Asp Gln
Glu Phe Arg Val 1 5 10 15 Gly Leu Thr Pro Ser Ser Val Arg Ala Leu
Leu Ser Gln Gly His Gln 20 25 30 Val Phe Val Glu Glu Gly Ala Gly
Val Gly Ser Gly Phe Pro Asp Gly 35 40 45 Ala Tyr Ala Lys Ala Gly
Ala Glu Leu Val Ala Thr Ala Lys Glu Ala 50 55 60 Trp Asn Arg Glu
Leu Val Val Lys Val Lys Glu Pro Leu Pro Glu Glu 65 70 75 80 Tyr Glu
Tyr Leu Thr Leu Pro Lys Leu Leu Phe Thr Tyr Leu His Leu 85 90 95
Ala Ala Glu Arg Thr Leu Thr Glu Ala Leu Ile Lys Ser Gly Ile Thr 100
105 110 Ala Ile Ala Tyr Glu Thr Val Glu Leu Ala Asp Gly Gln Leu Pro
Leu 115 120 125 Leu Ala Pro Met Ser Arg Ile Ala Gly Arg Leu Ala Val
Gln Met Gly 130 135 140 Ala His Tyr Leu Glu Lys Gln Gln Gly Gly Arg
Gly Val Leu Leu Gly 145 150 155 160 Gly Val Pro Gly Val Lys Ala Gly
Gln Val Thr Ile Leu Gly Gly Gly 165 170 175 Val Val Gly Thr Glu Ala
Ala Lys Met Ala Ile Gly Leu Gly Ala Met 180 185 190 Val Thr Ile Leu
Asp Ile Asn Val Asp Arg Leu Asn Gln Leu Gly Glu 195 200 205 Leu Phe
Gly Ser Arg Val Asp Leu Arg Tyr Ser Asn Ala Ser Gln Ile 210 215 220
Glu Asp Leu Leu Pro His Thr Asp Leu Leu Ile Gly Ala Val Leu Ile 225
230 235 240 Thr Gly Lys Arg Ala Pro Val Leu Val Ser Arg Gln Glu Val
Glu Gln 245 250 255 Met Leu Pro Gly Ala Val Ile Met Asp Val Ala Ile
Asp Gln Gly Gly 260 265 270 Cys Val Glu Thr Leu Arg Val Thr Ser His
Ser Gln Pro Ser Tyr Ile 275 280 285 Glu Ala Glu Val Val His Val Gly
Ile Pro Asn Met Pro Gly Ala Thr 290 295 300 Pro Trp Thr Ala Thr Gln
Ala Leu Asn Asn Ser Thr Leu Arg Tyr Val 305 310 315 320 Leu Lys Leu
Ala Asn Leu Gly Glu Gln Ala Trp Glu Asn Asp Leu Pro 325 330 335 Leu
Ala Lys Gly Val Asn Val Gln Ala Gly Lys Leu Val Gln Gly Ala 340 345
350 Val Lys Thr Val Phe Pro Asp Leu 355 360
62292DNAartificialconstruct pGEM-T/ald-KManti 6gggattggct
gacccccagt agtgtacggg cattgctgag ccagggccat caagtatttg 60tggaagaagg
ggccggagtc gggtctggct tccccgatgg agcctacgca aaggcgggag
120ctgagttagt tgccactgcc aaagaggctt ggaacaggga attggtggtg
aaagtgaaag 180agcctctccc tgaagagtat gaatatttaa ctttgcctaa
gttgttgttc acttatctcc 240atttggcagc ggaacgtacc ctcaccgaag
ctctaattaa atctggcatt acggcgatcg 300cctatgaaac ggtggaattg
gctgatggtc aattgccatt gttggccccc atgagccgca 360ttgccggacg
attggcggtg cagatgggtg cccattattt ggaaaaacaa cagggaggcc
420ggggagttct gttgggaggc gtacccggag tcaaggccgg acaagtaact
atcctcggcg 480gtggcgtagt cggtacagag gcggccaaaa tggcgatcgg
actgggggcc atggtgacca 540tcctagacat caatgtagac cgtttaaacc
aattgggaga actgttcggt tcccgaattc 600cccggatccg tcgacctgca
gggggggggg ggcgctgagg tctgcctcgt gaagaaggtg 660ttgctgactc
ataccaggcc tgaatcgccc catcatccag ccagaaagtg agggagccac
720ggttgatgag agctttgttg taggtggacc agttggtgat tttgaacttt
tgctttgcca 780cggaacggtc tgcgttgtcg ggaagatgcg tgatctgatc
cttcaactca gcaaaagttc 840gatttattca acaaagccgc cgtcccgtca
agtcagcgta atgctctgcc agtgttacaa 900ccaattaacc aattctgatt
agaaaaactc atcgagcatc aaatgaaact gcaatttatt 960catatcagga
ttatcaatac catatttttg aaaaagccgt ttctgtaatg aaggagaaaa
1020ctcaccgagg cagttccata ggatggcaag atcctggtat cggtctgcga
ttccgactcg 1080tccaacatca atacaaccta ttaatttccc ctcgtcaaaa
ataaggttat caagtgagaa 1140atcaccatga gtgacgactg aatccggtga
gaatggcaaa agcttatgca tttctttcca 1200gacttgttca acaggccagc
cattacgctc gtcatcaaaa tcactcgcat caaccaaacc 1260gttattcatt
cgtgattgcg cctgagcgag acgaaatacg cgatcgctgt taaaaggaca
1320attacaaaca ggaatcgaat gcaaccggcg caggaacact gccagcgcat
caacaatatt 1380ttcacctgaa tcaggatatt cttctaatac ctggaatgct
gttttcccgg ggatcgcagt 1440ggtgagtaac catgcatcat caggagtacg
gataaaatgc ttgatggtcg gaagaggcat 1500aaattccgtc agccagttta
gtctgaccat ctcatctgta acatcattgg caacgctacc 1560tttgccatgt
ttcagaaaca actctggcgc atcgggcttc ccatacaatc gatagattgt
1620cgcacctgat tgcccgacat tatcgcgagc ccatttatac ccatataaat
cagcatccat 1680gttggaattt aatcgcggcc tcgagcaaga cgtttcccgt
tgaatatggc tcataacacc 1740ccttgtatta ctgtttatgt aagcagacag
ttttattgtt catgatgata tatttttatc 1800ttgtgcaatg taacatcaga
gattttgaga cacaacgtgg ctttcccccc cccccctgca 1860ggtcgacgga
tccggggaat tcgggtggat ctgcgctaca gcaatgccag ccaaatcgaa
1920gacctgttgc cccatacaga tttgctcatc ggcgcagtat tgatcacagg
caagcgggcc 1980ccagtgttgg tttcccgcca ggaagtggag caaatgttgc
caggggcggt gattatggat 2040gtggcgatcg accaaggggg ctgtgtggag
actttgcggg taacttctca tagtcaaccc 2100agttacatcg aagcagaagt
agttcatgtg ggcattccca atatgccagg agccactccc 2160tggacagcaa
cccaagcgtt gaataatagt acattgcgct atgtgttgaa attggccaat
2220ctgggggaac aggcttggga aaatgatttg ccgttggcga aaggagtcaa
tgttcaagcc 2280ggaaaataat ca 22927439PRTSynechocystis sp. strain
PCC6803 7Met Cys Cys Trp Gln Ser Arg Gly Leu Leu Val Lys Arg Val
Leu Ala 1 5 10 15 Ile Ile Leu Gly Gly Gly Ala Gly Thr Arg Leu Tyr
Pro Leu Thr Lys 20 25 30 Leu Arg Ala Lys Pro Ala Val Pro Leu Ala
Gly Lys Tyr Arg Leu Ile 35 40 45 Asp Ile Pro Val Ser Asn Cys Ile
Asn Ser Glu Ile Val Lys Ile Tyr 50 55 60 Val Leu Thr Gln Phe Asn
Ser Ala Ser Leu Asn Arg His Ile Ser Arg 65 70 75 80 Ala Tyr Asn Phe
Ser Gly Phe Gln Glu Gly Phe Val Glu Val Leu Ala 85 90 95 Ala Gln
Gln Thr Lys Asp Asn Pro Asp Trp Phe Gln Gly Thr Ala Asp 100 105 110
Ala Val Arg Gln Tyr Leu Trp Leu Phe Arg Glu Trp Asp Val Asp Glu 115
120 125 Tyr Leu Ile Leu Ser Gly Asp His Leu Tyr Arg Met Asp Tyr Ala
Gln 130 135 140 Phe Val Lys Arg His Arg Glu Thr Asn Ala Asp Ile Thr
Leu Ser Val 145 150 155 160 Val Pro Val Asp Asp Arg Lys Ala Pro Glu
Leu Gly Leu Met Lys Ile 165 170 175 Asp Ala Gln Gly Arg Ile Thr Asp
Phe Ser Glu Lys Pro Gln Gly Glu 180 185 190 Ala Leu Arg Ala Met Gln
Val Asp Thr Ser Val Leu Gly Leu Ser Ala 195 200 205 Glu Lys Ala Lys
Leu Asn Pro Tyr Ile Ala Ser Met Gly Ile Tyr Val 210 215 220 Phe Lys
Lys Glu Val Leu His Asn Leu Leu Glu Lys Tyr Glu Gly Ala 225 230 235
240 Thr Asp Phe Gly Lys Glu Ile Ile Pro Asp Ser Ala Ser Asp His Asn
245 250 255 Leu Gln Ala Tyr Leu Phe Asp Asp Tyr Trp Glu Asp Ile Gly
Thr Ile 260 265 270 Glu Ala Phe Tyr Glu Ala Asn Leu Ala Leu Thr Lys
Gln Pro Ser Pro 275 280 285 Asp Phe Ser Phe Tyr Asn Glu Lys Ala Pro
Ile Tyr Thr Arg Gly Arg 290 295 300 Tyr Leu Pro Pro Thr Lys Met Leu
Asn Ser Thr Val Thr Glu Ser Met 305 310 315 320 Ile Gly Glu Gly Cys
Met Ile Lys Gln Cys Arg Ile His His Ser Val 325 330 335 Leu Gly Ile
Arg Ser Arg Ile Glu Ser Asp Cys Thr Ile Glu Asp Thr 340 345 350 Leu
Val Met Gly Asn Asp Phe Tyr Glu Ser Ser Ser Glu Arg Asp Thr 355 360
365 Leu Lys Ala Arg Gly Glu Ile Ala Ala Gly Ile Gly Ser Gly Thr Thr
370 375 380 Ile Arg Arg Ala Ile Ile Asp Lys Asn Ala Arg Ile Gly Lys
Asn Val 385 390 395 400 Met Ile Val Asn Lys Glu Asn Val Gln Glu Ala
Asn Arg Glu Glu Leu 405 410 415 Gly Phe Tyr Ile Arg Asn Gly Ile Val
Val Val Ile Lys Asn Val Thr 420 425 430 Ile Ala Asp Gly Thr Val Ile
435 82522DNAartificialconstruct pGEM-T/glgC-KManti 8gggattgttg
ttggcaatcg agaggtctgc ttgtgaaacg tgtcttagcg attatcctgg 60gcggtggggc
cgggacccgc ctctatcctt taaccaaact cagagccaaa cccgcagttc
120ccttggccgg aaagtatcgc ctcatcgata ttcccgtcag taattgcatc
aactcagaaa 180tcgttaaaat ttacgtcctt acccagttta attccgcctc
ccttaaccgt cacatcagcc 240gggcctataa tttttccggc ttccaagaag
gatttgtgga agtcctcgcc gcccaacaaa 300ccaaagataa tcctgattgg
tttcagggca ctgctgatgc ggtacggcaa tacctctggt 360tgtttaggga
atgggacgta gatgaatatc ttattctgtc cggcgaccat ctctaccgca
420tggattacgc ccaatttgtt aaaagacacc gggaaaccaa tgccgacata
accctttccg 480ttgtgcccgt ggatgacaga aaggcacccg agctgggctt
aatgaaaatc gacgcccagg 540gcagaattac tgacttttct gaaaagcccc
agggggaagc cctccgggcc atgcaggtgg 600acaccagcgt tttgggccta
agtgcggaga aggctaagct taatccttac attgcctcca 660tgggcattta
cgttttcaag aaggaagtat tgcacaacct cctggaaaaa tatgaagggg
720caacggactt tggcaaagaa atcattcctg attcagccag tgatcacaat
ctgcaagcct 780atctctttga tgactattgg gaagacattg gtaccattga
agccttctat gaggctaatt 840tagccctgac caaacaacct agtcccgact
ttagttttta taacgaaaaa gcccccatct 900ataccagggg tcgttatctt
ccccccacca aaatgttgaa ttccccggat ccgtcgacct 960gcaggggggg
gggggcgctg aggtctgcct cgtgaagaag gtgttgctga ctcataccag
1020gcctgaatcg ccccatcatc cagccagaaa gtgagggagc cacggttgat
gagagctttg 1080ttgtaggtgg accagttggt gattttgaac ttttgctttg
ccacggaacg gtctgcgttg 1140tcgggaagat gcgtgatctg atccttcaac
tcagcaaaag ttcgatttat tcaacaaagc 1200cgccgtcccg tcaagtcagc
gtaatgctct gccagtgtta caaccaatta accaattctg 1260attagaaaaa
ctcatcgagc atcaaatgaa actgcaattt attcatatca ggattatcaa
1320taccatattt ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg
aggcagttcc 1380ataggatggc aagatcctgg tatcggtctg cgattccgac
tcgtccaaca tcaatacaac 1440ctattaattt cccctcgtca aaaataaggt
tatcaagtga gaaatcacca tgagtgacga 1500ctgaatccgg tgagaatggc
aaaagcttat gcatttcttt ccagacttgt tcaacaggcc 1560agccattacg
ctcgtcatca aaatcactcg catcaaccaa accgttattc attcgtgatt
1620gcgcctgagc gagacgaaat acgcgatcgc tgttaaaagg acaattacaa
acaggaatcg 1680aatgcaaccg gcgcaggaac actgccagcg catcaacaat
attttcacct gaatcaggat 1740attcttctaa tacctggaat gctgttttcc
cggggatcgc agtggtgagt aaccatgcat 1800catcaggagt acggataaaa
tgcttgatgg tcggaagagg cataaattcc gtcagccagt 1860ttagtctgac
catctcatct gtaacatcat tggcaacgct acctttgcca tgtttcagaa
1920acaactctgg cgcatcgggc ttcccataca atcgatagat tgtcgcacct
gattgcccga 1980cattatcgcg agcccattta tacccatata aatcagcatc
catgttggaa tttaatcgcg 2040gcctcgagca agacgtttcc cgttgaatat
ggctcataac accccttgta ttactgttta 2100tgtaagcaga cagttttatt
gttcatgatg atatattttt atcttgtgca atgtaacatc 2160agagattttg
agacacaacg tggctttccc ccccccccct gcaggtcgac ggatccgggg
2220aattccaccg tgacggaatc catgatcggg gaaggttgca tgattaagca
atgtcgcatc 2280caccactcag ttttaggcat tcgcagtcgc attgaatctg
attgcaccat tgaggatact 2340ttggtgatgg gcaatgattt ctacgaatct
tcatcagaac gagacaccct caaagcccgg 2400ggggaaattg ccgctggcat
aggttccggc accactatcc gccgagccat catcgacaaa 2460aatgcccgca
tcggcaaaaa cgtcatgatt gtcaacaagg aaaatgtcca ggaggctaat 2520ca
252292586DNAartificialconstruct pDrive/glgC-CMantisense 9aattcgtgat
taccccatca tcatacgaag ccagggacag tttactcagc ggcagtttcc 60gacctttgcc
atttcggtta tccgtacccc cacagtgatc tgacaactca gctccgaatc
120ccaacggcga tcgccattct tgcttggggc attaaaaccc gctggttagc
cggaatttcc 180gtccagattc cctttccaga tgtccccctc ggttctaaac
ttgacttcga agtgtgttgt 240tggcaatcga gaggtctgct tgtgaaacgt
gtcttagcga ttatcctggg cggtggggcc 300gggacccgcc tctatccttt
aaccaaactc agagccaaac ccgcagttcc cttggccgga 360aagtatcgcc
tcatcgatat tcccgtcagt aattgcatca actcagaaat cgttaaaatt
420tacgtcctta cccagtttaa ttccgcctcc cttaaccgtc acatcagccg
ggcctataat 480ttttccggct tccaagaagg atttgtggaa gtcctcgccg
cccaacaaac caaagataat 540cctgattggt ttcagggcac tgctgatgcg
gtacggcaat acctctggtt gtttagggaa 600tgggacgtag atgatacgtg
ctgctgaagt tgcccgcaac agagagtgga accaaccggt 660gataccacga
tactatgact gagagtcaac gccatgagcg gcctcatttc ttattctgag
720ttacaacagt ccgcaccgct gtccggtagc tccttccggt gggcgcgggg
catgactatc 780gtcgccgcac ttatgactgt cttctttatc atgcaactcg
taggacaggt gccggcagcg 840cccaacagtc ccccggccac ggggcctgcc
accataccca cgccgaaaca agcgccctgc 900accattatgt tccggatctg
catcgcagga tgctgctggc taccctgtgg aacacctaca 960tctgtattaa
cgaagcgcta accgttttta tcaggctctg ggaggcagaa taaatgatca
1020tatcgtcaat tattacctcc acggggagag cctgagcaaa ctggcctcag
gcatttgaga 1080agcacacggt cacactgctt ccggtagtca ataaaccggt
aaaccagcaa tagacataag 1140cggctattta acgaccctgc cctgaaccga
cgaccgggtc gaatttgctt tcgaatttct 1200gccattcatc cgcttattat
cacttattca ggcgtagcac caggcgttta agggcaccaa 1260taactgcctt
aaaaaaatta cgccccgccc tgccactcat cgcagtactg ttgtaattca
1320ttaagcattc tgccgacatg gaagccatca cagacggcat gatgaacctg
aatcgccagc 1380ggcatcagca ccttgtcgcc ttgcgtataa tatttgccca
tggtgaaaac gggggcgaag 1440aagttgtcca tattggccac gtttaaatca
aaactggtga aactcaccca gggattggct 1500gagacgaaaa acatattctc
aataaaccct ttagggaaat aggccaggtt ttcaccgtaa 1560cacgccacat
cttgcgaata tatgtgtaga aactgccgga aatcgtcgtg gtattcactc
1620cagagcgatg aaaacgtttc agtttgctca tggaaaacgg tgtaacaagg
gtgaacacta 1680tcccatatca ccagctcacc gtctttcatt gccatacgga
attccggatg agcattcatc 1740aggcgggcaa gaatgtgaat aaaggccgga
taaaacttgt gcttattttt ctttacggtc 1800tttaaaaagg ccgtaatatc
cagctgaacg gtctggttat aggtacattg agcaactgac 1860tgaaatgcct
caaaatgttc tttacgatgc cattgggata tatcaacggt ggtatatcca
1920gtgatttttt tctccatttt agcttcctta gctcctgaaa atctcgataa
ctcaaaaaat 1980acgcccggta gtgatcttat ttcattatgg tgaaagttgg
aacctcttac gtgatatctt 2040attctgtccg gcgaccatct ctaccgcatg
gattacgccc aatttgttaa aagacaccgg 2100gaaaccaatg ccgacataac
cctttccgtt gtgcccgtgg atgacagaaa ggcacccgag 2160ctgggcttaa
tgaaaatcga cgcccagggc agaattactg acttttctga aaagccccag
2220ggggaagccc tccgggccat gcaggtggac accagcgttt tgggcctaag
tgcggagaag 2280gctaagctta atccttacat tgcctccatg ggcatttacg
ttttcaagaa ggaagtattg 2340cacaacctcc tggaaaaata tgaaggggca
acggactttg gcaaagaaat cattcctgat 2400tcagccagtg atcacaatct
gcaagcctat ctctttgatg actattggga agacattggt 2460accattgaag
ccttctatga ggctaattta gccctgacca aacaacctag tcccgacttt
2520agtttttata acgaaaaagc ccccatctat accaggggtc gttatcttcc
ccccaccaaa 2580atgttg
258610818PRTSynechocystis sp. strain PCC6803 10Met Val Ser Ser Val
Val Glu Lys Thr Ser Val Ala His Lys Glu Thr 1 5 10 15 Ala Leu Ile
Leu Trp Phe Glu Glu Val Gly Thr His Asp Val Gly Leu 20 25 30 Val
Gly Gly Lys Asn Ser Ser Leu Gly Glu Met Ile Gln Gln Leu Thr 35 40
45 Asn Lys Gly Val Asn Val Pro Ser Gly Phe Ala Thr Thr Ala Tyr Ala
50 55 60 Tyr Arg Tyr Phe Ile Gln Glu Ala Gly Leu Glu Gln Lys Leu
Arg Asp 65 70 75 80 Leu Phe Thr Asp Leu Asp Val Asn Asp Met Ala Asn
Leu Gln Glu Arg 85 90 95 Gly His Leu Ala Arg Gln Leu Ile Leu Asp
Thr Pro Phe Pro Gln Asn 100 105 110 Leu Gln Thr Ala Ile Ala Glu Ala
Tyr Gly Ala Met Cys Glu Arg Tyr 115 120 125 Gly Gln Lys Met Gly Arg
Thr Gly Val Asp Val Ala Val Arg Ser Ser 130 135 140 Ala Thr Ala Glu
Asp Leu Pro Glu Ala Ser Phe Ala Gly Gln Gln Glu 145 150 155 160 Thr
Tyr Leu Asn Val His Ser Leu Ser Cys Val Leu Glu Ser Cys His 165 170
175 Lys Cys Phe Ala Ser Leu Phe Thr Asp Arg Ala Ile Ser Tyr Arg His
180 185 190 His Asn Gly Phe Asp His Phe Ala Val Ala Leu Ser Val Gly
Val Gln 195 200 205 Lys Met Val Arg Ser Asp Leu Ala Thr Ser Gly Val
Met Phe Ser Ile 210 215 220 Asp Thr Glu Thr Gly Phe Lys Asn Ala Ala
Leu Ile Thr Ala Ala Tyr 225 230 235 240 Gly Leu Gly Glu Asn Val Val
Gln Gly Ala Val Asn Pro Asp Glu Tyr 245 250 255 Phe Val Phe Lys Pro
Thr Leu Lys Glu Gly Phe Lys Pro Ile Leu Glu 260 265 270 Lys Arg Leu
Gly Ser Lys Ala Ile Lys Met Val Tyr Asp Val Gly Gly 275 280 285 Ser
Lys Leu Thr Lys Asn Val Glu Val Ala Glu Pro Glu Arg Glu Lys 290 295
300 Tyr Cys Ile Asn Asp Glu Glu Ile Leu Gln Leu Ala Arg Trp Ala Cys
305 310 315 320 Ile Ile Glu Asp His Tyr Ser Gly Val Arg Gly Val Tyr
Thr Pro Met 325 330 335 Asp Ile Glu Trp Ala Lys Asp Gly Gln Thr Gly
Glu Leu Phe Ile Val 340 345 350 Gln Ala Arg Pro Glu Thr Val Gln Ser
Gln Lys Ser Ala Asn Val Ile 355 360 365 Lys Thr Tyr Glu Leu Lys Asp
His Ser Gln Val Leu Ala Thr Gly Arg 370 375 380 Ser Val Gly Ala Ala
Ile Gly Gln Gly Lys Ala Gln Val Ile Arg Asn 385 390 395 400 Val Ser
Gln Ile Asn Gln Phe Arg Pro Gly Glu Val Leu Ile Thr Asn 405 410 415
Arg Thr Asp Pro Asp Trp Glu Pro Ile Met Lys Gln Ala Ser Ala Ile 420
425 430 Val Thr Asn Gln Gly Gly Lys Thr Cys His Ala Ala Ile Ile Ala
Arg 435 440 445 Glu Met Gly Ile Pro Ala Ile Val Gly Cys Gly Asp Ala
Thr Asp Thr 450 455 460 Ile Lys Thr Gly Glu Asp Val Thr Ile Cys Cys
Ser Glu Gly Asp Glu 465 470 475 480 Gly Ser Val Tyr Ser Gly Ile Leu
Asn Tyr Glu Val His Glu Thr Glu 485 490 495 Leu Ser Asn Leu Pro Arg
Thr Lys Thr Gln Ile Leu Met Asn Val Gly 500 505 510 Asn Pro Glu Gln
Ala Phe Gly Phe Ala Ser Tyr Pro Ala Asp Gly Val 515 520 525 Gly Leu
Ala Arg Leu Glu Phe Ile Ile Ala Asn His Ile Lys Ala His 530 535 540
Pro Leu Ala Leu Met Lys Phe Asp Glu Leu Glu Asp Pro Leu Ala Lys 545
550 555 560 Ala Glu Ile Ala Glu Leu Thr Lys Leu Tyr Ala Gly Asp Arg
Pro Arg 565 570 575 Phe Phe Val Asp Lys Leu Ala His Gly Ile Ala Met
Ile Ala Ala Ala 580 585 590 Phe Tyr Pro Lys Pro Val Val Val Arg Met
Ser Asp Phe Lys Ser Asn 595 600 605 Glu Tyr Ala Asn Leu Leu Gly Gly
Arg Gln Phe Glu Pro Lys Glu Glu 610 615 620 Asn Pro Met Ile Gly Trp
Arg Gly Ala Ser Arg Tyr Tyr Asp Pro Asn 625 630 635 640 Tyr Arg Glu
Ala Tyr Ala Leu Glu Cys Gln Ala Leu Lys Arg Val Arg 645 650 655 Asp
Glu Met Gly Leu Thr Asn Val Ile Pro Met Ile Pro Phe Cys Arg 660 665
670 Thr Pro Asp Glu Gly Arg Lys Val Ile Ala Glu Met Ala Lys His Gly
675 680 685 Leu Lys Gln Gly Lys Asn Gly Leu Glu Ile Tyr Val Met Cys
Glu Leu 690 695 700 Pro Ser Asn Val Ile Leu Ala Asp Glu Phe Ser Glu
Val Phe Asp Gly 705 710 715 720 Phe Ser Ile Gly Ser Asn Asp Leu Thr
Gln Leu Thr Leu Gly Leu Asp 725 730 735 Arg Asp Ser Ser Leu Val Ala
His Leu Phe Asp Glu Arg Asn Leu Gly 740 745 750 Val Lys Arg Met Val
Lys Met Ala Ile Glu Thr Ala Lys Ala Asn Gly 755 760 765 Arg Lys Ile
Gly Ile Cys Gly Gln Ala Pro Ser Asp Tyr Pro Glu Phe 770 775 780 Ala
Glu Phe Leu Val Glu Leu Gly Ile Asp Ser Ile Ser Leu Asn Pro 785 790
795 800 Asp Ser Val Leu Lys Thr Val Leu Arg Ile Ala Glu Val Glu Lys
Ala 805 810 815 Leu Gly 113082DNAartificialconstruct
pGEM-T/ppsA-anti 11gggattttca ctgaccgggc tatttcctat cgccatcaca
atggttttga ccattttgcg 60gtggccctat cggtgggcgt acaaaagatg gtgcgttctg
atctggccac ctccggggtg 120atgttttcca ttgatacgga aacgggtttc
aaaaatgccg ctctgattac tgccgcctat 180ggcctagggg aaaatgtggt
gcaaggggcg gttaaccccg atgaatattt tgtgtttaaa 240cctactttga
aagagggttt taaaccaatt ctggaaaaac gtttgggtag taaagctatc
300aaaatggtct atgacgtggg cggttccaaa ctgaccaaaa atgtggaagt
agcggagccg 360gaacgggaaa aatattgcat taatgatgaa gaaattctcc
aattagcccg ctgggcctgc 420atcattgaag accattattc tggggtgcgg
ggagtttata cccccatgga tattgaatgg 480gctaaggatg ggcaaacggg
ggaattgttc attgtccaag cccgcccaga aacggtgcag 540tcgcaaaaat
ccgccaatgt gattaaaacc tatgagttaa aagatcacag ccaagtgtta
600gccacgggcc gcagtgtggg ggcggcgatc ggccagggta aagcccaggt
aattcgcaat 660gtgtcccaaa tcaatcagtt tcgtcccggc gaggtgttaa
tcaccaaccg cactgacccg 720gattgggaac cgattatgaa acaggcttcg
gcgatcgtca ctaaccaggg ggggaaaacc 780tgccacgccg caattattgc
ccgaattccc cggatccgtc gacctgcagg gggggggggg 840cgctgaggtc
tgcctcgtga agaaggtgtt gctgactcat accaggcctg aatcgcccca
900tcatccagcc agaaagtgag ggagccacgg ttgatgagag ctttgttgta
ggtggaccag 960ttggtgattt tgaacttttg ctttgccacg gaacggtctg
cgttgtcggg aagatgcgtg 1020atctgatcct tcaactcagc aaaagttcga
tttattcaac aaagccgccg tcccgtcaag 1080tcagcgtaat gctctgccag
tgttacaacc aattaaccaa ttctgattag aaaaactcat 1140cgagcatcaa
atgaaactgc aatttattca tatcaggatt atcaatacca tatttttgaa
1200aaagccgttt ctgtaatgaa ggagaaaact caccgaggca gttccatagg
atggcaagat 1260cctggtatcg gtctgcgatt ccgactcgtc caacatcaat
acaacctatt aatttcccct 1320cgtcaaaaat aaggttatca agtgagaaat
caccatgagt gacgactgaa tccggtgaga 1380atggcaaaag cttatgcatt
tctttccaga cttgttcaac aggccagcca ttacgctcgt 1440catcaaaatc
actcgcatca accaaaccgt tattcattcg tgattgcgcc tgagcgagac
1500gaaatacgcg atcgctgtta aaaggacaat tacaaacagg aatcgaatgc
aaccggcgca 1560ggaacactgc cagcgcatca acaatatttt cacctgaatc
aggatattct tctaatacct 1620ggaatgctgt tttcccgggg atcgcagtgg
tgagtaacca tgcatcatca ggagtacgga 1680taaaatgctt gatggtcgga
agaggcataa attccgtcag ccagtttagt ctgaccatct 1740catctgtaac
atcattggca acgctacctt tgccatgttt cagaaacaac tctggcgcat
1800cgggcttccc atacaatcga tagattgtcg cacctgattg cccgacatta
tcgcgagccc 1860atttataccc atataaatca gcatccatgt tggaatttaa
tcgcggcctc gagcaagacg 1920tttcccgttg aatatggctc ataacacccc
ttgtattact gtttatgtaa gcagacagtt 1980ttattgttca tgatgatata
tttttatctt gtgcaatgta acatcagaga ttttgagaca 2040caacgtggct
ttcccccccc cccctgcagg tcgacggatc cggggaattc gggaaatggg
2100tattccggcg attgtgggtt gtggagatgc caccgacaca atcaaaaccg
gggaagatgt 2160caccatctgt tgctccgaag gggatgaagg ttcggtttac
agcggcattt tgaactatga 2220agttcacgaa acggaactgt ccaatttgcc
ccgcaccaag actcaaattt tgatgaatgt 2280gggtaaccca gaacaggcct
ttggatttgc tagttatccc gccgatggcg tgggtctagc 2340ccggttggaa
tttatcattg ctaaccacat taaggctcac cccctcgccc tgatgaaatt
2400tgatgagttg gaagatccct tggccaaggc agaaattgcc gaactaacca
aactctatgc 2460tggcgatcgc ccccggttct ttgtggacaa attggcccat
ggtattgcca tgattgcggc 2520ggcgttctat cccaaacctg tggtggtgcg
gatgtcggat tttaaatcca atgaatacgc 2580taacctcctg ggtggtcgtc
agtttgagcc gaaggaagaa aaccccatga tcggttggcg 2640gggcgcttcc
cgttactacg atcccaatta ccgagaagcc tacgctttgg aatgccaagc
2700tctgaaacga gtgcgggacg aaatgggttt aaccaacgtc attcccatga
ttcccttctg 2760tcgtaccccc gatgaaggtc gcaaagttat tgcggaaatg
gctaaacatg gcttgaaaca 2820agggaaaaac ggcttggaaa tctacgttat
gtgtgaattg cccagtaacg tcattctggc 2880cgatgaattt agcgaggtat
ttgacggctt ctccattggc tccaatgatt taacccaatt 2940aactttaggt
ttagaccggg attcttccct cgttgcccat ctgtttgatg aacgcaatct
3000aggggtcaaa cggatggtca aaatggccat tgaaacggcg aaagctaacg
gtcgcaaaat 3060cggtatctgt ggccaagaat ca 308212333PRTSynechocystis
sp. strain PCC6803 12Met Lys Ile Ala Phe Phe Ser Ser Lys Ala Tyr
Asp Arg Gln Phe Phe 1 5 10 15 Gln Gln Ala Asn His Pro His Gln Arg
Glu Met Val Phe Phe Asp Ala 20 25 30 Gln Leu Asn Leu Asp Thr Ala
Ile Leu Ala Glu Asp Cys Pro Val Ile 35 40 45 Cys Leu Phe Val Asn
Asp Gln Ala Pro Ala Pro Val Leu Glu Lys Leu 50 55 60 Ala Ala Gln
Gly Thr Lys Leu Ile Ala Leu Arg Ser Ala Gly Tyr Asn 65 70 75 80 Asn
Val Asp Leu Lys Thr Ala Ala Asp Leu Gly Leu Lys Val Val His 85 90
95 Val Pro Ser Tyr Ser Pro His Ala Val Ala Glu His Thr Val Gly Leu
100 105 110 Ile Leu Ala Leu Asn Arg Lys Leu Tyr Arg Ala Tyr Asn Arg
Val Arg 115 120 125 Asp Asp Asn Phe Ser Leu Glu Gly Leu Leu Gly Phe
Asp Leu His Gly 130 135 140 Thr Thr Val Gly Val Ile Gly Thr Gly Lys
Ile Gly Leu Ala Phe Ala 145 150 155 160 Gln Ile Met Asn Gly Phe Gly
Cys His Leu Leu Gly Tyr Asp Ala Phe 165 170 175 Pro Asn Asp Lys Phe
Thr Ala Ile Gly Gln Ala Leu Tyr Val Ser Leu 180 185 190 Asn Glu Leu
Leu Ala His Ser Asp Ile Ile Ser Leu His Cys Pro Leu 195 200 205 Leu
Pro Glu Thr His Tyr Leu Ile Asn Thr Asn Thr Ile Ala Gln Met 210 215
220 Lys Pro Gly Val Met Leu Ile Asn Thr Ser Arg Gly His Leu Ile Asp
225 230 235 240 Thr Gln Ala Val Ile Gln Gly Ile Lys Ser His Lys Ile
Gly Phe Leu 245 250 255 Gly Ile Asp Val Tyr Glu Glu Glu Glu Glu Leu
Phe Phe Thr Asp His 260 265 270 Ser Asp Thr Ile Ile Gln Asp Asp Thr
Phe Gln Leu Leu Gln Ser Phe 275 280 285 Pro Asn Val Met Ile Thr Ala
His Gln Gly Phe Phe Thr His Asn Ala 290 295 300 Leu Gln Thr Ile Ala
Ala Thr Thr Leu Ala Asn Ile Ala Glu Phe Glu 305 310 315 320 Gln Asn
Lys Pro Leu Thr Tyr Gln Val Ile Cys Pro His 325 330
132520DNAartificialconstruct pBlue ldh-Kan-a 13cgggcccccc
ctcgaggtcg acggtatcga taagcttgat atccctggga gcggagacct 60ttaagctcaa
atttggtcat cgagggctca atcaaccctg tggcctggag caacaggtgg
120aaattaccag ccagaaccat ggttttgcgg tgacggaagg ttccctggcc
gaagaagtgg 180aaattaccca tttcaacctc aacgataaaa cggtggcggg
gctacgccat aaagaattgc 240cctttttctc ggtgcagtac cacccggagg
ccagccctgg accccatgat gccgattatc 300tgttcgagaa gttcgtcaag
ttgatgcgac aacaaaaggc agaagtcgcc ggttagtaaa 360acctagtaac
agctacgatt tatcgtacta tcgatcacca aggtagtgcc gttaatacta
420tcgttgcttc atttttagga aaaatatctg gtaataaaag gctaaaaaat
ttaacgttat 480ctttagttaa cattgatatt ttctaacctt aatggggaca
gattacttgg taagttaaag 540gtttattctg ctcaaattca gcaatatttg
ccagtgtcgt tgcggcaatg gtttgcagag 600cgttgtgggt aaagaatccc
tgatgagctg tgatccgtcg acctgcaggg gggggggggc 660gctgaggtct
gcctcgtgaa gaaggtgttg ctgactcata ccaggcctga atcgccccat
720catccagcca gaaagtgagg gagccacggt tgatgagagc tttgttgtag
gtggaccagt 780tggtgatttt gaacttttgc tttgccacgg aacggtctgc
gttgtcggga agatgcgtga 840tctgatcctt caactcagca aaagttcgat
ttattcaaca aagccgccgt cccgtcaagt 900cagcgtaatg ctctgccagt
gttacaacca attaaccaat tctgattaga aaaactcatc 960gagcatcaaa
tgaaactgca atttattcat atcaggatta tcaataccat atttttgaaa
1020aagccgtttc tgtaatgaag gagaaaactc accgaggcag ttccatagga
tggcaagatc 1080ctggtatcgg tctgcgattc cgactcgtcc aacatcaata
caacctatta atttcccctc 1140gtcaaaaata aggttatcaa gtgagaaatc
accatgagtg acgactgaat ccggtgagaa 1200tggcaaaagc ttatgcattt
ctttccagac ttgttcaaca ggccagccat tacgctcgtc 1260atcaaaatca
ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct gagcgagacg
1320aaatacgcga tcgctgttaa aaggacaatt acaaacagga atcgaatgca
accggcgcag 1380gaacactgcc agcgcatcaa caatattttc acctgaatca
ggatattctt ctaatacctg 1440gaatgctgtt ttcccgggga tcgcagtggt
gagtaaccat gcatcatcag gagtacggat 1500aaaatgcttg atggtcggaa
gaggcataaa ttccgtcagc cagtttagtc tgaccatctc 1560atctgtaaca
tcattggcaa cgctaccttt gccatgtttc agaaacaact ctggcgcatc
1620gggcttccca tacaatcgat agattgtcgc acctgattgc ccgacattat
cgcgagccca 1680tttataccca tataaatcag catccatgtt ggaatttaat
cgcggcctcg agcaagacgt 1740ttcccgttga atatggctca taacacccct
tgtattactg tttatgtaag cagacagttt 1800tattgttcat gatgatatat
ttttatcttg tgcaatgtaa catcagagat tttgagacac 1860aacgtggctt
tccccccccc ccctgcaggt cgacggatct gcggctgttt tgaggtcaac
1920attattatag cccgcactgc gcagagcgat taattttgtg ccctgggcag
ctaacttttc 1980tagcaccggg gcaggagctt ggtcattaac gaagaggcaa
ataacggggc aatcctccgc 2040taaaatagcg gtatcaaggt tgagttgggc
atcaaaaaag accatttccc gttgatgggg 2100gtggtttgct tgttggaaaa
attgacgatc ataggcttta ctgctaaaaa aagcgatttt 2160catgacgatt
atgggaagta gtttagaacg tgtttggaaa gttttattcc gccccctaaa
2220tcccccaata atggggtact ttcacccagc ttcaccccaa atttgggggc
caggggggct 2280tgttaaacag gctcttagag cactatttag tcaagggtat
tttttccaat cttaaaagaa 2340attttctgat tcttcctgca tttgtattat
ttaatactcc cctagctctg gcgattgccg 2400tagcggtaga tattgactca
cactggggca actgtagggt ttggcctcca actcctgtgt 2460ttggctgtga
tgcattttca gcttcccttt agctagagcg gccgccaccg cggtggagct
252014413PRTSynechocystis PCC6803 14Met Lys Phe Leu Ile Leu Asn Ala
Gly Ser Ser Ser Gln Lys Ser Cys 1 5 10 15 Leu Tyr Glu Leu Thr Gly
Asp Arg Leu Pro Glu Thr Ile Pro Glu Pro 20 25 30 Leu Trp Glu Ala
Phe Ile Asp Trp Thr Val Leu Ala Asn Gln Gly Arg 35 40 45 Leu Thr
Val Glu Thr Ala Gly Gln Lys Gln Val Ile Ile Leu Glu Thr 50 55 60
Gly Asp Arg Gln Gln Gly Ile Ala Arg Met Leu Asp Thr Leu Val Thr 65
70 75 80 Gly Asp Asp Ala Val Leu Lys Ser Leu Ala Glu Ile Asp Leu
Val Gly 85 90 95 His Arg Val Val His Gly Gly Thr Asp His Ala Glu
Ala Thr Leu Ile 100 105 110 Thr Pro Glu Val Gln Gln Ala Ile Ala Asp
Leu Ile Pro Leu Ala Pro 115 120 125 Ala His Asn Pro Ala His Leu Glu
Gly Ile Glu Ala Ile Ser Ala Leu 130 135 140 Leu Val Leu Gly Glu Val
Pro Gln Ile Ala Val Phe Asp Thr Ala Phe 145 150 155 160 His Arg Thr
Ile Pro Thr Pro Ala Ala Glu Tyr Pro Ile Pro Gln Ala 165 170 175 Trp
Thr Asn Leu Gly Ile Arg Arg Tyr Gly Phe His Gly Thr Ser His 180 185
190 Lys Tyr Cys Ala Gln Lys Thr Ala Glu Ile Leu Gly Lys Pro Leu Ala
195 200 205 Asp Leu Lys Leu Ile Thr Cys His Ile Gly Asn Gly Ala Ser
Leu Thr 210 215 220 Ala Ile Lys Asn Gly Val Ser Ile Asp Thr Thr Met
Gly Phe Thr Pro 225 230 235 240 Leu Glu Gly Leu Met Met Gly Ala Arg
Ser Gly Ser Ile Asp Pro Ala 245 250 255 Ile Leu Leu Phe Leu Gln Glu
Thr Gln
Gly Leu Thr Pro Ala Glu Ile 260 265 270 Asn Thr Thr Leu Asn Lys Lys
Ser Gly Leu Leu Gly Val Ser Gly Leu 275 280 285 Ser Ala Asp Leu Arg
Thr Ile Leu Gln Ala Lys Ala Glu Gly Asn Glu 290 295 300 Gln Ala Gln
Leu Ala Tyr Val Met Tyr Ile His Arg Phe Arg Ser Cys 305 310 315 320
Leu Gly Gln Met Ile Ala Ser Leu Glu Gly Leu Asp Thr Leu Val Phe 325
330 335 Thr Ala Gly Val Gly Glu Asn Ala Ala Thr Val Arg Ala Asp Val
Cys 340 345 350 Gln Ala Phe Glu Phe Leu Gly Leu Lys Leu Asp Pro Glu
Leu Asn Asn 355 360 365 Arg Ser Pro Arg Asp Thr Val Ile Ser His Ser
Asp Ser Leu Val Thr 370 375 380 Val Leu Ile Val His Thr Glu Glu Asp
Trp Ala Ile Ala Gln Asp Cys 385 390 395 400 Trp His Trp Trp His Ser
Gln Gly Gln Arg Lys Gln Ser 405 410 152754DNAartificialconstruct
pBlue-ack-Kan-b 15ctagtagtgc agaaattttg agcgatctgg aagccaccat
tgcctacgcc caaactttac 60ccaacgttaa accggaagaa gtaggattaa ttggtttttg
ttttggtggt tggattgtct 120atttaggggc tagtttaccc acagtcaagg
ccacggcttc cttttacggc gcgggtattc 180cccattgggc tccagggaca
gcggaaccgc ccattaccta taccgataaa attcagggca 240ctttatacgc
cttcttcggc ttggaagata ccagcattcc catggcagat acggagcaga
300ttgaacaggc tttaaccaag tatcaggtga accataaaat tttccgttac
ccaggcgcag 360accatggctt tttctgtgac caaagggcta gctataacgc
cgaagcggcc gccgatgctt 420ggcaaaaagt gaaacaactt ttccaaaccg
aattgaaatg aaattcctga ttctcaatgc 480cggttccagc agtcaaaaaa
gttgtcttta tgagctgact ggcgatcgcc taccggagac 540gataccggag
cccttatggg aggctttcat tgattggacg gtgttggcaa atcaggggcg
600gttgacctgc aggggggggg gggcgctgag gtctgcctcg tgaagaaggt
gttgctgact 660cataccaggc ctgaatcgcc ccatcatcca gccagaaagt
gagggagcca cggttgatga 720gagctttgtt gtaggtggac cagttggtga
ttttgaactt ttgctttgcc acggaacggt 780ctgcgttgtc gggaagatgc
gtgatctgat ccttcaactc agcaaaagtt cgatttattc 840aacaaagccg
ccgtcccgtc aagtcagcgt aatgctctgc cagtgttaca accaattaac
900caattctgat tagaaaaact catcgagcat caaatgaaac tgcaatttat
tcatatcagg 960attatcaata ccatattttt gaaaaagccg tttctgtaat
gaaggagaaa actcaccgag 1020gcagttccat aggatggcaa gatcctggta
tcggtctgcg attccgactc gtccaacatc 1080aatacaacct attaatttcc
cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg 1140agtgacgact
gaatccggtg agaatggcaa aagcttatgc atttctttcc agacttgttc
1200aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac
cgttattcat 1260tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg
ttaaaaggac aattacaaac 1320aggaatcgaa tgcaaccggc gcaggaacac
tgccagcgca tcaacaatat tttcacctga 1380atcaggatat tcttctaata
cctggaatgc tgttttcccg gggatcgcag tggtgagtaa 1440ccatgcatca
tcaggagtac ggataaaatg cttgatggtc ggaagaggca taaattccgt
1500cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac
ctttgccatg 1560tttcagaaac aactctggcg catcgggctt cccatacaat
cgatagattg tcgcacctga 1620ttgcccgaca ttatcgcgag cccatttata
cccatataaa tcagcatcca tgttggaatt 1680taatcgcggc ctcgagcaag
acgtttcccg ttgaatatgg ctcataacac cccttgtatt 1740actgtttatg
taagcagaca gttttattgt tcatgatgat atatttttat cttgtgcaat
1800gtaacatcag agattttgag acacaacgtg gctttccccc ccccccctgc
aggtcaaccc 1860cggcggaaat taacaccacc ctcaataaaa aatccggttt
gctcggagtc tctgggctgt 1920cggcggatct tcgtaccatt ttgcaggcca
aagcagaggg taatgaacaa gctcaattgg 1980cttatgtcat gtatatccat
cgcttccgga gttgtttggg gcaaatgatt gcttccttgg 2040aaggtttgga
tacgttggtg tttaccgccg gggtggggga aaatgccgcc actgtgcggg
2100cagatgtttg ccaagctttt gaatttctag gtttaaaact tgatccagag
ttgaataacc 2160gatcgccaag ggatactgtc atttctcact ccgactcctt
ggtgacggtg ttgattgtcc 2220acaccgaaga agattgggcg atcgcccagg
attgttggca ctggtggcat agccagggac 2280agagaaagca atcgtaaatt
gcgaaaatgt tagaaaatgg ctgtgaagat aaatgttgaa 2340ttaggctaaa
tttccttggc tagagtccgc atccgccaac acgtcaaccc cctcagtgaa
2400aaatatcggc aggtgttggc ctgtcccgat tgggccaccg tttatgacga
tgtccaacga 2460ccattgcatc tagatattgg ctgtgcccgg ggtcgctttc
ccctcaaaat ggctcaacaa 2520caccccgact ggaatttttt aggggtggaa
atccgtcaac ccttggtgct agaggccaac 2580gaaaccggcg atcgtctggg
gttaaaaaat ctccattacc tgtttggcaa catcaatgtg 2640gagccagaaa
aattcttttc cgcctttccc cccactctgc aacgggtcag catccaattt
2700cccgatccct ggtttaagca acgacataat aaacgccgag tggcccaacc agaa
275416697PRTSynechocystis sp. strain PCC6803 16Met Thr Ser Ser Leu
Tyr Leu Ser Thr Thr Glu Ala Arg Ser Gly Lys 1 5 10 15 Ser Leu Val
Val Leu Gly Ile Leu Asp Leu Ile Leu Lys Lys Thr Thr 20 25 30 Arg
Ile Ala Tyr Phe Arg Pro Ile Ile Gln Asp Pro Val Asn Gly Lys 35 40
45 His Asp Asn Asn Ile Ile Leu Val Leu Glu Asn Phe Arg Leu Gln Gln
50 55 60 Thr Tyr Thr Asp Ser Phe Gly Leu Tyr Phe His Glu Ala Val
Ser Leu 65 70 75 80 Ala Ser Asp Gly Ala Ile Asp Gln Val Leu Asp Arg
Ile Leu Ala Lys 85 90 95 Tyr Arg His Leu Ala Asp Gln Val Asp Phe
Ile Leu Cys Glu Gly Ser 100 105 110 Asp Tyr Leu Gly Glu Glu Ser Ala
Phe Glu Phe Asp Leu Asn Thr Thr 115 120 125 Ile Ala Lys Met Leu Asn
Cys Pro Ile Leu Leu Leu Gly Asn Ala Met 130 135 140 Gly Asn Thr Ile
Ala Asp Ser Leu Gln Pro Ile Asp Met Ala Leu Asn 145 150 155 160 Ser
Tyr Asp Gln Glu Ser Cys Gln Val Val Gly Val Ile Ile Asn Arg 165 170
175 Val Gln Pro Glu Leu Ala Thr Glu Ile Gln Ala Gln Leu Glu Gln Arg
180 185 190 Tyr Gly Asp Arg Pro Met Val Leu Gly Thr Ile Pro Gln Asp
Ile Met 195 200 205 Leu Lys Ser Leu Arg Leu Arg Glu Ile Val Ser Gly
Leu Asn Ala Gln 210 215 220 Val Leu Ser Gly Ala Asp Leu Leu Asp Asn
Leu Val Tyr His His Leu 225 230 235 240 Val Val Ala Met His Ile Ala
His Ala Leu His Trp Leu His Glu Lys 245 250 255 Asn Thr Leu Ile Ile
Thr Pro Gly Asp Arg Gly Asp Ile Ile Leu Gly 260 265 270 Val Met Gln
Ala His Arg Ser Leu Asn Tyr Pro Ser Ile Ala Gly Ile 275 280 285 Leu
Leu Thr Ala Asp Tyr His Pro Glu Pro Ala Ile Met Lys Leu Ile 290 295
300 Glu Gly Leu Pro Asp Ala Pro Pro Leu Leu Leu Thr Ser Thr His Thr
305 310 315 320 His Glu Thr Ser Ala Arg Leu Glu Thr Leu His Pro Ala
Leu Ser Pro 325 330 335 Thr Asp Asn Tyr Lys Ile Arg His Ser Ile Ala
Leu Phe Gln Gln Gln 340 345 350 Ile Asp Gly Glu Lys Leu Leu Asn Tyr
Leu Lys Thr Ile Arg Ser Lys 355 360 365 Gly Ile Thr Pro Lys Leu Phe
Leu Tyr Asn Leu Val Gln Ala Ala Thr 370 375 380 Ala Ala Gln Arg His
Ile Val Leu Pro Glu Gly Glu Glu Ile Arg Ile 385 390 395 400 Leu Lys
Ala Ala Ala Ser Leu Ile Asn His Gly Ile Val Arg Leu Thr 405 410 415
Leu Leu Gly Asn Ile Glu Ala Ile Glu Gln Thr Val Lys Ile Asn His 420
425 430 Ile Asp Leu Asp Leu Ser Lys Val Arg Leu Ile Asn Pro Lys Thr
Ser 435 440 445 Pro Asp Arg Glu Arg Tyr Ala Glu Thr Tyr Tyr Gln Leu
Arg Lys His 450 455 460 Lys Gly Val Thr Leu Ala Met Ala Arg Asp Ile
Leu Thr Asp Ile Ser 465 470 475 480 Tyr Phe Gly Thr Met Met Val His
Leu Gly Glu Ala Asp Gly Met Val 485 490 495 Ser Gly Ser Val Asn Thr
Thr Gln His Thr Val Arg Pro Ala Leu Gln 500 505 510 Ile Ile Lys Thr
Gln Pro Gly Phe Ser Leu Val Ser Ser Val Phe Phe 515 520 525 Met Cys
Leu Glu Asp Arg Val Leu Val Tyr Gly Asp Cys Ala Val Asn 530 535 540
Pro Asp Pro Asn Ala Glu Gln Leu Ala Glu Ile Ala Leu Thr Ser Ala 545
550 555 560 Ala Thr Ala Lys Asn Phe Gly Ile Glu Pro Arg Val Ala Leu
Leu Ser 565 570 575 Tyr Ser Ser Gly Ser Ser Gly Gln Gly Ala Asp Val
Glu Lys Val Arg 580 585 590 Gln Ala Thr Ala Ile Ala Lys Glu Arg Glu
Pro Asp Leu Ala Leu Glu 595 600 605 Gly Pro Ile Gln Tyr Asp Ala Ala
Val Asp Ser Thr Val Ala Ala Gln 610 615 620 Lys Met Pro Gly Ser Ala
Val Ala Gly Lys Ala Thr Val Phe Ile Phe 625 630 635 640 Pro Asp Leu
Asn Thr Gly Asn Asn Thr Tyr Lys Ala Val Gln Arg Glu 645 650 655 Thr
Lys Ala Ile Ala Ile Gly Pro Ile Leu Gln Gly Leu Asn Lys Pro 660 665
670 Val Asn Asp Leu Ser Arg Gly Cys Leu Val Glu Asp Ile Ile Asn Thr
675 680 685 Val Val Ile Thr Ala Leu Gln Val Lys 690 695
173654DNAartificialconstruct pUC pta-Cm 17ttggccaaaa aacaaggttt
actgggtttt accgctgatg ttttactgga aaatcgagcc 60atgttgcatc tatttgagaa
gatgaacttt cgcatggaac gacgtatgag cgaaggggtt 120tacgaattaa
aaatgttttt tagttgagcc gtcttctttc tgctaattta ttgaaggaat
180ttttgatgct ggcgttagta attttaccgc ttcttagatt tattaaaatc
tcgtcataaa 240actttactga ctagcggttt attttctggc taaaagcgct
atcacttaag taggtggaat 300tggcagattt gtagtagttg atacttaact
ttttagggaa tatcgctgtg ggaaaaatcg 360agatcatttt cccagaaaaa
tcattgctgg atacgttgag gttatttaaa ttatgacgag 420ttccctttat
ttaagcacca ccgaagcccg cagcggtaaa tctctagtag tattgggcat
480tttagactta attctcaaaa aaaccacccg tattgcctat tttcgtccca
ttattcaaga 540cccagttaat ggcaaacatg ataacaacat tattctggtg
ctggaaaatt ttcgtctcca 600acaaacctat accgattcct ttggtttgta
tttccatgaa gcggtgagtt tagcctccga 660tggagctatt gatcaggtat
tagaccgaat tttggctaaa tatcgccatt tggcagatca 720agtagatttt
attctctgtg aaggctcaga ctatttgggg gaggaatcgg cttttgaatt
780tgatctcaac accacgatcg ccaagatgtt gaactgcccc attttgctgt
tgggcaatgc 840catgggcaac accattgccg atagtttgca acccatcgat
tttccatggc agctgagaat 900attgtaggag atcttctaga aagatcctgt
gacggaagtt aacttcgcag aataaataaa 960tcctggtgtc cctgttgata
ccgggaagcc ctgggccaac ttttggcgaa aatgagacgt 1020tgatcggcac
gtaagaggtt ccaactttca ccataatgaa ataagatcac taccgggcgt
1080attttttgag ttatcgagat tttcaggagc taaggaagct aaaatggaga
aaaaaatcac 1140tggatatacc accgttgata tatcccaatg gcatcgtaaa
gaacattttg aggcatttca 1200gtcagttgct caatgtacct ataaccagac
cgttcagctg gatattacgg cctttttaaa 1260gaccgtaaag aaaaataagc
acaagtttta tccggccttt attcacattc ttgcccgcct 1320gatgaatgct
catccggaat tccgtatggc aatgaaagac ggtgagctgg tgatatggga
1380tagtgttcac ccttgttaca ccgttttcca tgagcaaact gaaacgtttt
catcgctctg 1440gagtgaatac cacgacgatt tccggcagtt tctacacata
tattcgcaag atgtggcgtg 1500ttacggtgaa aacctggcct atttccctaa
agggtttatt gagaatatgt ttttcgtctc 1560agccaatccc tgggtgagtt
tcaccagttt tgatttaaac gtggccaata tggacaactt 1620cttcgccccc
gttttcacca tgggcaaata ttatacgcaa ggcgacaagg tgctgatgcc
1680gctggcgatt caggttcatc atgccgtttg tgatggcttc catgtcggca
gaatgcttaa 1740tgaattacaa cagtactgcg atgagtggca gggcggggcg
taattttttt aaggcagtta 1800ttggtgccct taaacgcctg gttgctacgc
ctgaataagt gataataagc ggttgactgg 1860cagaaattcg atcttgctga
aaaactcgag ccatccggaa gatctggcgg ccgctctccc 1920tatagtgagt
cgtattacgc cggatggata tggtgttcag gcacaagtgt taaagcagtt
1980gattttattc actatgatga aaaaaacaat gaatggaacc tgctccaagt
taaaaataga 2040gataataccg aaaactcatc gagtagtaag attagagata
atacaacaat aaaaaaatgg 2100tttagaactt actcacagcg tgatgctact
aattgggaca attttccaga tgaagtatca 2160tctaagaatt taaatgaaga
agacttcaga gcttttgtta aaaattattt ggcaaaaata 2220atataattcg
gctgcagatt accatcccga accggccatt atgaaactaa ttgaagggct
2280acccgacgcc cctcccctgt tgctgactag cacccacacc catgaaactt
ccgcccgttt 2340ggaaactctc caccctgccc tgagccctac ggataattat
aaaattcgcc acagtattgc 2400gctgtttcaa caacaaattg atggggagaa
attactcaat taccttaaaa ccatccgcag 2460taaaggtatt acccccaaac
tgtttctcta caatttagtt caagccgcca ccgccgccca 2520acgacatatt
gtcctaccgg aaggggaaga aattcgtatt ctcaaggcgg ccgctagctt
2580aattaaccac ggcattgtcc gtttgacttt actcggtaac attgaggcga
tcgagcaaac 2640ggtaaaaatt aatcacattg acttagattt gagcaaagtt
cgcctcatta atcctaaaac 2700tagcccagac cgagagcgct acgccgaaac
ctattaccag ctacgtaaac ataagggggt 2760aaccctggcc atggctcggg
atatcctcac cgatatttcc tattttggaa cgatgatggt 2820gcatttggga
gaggccgatg gcatggtttc tggctccgtc aataccaccc aacataccgt
2880gcgtcctgct ttacaaatta ttaaaaccca gccaggtttt tccttggttt
cttcagtctt 2940ttttatgtgt ttagaagacc gagttttggt ctatggagat
tgtgctgtta atcccgatcc 3000caatgcagaa cagttagcag aaattgccct
tacttctgcg gctacggcca agaattttgg 3060cattgagccc agggtagctc
tattgtccta ttcttccggt tcttctgggc aaggggccga 3120tgtggaaaaa
gtgcggcaag ccacggcgat cgccaaggaa agagagccag atttagcatt
3180ggaagggccg atccagtatg atgcggcggt ggattccaca gtggcggccc
aaaaaatgcc 3240tgggtcagcg gtggcgggta aagcaacggt gtttattttt
cccgatttaa ataccggtaa 3300caatacttac aaggcagtgc aaagagaaac
aaaggcgatc gccattggcc ccattttaca 3360aggattaaat aaaccagtta
atgatctaag tcggggttgt ttagtggagg atattattaa 3420tacggtggta
attacagctt tgcaagttaa ataattttac tcttaattag ttaaaatgat
3480cccttgaatt accttgattt tgccctccaa actaccaata gctgggccga
aaattggcat 3540catttaaaat caccaacgtg tccccggacg gagctagcac
aaacagaccc ttaccatagg 3600catagctgac cacttcttgg cttaacacca
tggctgccac tgcacctaaa gctt 365418378PRTSynechocystis sp. strain
PCC6803 18Met Phe Leu Leu Phe Phe Ile Val His Trp Leu Lys Ile Met
Leu Pro 1 5 10 15 Phe Phe Ala Gln Val Gly Leu Glu Glu Asn Leu His
Glu Thr Leu Asp 20 25 30 Phe Thr Glu Lys Phe Leu Ser Gly Leu Glu
Asn Leu Gln Gly Leu Asn 35 40 45 Glu Asp Asp Ile Gln Val Gly Phe
Thr Pro Lys Glu Ala Val Tyr Gln 50 55 60 Glu Asp Lys Val Ile Leu
Tyr Arg Phe Gln Pro Val Val Glu Asn Pro 65 70 75 80 Leu Pro Ile Pro
Val Leu Ile Val Tyr Ala Leu Val Asn Arg Pro Tyr 85 90 95 Met Val
Asp Leu Gln Glu Gly Arg Ser Leu Val Ala Asn Leu Leu Lys 100 105 110
Leu Gly Leu Asp Val Tyr Leu Ile Asp Trp Gly Tyr Pro Ser Arg Gly 115
120 125 Asp Arg Trp Leu Thr Leu Glu Asp Tyr Leu Ser Gly Tyr Leu Asn
Asn 130 135 140 Cys Val Asp Ile Ile Cys Gln Arg Ser Gln Gln Glu Lys
Ile Thr Leu 145 150 155 160 Leu Gly Val Cys Gln Gly Gly Thr Phe Ser
Leu Cys Tyr Ala Ser Leu 165 170 175 Phe Pro Asp Lys Val Lys Asn Leu
Val Val Met Val Ala Pro Val Asp 180 185 190 Phe Glu Gln Pro Gly Thr
Leu Leu Asn Ala Arg Gly Gly Cys Thr Leu 195 200 205 Gly Ala Glu Ala
Val Asp Ile Asp Leu Met Val Asp Ala Met Gly Asn 210 215 220 Ile Pro
Gly Asp Tyr Leu Asn Leu Glu Phe Leu Met Leu Lys Pro Leu 225 230 235
240 Gln Leu Gly Tyr Gln Lys Tyr Leu Asp Val Pro Asp Ile Met Gly Asp
245 250 255 Glu Ala Lys Leu Leu Asn Phe Leu Arg Met Glu Lys Trp Ile
Phe Asp 260 265 270 Ser Pro Asp Gln Ala Gly Glu Thr Tyr Arg Gln Phe
Leu Lys Asp Phe 275 280 285 Tyr Gln Gln Asn Lys Leu Ile Lys Gly Glu
Val Met Ile Gly Asp Arg 290 295 300 Leu Val Asp Leu His Asn Leu Thr
Met Pro Ile Leu Asn Leu Tyr Ala 305 310 315 320 Glu Lys Asp His Leu
Val Ala Pro Ala Ser Ser Leu Ala Leu Gly Asp 325 330 335 Tyr Leu Pro
Glu Asn Cys Asp Tyr Thr Val Gln Ser Phe Pro Val Gly 340 345 350 His
Ile Gly Met Tyr Val Ser Gly Lys Val Gln Arg Asp Leu Pro Pro 355 360
365 Ala Ile Ala His Trp Leu Ser Glu Arg Gln 370 375
192121DNAartificialconstruct pIC2OH/deltaphaC-KM 19tctagataat
tcaccatcaa tgtttttact attttttatc gttcattggt taaaaattat 60gttgcctttt
tttgctcagg tggggttaga agaaaatctc catgaaaccc tagattttac
120tgaaaaattt ctctctggct tggaaaattt gcagggtttg aatgaagatg
acatccaggt 180gggctttacc cccaaagaag cagtttacca ggaagataag
gttattcttt accgtttcca 240accggtggtg gaaaatccct tacctatccc
ggttttaatt gtttacgccc tggtaaatcg 300cccctacatg gtggatttgc
aggaaggacg ctccctggtg gccaacctcc tcaaactggg 360tttggacgtg
tatttaattg attggggtta tccctcccgg
ggcgatcgtt ggatccgtcg 420acctgcaggg gggggggggc gctgaggtct
gcctcgtgaa gaaggtgttg ctgactcata 480ccaggcctga atcgccccat
catccagcca gaaagtgagg gagccacggt tgatgagagc 540tttgttgtag
gtggaccagt tggtgatttt gaacttttgc tttgccacgg aacggtctgc
600gttgtcggga agatgcgtga tctgatcctt caactcagca aaagttcgat
ttattcaaca 660aagccgccgt cccgtcaagt cagcgtaatg ctctgccagt
gttacaacca attaaccaat 720tctgattaga aaaactcatc gagcatcaaa
tgaaactgca atttattcat atcaggatta 780tcaataccat atttttgaaa
aagccgtttc tgtaatgaag gagaaaactc accgaggcag 840ttccatagga
tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc aacatcaata
900caacctatta atttcccctc gtcaaaaata aggttatcaa gtgagaaatc
accatgagtg 960acgactgaat ccggtgagaa tggcaaaagc ttatgcattt
ctttccagac ttgttcaaca 1020ggccagccat tacgctcgtc atcaaaatca
ctcgcatcaa ccaaaccgtt attcattcgt 1080gattgcgcct gagcgagacg
aaatacgcga tcgctgttaa aaggacaatt acaaacagga 1140atcgaatgca
accggcgcag gaacactgcc agcgcatcaa caatattttc acctgaatca
1200ggatattctt ctaatacctg gaatgctgtt ttcccgggga tcgcagtggt
gagtaaccat 1260gcatcatcag gagtacggat aaaatgcttg atggtcggaa
gaggcataaa ttccgtcagc 1320cagtttagtc tgaccatctc atctgtaaca
tcattggcaa cgctaccttt gccatgtttc 1380agaaacaact ctggcgcatc
gggcttccca tacaatcgat agattgtcgc acctgattgc 1440ccgacattat
cgcgagccca tttataccca tataaatcag catccatgtt ggaatttaat
1500cgcggcctcg agcaagacgt ttcccgttga atatggctca taacacccct
tgtattactg 1560tttatgtaag cagacagttt tattgttcat gatgatatat
ttttatcttg tgcaatgtaa 1620catcagagat tttgagacac aacgtggctt
tccccccccc ccctgcaggt cgacggatcc 1680tcttaaccta gaatttctca
tgcttaaacc cctgcaatta ggttaccaaa agtatcttga 1740tgtgcccgat
attatggggg atgaagcgaa attgttaaac tttctacgca tggaaaaatg
1800gatttttgat agtcccgatc aagcggggga aacttaccgt caattcctca
aggattttta 1860tcaacaaaat aaattgatca aaggggaagt gatgattggc
gatcgcctgg tggatctgca 1920taatttgacc atgcccatat tgaatttata
tgcggaaaaa gaccacttgg tggcccctgc 1980ttcttcccta gctttggggg
actatttgcc ggaaaactgt gactacaccg tccaatcttt 2040ccccgtgggt
catattggca tgtatgtcag tggtaaagta caacgggatc tgcccccggc
2100gatcgcccat tggctatcga t 212120429PRTSynechocystis PCC6701 20Met
Lys Lys Val Leu Ala Ile Ile Leu Gly Gly Gly Ala Gly Thr Arg 1 5 10
15 Leu Tyr Pro Leu Thr Lys Leu Arg Ala Lys Pro Ala Val Pro Val Ala
20 25 30 Gly Lys Tyr Arg Leu Ile Asp Ile Pro Val Ser Asn Cys Ile
Asn Ser 35 40 45 Glu Ile Phe Lys Ile Tyr Val Leu Thr Gln Phe Asn
Ser Ala Ser Leu 50 55 60 Asn Arg His Ile Ala Arg Thr Tyr Asn Phe
Ser Gly Phe Ser Glu Gly 65 70 75 80 Phe Val Glu Val Leu Ala Ala Gln
Gln Thr Pro Glu Asn Pro Asn Trp 85 90 95 Phe Gln Gly Thr Ala Asp
Ala Val Arg Gln Tyr Leu Trp Met Leu Gln 100 105 110 Glu Trp Asp Val
Asp Glu Phe Leu Ile Leu Ser Gly Asp His Leu Tyr 115 120 125 Arg Met
Asp Tyr Arg Leu Phe Ile Gln Arg His Arg Glu Thr Asn Ala 130 135 140
Asp Ile Thr Leu Ser Val Ile Pro Ile Asp Asp Arg Arg Ala Ser Asp 145
150 155 160 Phe Gly Leu Met Lys Ile Asp Asn Ser Gly Arg Val Ile Asp
Phe Ser 165 170 175 Glu Lys Pro Lys Gly Glu Ala Leu Thr Lys Met Arg
Val Asp Thr Thr 180 185 190 Val Leu Gly Leu Thr Pro Glu Gln Ala Ala
Ser Gln Pro Tyr Ile Ala 195 200 205 Ser Met Gly Ile Tyr Val Phe Lys
Lys Asp Val Leu Ile Lys Leu Leu 210 215 220 Lys Glu Ala Leu Glu Arg
Thr Asp Phe Gly Lys Glu Ile Ile Pro Asp 225 230 235 240 Ala Ala Lys
Asp His Asn Val Gln Ala Tyr Leu Phe Asp Asp Tyr Trp 245 250 255 Glu
Asp Ile Gly Thr Ile Glu Ala Phe Tyr Asn Ala Asn Leu Ala Leu 260 265
270 Thr Gln Gln Pro Met Pro Pro Phe Ser Phe Tyr Asp Glu Glu Ala Pro
275 280 285 Ile Tyr Thr Arg Ala Arg Tyr Leu Pro Pro Thr Lys Leu Leu
Asp Cys 290 295 300 His Val Thr Glu Ser Ile Ile Gly Glu Gly Cys Ile
Leu Lys Asn Cys 305 310 315 320 Arg Ile Gln His Ser Val Leu Gly Val
Arg Ser Arg Ile Glu Thr Gly 325 330 335 Cys Met Ile Glu Glu Ser Leu
Leu Met Gly Ala Asp Phe Tyr Gln Ala 340 345 350 Ser Val Glu Arg Gln
Cys Ser Ile Asp Lys Gly Asp Ile Pro Val Gly 355 360 365 Ile Gly Pro
Asp Thr Ile Ile Arg Arg Ala Ile Ile Asp Lys Asn Ala 370 375 380 Arg
Ile Gly His Asp Val Lys Ile Ile Asn Lys Asp Asn Val Gln Glu 385 390
395 400 Ala Asp Arg Glu Ser Gln Gly Phe Tyr Ile Arg Ser Gly Ile Val
Val 405 410 415 Val Leu Lys Asn Ala Val Ile Thr Asp Gly Thr Ile Ile
420 425 215916DNAartificialinsert of pRL271 agp (all4645)::C.K3 -
PpetE-pdc-adhII 21gagctctgtt aacagtcaac agtcatttca caaattaagg
caagattaag aaaatactgt 60aaccattaac atatctaata tttttaatca tgagtgcaaa
ttaatacagt ggaaattgtt 120ttctgatcaa tggctgcacg atacgtcacc
agtaaggttt tttaaaattc attcaagata 180atctttgatc cccccttacc
agctgccaca gacagtccta aactgtaggt gggagttgaa 240aggcagttgg
gagaaatctt gtgaaaaaag tcttagcaat tattcttggt ggtggtgcgg
300gtactcgcct ttacccacta accaaactcc gcgctaaacc ggcagtacca
gtggcaggga 360aataccgcct aatagatatc cctgtcagta actgcattaa
ttcggaaatt tttaaaatct 420acgtattaac acaatttaac tcagcttctc
tcaatcgcca cattgcccgt acctacaact 480ttagtggttt tagcgagggt
tttgtggaag tgctggccgc ccagcagaca ccagagaacc 540ctaactggtt
ccaaggtaca gccgatgctg tacgtcagta tctctggatg ttacaagagt
600gggacgtaga tgaatttttg atcctgtcgg gggatcacct gtaccggatg
gactatcgcc 660tatttatcca gcgccatcta gaggatcccc aaatggcaaa
ttatttatga cggtaggctt 720aatagcctgt aaaaatttgt aacaatattt
tttgtttttg caataaacaa aaacaaatgc 780ctccgattag aaatcggagg
cattgtttgc ttgaaaatca agacaggacg gaaaaccgtt 840ttcctgtttt
gaaattagaa agcgctcagg aagagttctt caacttcttt ctgatcaccc
900tgacgcgggt tggtcagagc acaagcatct ttcagagcgt ggtcagcaag
aagcggcaca 960tcttctttct tagcacccag ctcggtcaga tttgctggaa
taccaatgga agcagccaga 1020tcgcgaacag cctgaatggt ggcttctgcg
ccttctttat caccgagatt ggcgatatcg 1080agacccatag caacaccaac
gtctttcaga cgaccagcaa cgacagaggc gttataagcc 1140agaacatgcg
gaagcagaac agcgttgcag acaccatgcg gcaggttgta gtagccgccc
1200aactggtgag ccatagcatg gacataacca agcgaagcgt tgttgaaggc
cataccagcg 1260aggaattggg cataagccat agcttcacga gctggcatat
ccttaccgtt gtcgcaagcg 1320gtcttcagat tcttagcgat catggacgca
gccttcaagg cgcaagcatc ggtgatcgga 1380gtagctgccg ttgaagaata
agcttcaaat gcgtgggtca gagcatccat accggtggcg 1440gcggtcaggc
cttttggcat accaaccatc aacagaggat cgttgacgga aaccatcggg
1500gtaacgtgac ggtcaacaat ggccatctta acgtgacgga cttcatcagt
gatgatgcag 1560aaacgcgtca tttcagaagc cgtaccagcc gtcgtgttga
ttgacatcaa aggcagggca 1620ggtttcttag atttgtcgat accttcgtag
tctttgactt caccaccatt ggttgcgacc 1680agagcgatgg ctttggcgca
gtcatgggga gaaccaccac cgagggagat gacgaagtct 1740gaattgttat
ccttcaggat cttaaggcct tccagaactg cggtaacagt cgggttcggc
1800ataacgccat cataaacagc agaattaata ccctgtgctt tcaacaggtc
agcaacctgc 1860ttcacaacac cggatttgtt catgaaagca tcagaaacga
tcagcgcatt tttaaagccg 1920ctgccgttaa gatccttgat tgctttttca
agcgaacctt cgcccatttc gttgacgaaa 1980ggaatataaa aagttgaaga
agccatagct ataacctcac cctacatact agtttgggta 2040ccgagctcga
attgatcccc aaaaactaga ggagcttgtt aacaggctta cggctgttgg
2100cggcagcaac gcgcttaccc catttgacca attcttcagt gcagtcttca
cgaccgatga 2160agcattcgat cagggttggg ccgtcggtgt ttgccagagc
aaccttgata gcttctgcca 2220gttcgccacc ggttttagcc ttcaggcctt
taccagcacc gctgtcataa ccaccgttac 2280cgttgaacac ttccatcaga
ccggcataat cccagttctt gatgttgttg tacggaccat 2340catggatcat
aacttcgatg gtgtaaccat agttattgat caagaagatg ataaccggca
2400gtttcaggcg aaccatctga gcgacttcct gagccgtcag ctggaaggaa
ccatcaccaa 2460ccatgaggat gttgcgacgt tccggagcac cgacggcata
accgaaggcg gcaggaacgg 2520accaaccgat gtgaccccac tgcatttcat
attcaacgcg agcaccgttc gggagcttca 2580tgcgctgagc attgaaccaa
gagtcaccgg tttcagcaat aaccgtcgtg ttcggggtca 2640gaagagcttc
gacctgacgg gcgatttctg cgttgaccaa cggagcactc ggatcagccg
2700gagcggcttt cttcagttca cctgcattga gggatttgaa gaagtccaaa
gcaccggttt 2760tcttggaaac tttctgagcc aaacgggtca gatagtcttt
cagatgaacg ctggggaagc 2820gaacgccgtt aacgacgaca gaacgcggtt
cagcgagaac cagtttctta ggatcaggaa 2880tatccgtcca accagtggtg
gagtagtcgt tgaagacagg agccagagcg ataaccgcat 2940cggcttcttt
catcgtcttt tcaacgcccg gatagctgac ttcaccccat gaggtaccga
3000tgtaatgcgg gttttcttct gggaagaagc tttttgcagc agccatggta
gcaactgcgc 3060caccgagagc atcagcaaat ttgacagcag cttcttcagc
accagctgcg cgcagcttgc 3120tgccgacgag gacggcaact ttgtcgcggt
tggcgatgaa tttcagggtt tcttcaaccg 3180ctgcattcaa agaagcttcg
tcgctggctt cgtcattgaa caatgcgctt gccggtccag 3240gagcggcgca
gggcatggaa gcaatgttgc aagcgatttc gagataaacc ggcttcttct
3300cacgaagagc agttttaatc acgtgatcga ttttagccgg agcttcttct
ggggtgtaaa 3360tcgcttcagc tgcggccgtg atgttcttgg ccatttccaa
ctgatagtga tagtcggttt 3420tgccaagagc gtgatgcaac acgtgaccag
cagcgtgatc attgttgttc ggagcaccgg 3480agatcaggat aaccggaagg
ttttctgcat aggcgccacc gatagcatca aatgcggaaa 3540gcgcaccgac
gctgtaggta acgacggctg ctgctgcgcc tttggcacga gcataacctt
3600ctgcactgaa accgcagttc agttcgttac agcaataaac ctgctccatg
tttttgttca 3660aaagcaggtt gtcaagaagg acgaggttgt agtcgcccgc
gactgcgaag tgatgcttga 3720gaccaatctg gacaagccgc tccgctaaat
aggtaccgac agtataagaa ttcatggcgt 3780tctcctaacc tgtagtttta
tttttcttat ttcattttaa ataaaatcga caccaaatat 3840gacaatctgt
catacttcgg gcaaattttt tttgagatcg cgataaatcc agggaaaaat
3900gctctgcgat cgctaaaaat cactataatt acatggcact aaaaaactta
tggctaaaaa 3960tacatcaata tatgcagcca gagcgacaaa ttaaattttt
tatttatgtg atgtagataa 4020aagatatctt tcccctatac ccctacactc
tccttcaagt catagaggcg cggagattgc 4080ccaattttat tagctgtgta
ctcagtacct cagcaaaaaa gtcgacatat gtttctcggc 4140aaaaattaat
tatcgattgg ctggaacctg gtcaaaccag ggcttttcat ccattggaaa
4200agcgattttg atcatctagg gtcaggagca aagatccccg gtgggcgaag
aactccagca 4260tgagatcccc gcgctggagg atcatccagc cggcgtcccg
gaaaacgatt ccgaagccca 4320acctttcata gaaggcggcg gtggaatcga
aatctcgtga tggcaggttg ggcgtcgctt 4380ggtcggtcat ttcgaacccc
agagtcccgc tcagaagaac tcgtcaagaa ggcgatagaa 4440ggcgatgcgc
tgcgaatcgg gagcggcgat accgtaaagc acgaggaagc ggtcagccca
4500ttcgccgcca agctcttcag caatatcacg ggtagccaac gctatgtcct
gatagcggtc 4560cgccacaccc agccggccac agtcgatgaa tccagaaaag
cggccatttt ccaccatgat 4620attcggcaag caggcatcgc catgggtcac
gacgagatcc tcgccgtcgg gcatgcgcgc 4680cttgagcctg gcgaacagtt
cggctggcgc gagcccctga tgctcttcgt ccagatcatc 4740ctgatcgaca
agaccggctt ccatccgagt acgtgctcgc tcgatgcgat gtttcgcttg
4800gtggtcgaat gggcaggtag ccggatcaag cgtatgcagc cgccgcattg
catcagccat 4860gatggatact ttctcggcag gagcaaggtg agatgacagg
agatcctgcc ccggcacttc 4920gcccaatagc agccagtccc ttcccgcttc
agtgacaacg tcgagcacag ctgcgcaagg 4980aacgcccgtc gtggccagcc
acgatagccg cgctgcctcg tcctgcagtt cattcagggc 5040accggacagg
tcggtcttga caaaaagaac cgggcgcccc tgcgctgaca gccggaacac
5100ggcggcatca gagcagccga ttgtctgttg tgcccagtca tagccgaata
gcctctccac 5160ccaagcggcc ggagaacctg cgtgcaatcc atcttgttca
atcatgcgaa acgatcctca 5220tcctgtctct tgatcagatc cgtccttgtt
attcaacagt ataacatgtc ttatacgccc 5280gtgtcaacca atattcattg
agatcctcta gacgagtcat tgatttcagt gaaaaaccca 5340agggcgaagc
cttaaccaaa atgcgtgttg ataccacggt tttaggcttg acaccagaac
5400aggcggcatc acagccttac attgcctcga tggggattta cgtatttaaa
aaagacgttt 5460tgatcaagct gttgaaggaa gctttagaac gtactgattt
cggcaaagaa attattcctg 5520atgccgccaa agatcacaac gttcaagctt
acctattcga tgactactgg gaagatattg 5580ggacaatcga agctttttat
aacgccaatt tagcgttaac tcagcagccc atgccgccct 5640ttagcttcta
cgatgaagaa gcacctattt atacccgcgc tcgttactta ccacccacaa
5700aactattaga ttgccacgtt acagaatcaa tcattggcga aggctgtatt
ctgaaaaact 5760gtcgcattca acactcagta ttgggagtgc gatcgcgtat
tgaaactggc tgcatgatcg 5820aagaatcttt actcatgggt gccgacttct
accaagcttc agtggaacgc cagtgcagca 5880tcgataaagg agacatccct
gtaggcatcg ctcgag 591622429PRTAnabaena variabilis ATCC29314 22Met
Lys Lys Val Leu Ala Ile Ile Leu Gly Gly Gly Ala Gly Thr Arg 1 5 10
15 Leu Tyr Pro Leu Thr Lys Leu Arg Ala Lys Pro Ala Val Pro Val Ala
20 25 30 Gly Lys Tyr Arg Leu Ile Asp Ile Pro Val Ser Asn Cys Ile
Asn Ser 35 40 45 Glu Ile Phe Lys Ile Tyr Val Leu Thr Gln Phe Asn
Ser Ala Ser Leu 50 55 60 Asn Arg His Ile Ala Arg Thr Tyr Asn Phe
Ser Gly Phe Ser Glu Gly 65 70 75 80 Phe Val Glu Val Leu Ala Ala Gln
Gln Thr Pro Glu Asn Pro Asn Trp 85 90 95 Phe Gln Gly Thr Ala Asp
Ala Val Arg Gln Tyr Leu Trp Met Leu Gln 100 105 110 Glu Trp Asp Val
Asp Glu Phe Leu Ile Leu Ser Gly Asp His Leu Tyr 115 120 125 Arg Met
Asp Tyr Arg Leu Phe Ile Gln Arg His Arg Glu Thr Asn Ala 130 135 140
Asp Ile Thr Leu Ser Val Ile Pro Ile Asp Asp Arg Arg Ala Ser Asp 145
150 155 160 Phe Gly Leu Met Lys Ile Asp Asn Ser Gly Arg Val Ile Asp
Phe Ser 165 170 175 Glu Lys Pro Lys Gly Glu Ala Leu Thr Lys Met Arg
Val Asp Thr Thr 180 185 190 Val Leu Gly Leu Thr Pro Glu Gln Ala Ala
Ser Gln Pro Tyr Ile Ala 195 200 205 Ser Met Gly Ile Tyr Val Phe Lys
Lys Asp Val Leu Ile Lys Leu Leu 210 215 220 Lys Glu Ser Leu Glu Arg
Thr Asp Phe Gly Lys Glu Ile Ile Pro Asp 225 230 235 240 Ala Ser Lys
Asp His Asn Val Gln Ala Tyr Leu Phe Asp Asp Tyr Trp 245 250 255 Glu
Asp Ile Gly Thr Ile Glu Ala Phe Tyr Asn Ala Asn Leu Ala Leu 260 265
270 Thr Gln Gln Pro Met Pro Pro Phe Ser Phe Tyr Asp Glu Glu Ala Pro
275 280 285 Ile Tyr Thr Arg Ala Arg Tyr Leu Pro Pro Thr Lys Leu Leu
Asp Cys 290 295 300 His Val Thr Glu Ser Ile Ile Gly Glu Gly Cys Ile
Leu Lys Asn Cys 305 310 315 320 Arg Ile Gln His Ser Val Leu Gly Val
Arg Ser Arg Ile Glu Thr Gly 325 330 335 Cys Val Ile Glu Glu Ser Leu
Leu Met Gly Ala Asp Phe Tyr Gln Ala 340 345 350 Ser Val Glu Arg Gln
Cys Ser Ile Asp Lys Gly Asp Ile Pro Val Gly 355 360 365 Ile Gly Pro
Asp Thr Ile Ile Arg Arg Ala Ile Ile Asp Lys Asn Ala 370 375 380 Arg
Ile Gly His Asp Val Lys Ile Ile Asn Lys Asp Asn Val Gln Glu 385 390
395 400 Ala Asp Arg Glu Ser Gln Gly Phe Tyr Ile Arg Ser Gly Ile Val
Val 405 410 415 Val Leu Lys Asn Ala Val Ile Thr Asp Gly Thr Ile Ile
420 425 23324PRTSynechocystis sp. strain PCC6803 23Met Ala Glu Thr
Leu Leu Phe Ala Ala Leu Arg Gln Ala Leu Asp Glu 1 5 10 15 Glu Met
Gly Arg Asp Val Asn Val Leu Val Leu Gly Glu Asp Val Gly 20 25 30
Leu Tyr Gly Gly Ser Tyr Lys Val Thr Lys Asp Leu Tyr Glu Lys Tyr 35
40 45 Gly Glu Met Arg Val Leu Asp Thr Pro Ile Ala Glu Asn Ser Phe
Thr 50 55 60 Gly Met Ala Val Gly Ala Ala Met Thr Gly Leu Arg Pro
Val Ile Glu 65 70 75 80 Gly Met Asn Met Gly Phe Leu Leu Leu Ala Phe
Asn Gln Ile Ala Asn 85 90 95 Asn Ala Gly Met Leu Arg Tyr Thr Ser
Gly Gly Asn Tyr Gln Ile Pro 100 105 110 Met Val Ile Arg Gly Pro Gly
Gly Val Gly Arg Gln Leu Gly Ala Glu 115 120 125 His Ser Gln Arg Leu
Glu Ala Tyr Phe His Ala Val Pro Gly Leu Lys 130 135 140 Ile Val Ala
Cys Ser Thr Pro Tyr Asn Ala Lys Gly Leu Leu Lys Ala 145 150 155 160
Ala Ile Arg Asp Asn Asn Pro Val Leu Phe Phe Glu His Val Leu Leu 165
170 175 Tyr Asn Leu Lys Glu Asn Leu Pro Asp Tyr Glu Tyr Ile Val Pro
Leu 180 185 190 Asp Lys Ala Glu Val Val Arg Pro Gly Lys Asp Val Thr
Ile Leu Thr 195 200 205 Tyr Ser Arg Met Arg His His Cys Leu Gln Ala
Leu Lys Thr Leu Glu 210 215 220 Lys Glu Gly Tyr Asp Pro Glu Ile Ile
Asp Leu Ile Ser Leu Lys Pro 225 230 235 240 Phe Asp Met Glu
Thr Ile Ser Ala Ser Val Lys Lys Thr His Arg Val 245 250 255 Ile Ile
Val Glu Glu Cys Met Lys Thr Gly Gly Ile Gly Ala Glu Leu 260 265 270
Ile Ala Leu Ile Asn Asp His Leu Phe Asp Glu Leu Asp Gly Pro Val 275
280 285 Val Arg Leu Ser Ser Gln Asp Ile Pro Thr Pro Tyr Asn Gly Met
Leu 290 295 300 Glu Arg Leu Thr Ile Val Gln Pro Pro Gln Ile Val Asp
Ala Val Lys 305 310 315 320 Ala Ile Ile Gly
24257DNAartificialinsert for pSK9/pdhBanti 24cgatataatt tccgggtcgt
agccttcttt ttccaaagtt tttagtgcct gtaaacaatg 60gtgacgcatg cgggaatagg
tcaaaatagt gacatcctta ccggggcgca ccacttcggc 120tttatccaga
ggcacaatat attcgtagtc gggtaagttt tctttcaagt tgtacaaaag
180tacgtgctca aaaaataaca ctgggttatt atcccgaatg gctgccgctc
ggttgccgcc 240gggcgttttt tattcca 257251128DNAartificialinsert for
the construct pSK9/pdhB 25atatggctga gaccctactg tttgccgccc
tacgccaagc ccttgacgaa gaaatgggac 60gggatgtcaa cgtccttgtg ctgggagaag
atgtgggact ctatggcggt tcctataagg 120taaccaagga tttgtacgag
aagtatggcg aaatgcgggt gctggatacg cccatcgccg 180aaaacagttt
taccggcatg gcggtggggg cggccatgac aggattgcgc ccagtcattg
240aaggcatgaa tatgggtttt cttctgctgg cgtttaacca aattgccaat
aatgcgggga 300tgttgcgcta tacctccggc ggcaattacc aaattcccat
ggttatccgt ggtcctgggg 360gcgtaggtcg gcaattaggg gcagaacatt
cccaacggtt ggaggcctat ttccatgcgg 420tgccggggtt aaaaattgtg
gcttgctcca ccccctataa cgccaaggga ttgctcaaag 480cagccattcg
ggataataac ccagtgttat tttttgagca cgtacttttg tacaacttga
540aagaaaactt acccgactac gaatatattg tgcctctgga taaagccgaa
gtggtgcgcc 600ccggtaagga tgtcactatt ttgacctatt cccgcatgcg
tcaccattgt ttacaggcac 660taaaaacttt ggaaaaagaa ggctacgacc
cggaaattat tgatttgatt tccctcaagc 720cttttgacat ggaaaccatc
agcgcttcgg tgaagaaaac ccatcgggtc attattgtcg 780aagaatgtat
gaaaaccgga ggcatcggcg ctgagctcat tgccctaatc aatgatcatc
840tctttgatga gttagatggg ccggtggtgc gcctttcttc ccaagatatt
cccactccct 900acaacggtat gttggaacga ctgaccattg tgcaaccgcc
ccaaattgtg gacgcagtta 960aggcgatcat cggctaattg gcctcaaaac
agtacccctt gccgtaaacg gaattaatca 1020ggcggggggc attgggctgt
tcaattttgc gccgcagtaa tcgcaccagg gccgccagca 1080cgttactact
gggggggctt tcgccgggcc aaaggtggga atagatct
1128262441DNAartificialinsert of construct pGEM-T/?pdhB-KMantisense
26gggattaatc gacatccacc cttgtcccct gactaaaatc tccgctatgg tggccagcca
60gtgtgtcaaa atactagatt gttgtgcccc gatcgccctt gcttggtttg cctacaccgg
120accaacaagg agagatgcgg cggcggtcta aattgttatt aaaaactaag
ttttccccca 180gaaatcatgg ctgagaccct actgtttgcc gccctacgcc
aagcccttga cgaagaaatg 240ggacgggatg tcaacgtcct tgtgctggga
gaagatgtgg gactctatgg cggttcctat 300aaggtaacca aggatttgta
cgagaagtat ggcgaaatgc gggtgctgga tacgcccatc 360gccgaaaaca
gttttaccgg catggcggtg ggggcggcca tgacaggatt gcgcccagtc
420attgaaggca tgaatatggg ttttcttctg ctggcgttta accaaattgc
caataatgcg 480gggatgttgc gctatacctc cggcggcaat taccaaattc
ccatggttat ccgtggtcct 540gggggcgtag gtcggcaatt aggggcagaa
cattcccaac ggttggaggg aattccccgg 600atccgtcgac ctgcaggggg
gggggggcgc tgaggtctgc ctcgtgaaga aggtgttgct 660gactcatacc
aggcctgaat cgccccatca tccagccaga aagtgaggga gccacggttg
720atgagagctt tgttgtaggt ggaccagttg gtgattttga acttttgctt
tgccacggaa 780cggtctgcgt tgtcgggaag atgcgtgatc tgatccttca
actcagcaaa agttcgattt 840attcaacaaa gccgccgtcc cgtcaagtca
gcgtaatgct ctgccagtgt tacaaccaat 900taaccaattc tgattagaaa
aactcatcga gcatcaaatg aaactgcaat ttattcatat 960caggattatc
aataccatat ttttgaaaaa gccgtttctg taatgaagga gaaaactcac
1020cgaggcagtt ccataggatg gcaagatcct ggtatcggtc tgcgattccg
actcgtccaa 1080catcaataca acctattaat ttcccctcgt caaaaataag
gttatcaagt gagaaatcac 1140catgagtgac gactgaatcc ggtgagaatg
gcaaaagctt atgcatttct ttccagactt 1200gttcaacagg ccagccatta
cgctcgtcat caaaatcact cgcatcaacc aaaccgttat 1260tcattcgtga
ttgcgcctga gcgagacgaa atacgcgatc gctgttaaaa ggacaattac
1320aaacaggaat cgaatgcaac cggcgcagga acactgccag cgcatcaaca
atattttcac 1380ctgaatcagg atattcttct aatacctgga atgctgtttt
cccggggatc gcagtggtga 1440gtaaccatgc atcatcagga gtacggataa
aatgcttgat ggtcggaaga ggcataaatt 1500ccgtcagcca gtttagtctg
accatctcat ctgtaacatc attggcaacg ctacctttgc 1560catgtttcag
aaacaactct ggcgcatcgg gcttcccata caatcgatag attgtcgcac
1620ctgattgccc gacattatcg cgagcccatt tatacccata taaatcagca
tccatgttgg 1680aatttaatcg cggcctcgag caagacgttt cccgttgaat
atggctcata acaccccttg 1740tattactgtt tatgtaagca gacagtttta
ttgttcatga tgatatattt ttatcttgtg 1800caatgtaaca tcagagattt
tgagacacaa cgtggctttc cccccccccc ctgcaggtcg 1860acggatccgg
ggaattccct atttccatgc ggtgccgggg ttaaaaattg tggcttgctc
1920caccccctat aacgccaagg gattgctcaa agcagccatt cgggataata
acccagtgtt 1980attttttgag cacgtacttt tgtacaactt gaaagaaaac
ttacccgact acgaatatat 2040tgtgcctctg gataaagccg aagtggtgcg
ccccggtaag gatgtcacta ttttgaccta 2100ttcccgcatg cgtcaccatt
gtttacaggc actaaaaact ttggaaaaag aaggctacga 2160cccggaaatt
attgatttga tttccctcaa gccttttgac atggaaacca tcagcgcttc
2220ggtgaagaaa acccatcggg tcattattgt cgaagaatgt atgaaaaccg
gaggcatcgg 2280cgctgagctc attgccctaa tcaatgatca tctctttgat
gagttagatg ggccggtggt 2340gcgcctttct tcccaagata ttcccactcc
ctacaacggt atgttggaac gactgaccat 2400tgtgcaaccg ccccaaattg
tggacgcagt taaggcaatc a 2441273000DNAartificialvector pGEM-T
27gggcgaattg ggcccgacgt cgcatgctcc cggccgccat ggccgcggga tatcactagt
60gcggccgcct gcaggtcgac catatgggag agctcccaac gcgttggatg catagcttga
120gtattctata gtgtcaccta aatagcttgg cgtaatcatg gtcatagctg
tttcctgtgt 180gaaattgtta tccgctcaca attccacaca acatacgagc
cggaagcata aagtgtaaag 240cctggggtgc ctaatgagtg agctaactca
cattaattgc gttgcgctca ctgcccgctt 300tccagtcggg aaacctgtcg
tgccagctgc attaatgaat cggccaacgc gcggggagag 360gcggtttgcg
tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
420ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
tccacagaat 480caggggataa cgcaggaaag aacatgtgag caaaaggcca
gcaaaaggcc aggaaccgta 540aaaaggccgc gttgctggcg tttttccata
ggctccgccc ccctgacgag catcacaaaa 600atcgacgctc aagtcagagg
tggcgaaacc cgacaggact ataaagatac caggcgtttc 660cccctggaag
ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
720ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
aggtatctca 780gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
cgaacccccc gttcagcccg 840accgctgcgc cttatccggt aactatcgtc
ttgagtccaa cccggtaaga cacgacttat 900cgccactggc agcagccact
ggtaacagga ttagcagagc gaggtatgta ggcggtgcta 960cagagttctt
gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct
1020gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
tccggcaaac 1080aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
gcagattacg cgcagaaaaa 1140aaggatctca agaagatcct ttgatctttt
ctacggggtc tgacgctcag tggaacgaaa 1200actcacgtta agggattttg
gtcatgagat tatcaaaaag gatcttcacc tagatccttt 1260taaattaaaa
atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
1320gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
cgttcatcca 1380tagttgcctg actccccgtc gtgtagataa ctacgatacg
ggagggctta ccatctggcc 1440ccagtgctgc aatgataccg cgagacccac
gctcaccggc tccagattta tcagcaataa 1500accagccagc cggaagggcc
gagcgcagaa gtggtcctgc aactttatcc gcctccatcc 1560agtctattaa
ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
1620acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt
atggcttcat 1680tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
ccccatgttg tgcaaaaaag 1740cggttagctc cttcggtcct ccgatcgttg
tcagaagtaa gttggccgca gtgttatcac 1800tcatggttat ggcagcactg
cataattctc ttactgtcat gccatccgta agatgctttt 1860ctgtgactgg
tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
1920gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
ttaaaagtgc 1980tcatcattgg aaaacgttct tcggggcgaa aactctcaag
gatcttaccg ctgttgagat 2040ccagttcgat gtaacccact cgtgcaccca
actgatcttc agcatctttt actttcacca 2100gcgtttctgg gtgagcaaaa
acaggaaggc aaaatgccgc aaaaaaggga ataagggcga 2160cacggaaatg
ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
2220gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
caaatagggg 2280ttccgcgcac atttccccga aaagtgccac ctgatgcggt
gtgaaatacc gcacagatgc 2340gtaaggagaa aataccgcat caggaaattg
taagcgttaa tattttgtta aaattcgcgt 2400taaatttttg ttaaatcagc
tcatttttta accaataggc cgaaatcggc aaaatccctt 2460ataaatcaaa
agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc
2520cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat
cagggcgatg 2580gcccactacg tgaaccatca ccctaatcaa gttttttggg
gtcgaggtgc cgtaaagcac 2640taaatcggaa ccctaaaggg agcccccgat
ttagagcttg acggggaaag ccggcgaacg 2700tggcgagaaa ggaagggaag
aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag 2760cggtcacgct
gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt
2820ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg
cctcttcgct 2880attacgccag ctggcgaaag ggggatgtgc tgcaaggcga
ttaagttggg taacgccagg 2940gttttcccag tcacgacgtt gtaaaacgac
ggccagtgaa ttgtaatacg actcactata 3000283851DNAartificialvector
pDrive 28gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat
gcagctggca 60cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg
tgagttagct 120cactcattag gcaccccagg ctttacactt tatgcttccg
gctcgtatgt tgtgtggaat 180tgtgagcgga taacaatttc acacaggaaa
cagctatgac catgattacg ccaagctcta 240atacgactca ctatagggaa
agctcggtac cacgcatgct gcagacgcgt tacgtatcgg 300atccagaatt
cgtgatatct gaattcgtcg acaagcttct cgagcctagg ctagctctag
360accacacgtg tgggggcccg agctcgcggc cgctgtattc tatagtgtca
cctaaatggc 420cgcacaattc actggccgtc gttttacaac gtcgtgactg
ggaaaaccct ggcgttaccc 480aacttaatcg ccttgcagca catccccctt
tcgccagctg gcgtaatagc gaagaggccc 540gcaccgatcg cccttcccaa
cagttgcgca gcctgaatgg cgaatggaaa ttgtaagcgt 600taatattttg
ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata
660ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag
ggttgagtgt 720tgttccagtt tggaacaaga gtccactatt aaagaacgtg
gactccaacg tcaaagggcg 780aaaaaccgtc tatcagggcg atggcccact
acgtgaacca tcaccctaat caagtttttt 840ggggtcgagg tgccgtaaag
cactaaatcg gaaccctaaa gggagccccc gatttagagc 900ttgacgggga
aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg
960cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac
ccgccgcgct 1020taatgcgccg ctacagggcg cgtcaggtgg cacttttcgg
ggaaatgtgc gcggaacccc 1080tatttgttta tttttctaaa tacattcaaa
tatgtatccg ctcatgagac aataaccctg 1140ataaatgctt caataatatt
gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc 1200ccttattccc
ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt
1260gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
aactggatct 1320caacagcggt aagatccttg agagttttcg ccccgaagaa
cgttttccaa tgatgagcac 1380ttttaaagtt ctgctatgtg gcgcggtatt
atcccgtatt gacgccgggc aagagcaact 1440cggtcgccgc atacactatt
ctcagaatga cttggttgag tactcaccag tcacagaaaa 1500gcatcttacg
gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga
1560taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
taaccgcttt 1620tttgcacaac atgggggatc atgtaactcg ccttgatcgt
tgggaaccgg agctgaatga 1680agccatacca aacgacgagc gtgacaccac
gatgcctgta gcaatggcaa caacgttgcg 1740caaactatta actggcgaac
tacttactct agcttcccgg caacaattaa tagactggat 1800ggaggcggat
aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat
1860tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag
cactggggcc 1920agatggtaag ccctcccgta tcgtagttat ctacacgacg
gggagtcagg caactatgga 1980tgaacgaaat agacagatcg ctgagatagg
tgcctcactg attaagcatt ggtaactgtc 2040agaccaagtt tactcatata
tactttagat tgatttaaaa cttcattttt aatttaaaag 2100gatctaggtg
aagatccttt ttgataatct catgaacaat aaaactgtct gcttacataa
2160acagtaatac aaggggtgtt atgagccata ttcaacggga aacgtcttgc
tctaggccgc 2220gattaaattc caacatggat gctgatttat atgggtataa
atgggctcgc gataatgtcg 2280ggcaatcagg tgcgacaatc tatcgattgt
atgggaagcc cgatgcgcca gagttgtttc 2340tgaaacatgg caaaggtagc
gttgccaatg atgttacaga tgagatggtc agactaaact 2400ggctgacgga
atttatgcct cttccgacca tcaagcattt tatccgtact cctgatgatg
2460catggttact caccactgcg atccccggga aaacagcatt ccaggtatta
gaagaatatc 2520ctgattcagg tgaaaatatt gttgatgcgc tggcagtgtt
cctgcgccgg ttgcattcga 2580ttcctgtttg taattgtcct tttaacagcg
atcgcgtatt tcgtctcgct caggcgcaat 2640cacgaatgaa taacggtttg
gttgatgcga gtgattttga tgacgagcgt aatggctggc 2700ctgttgaaca
agtctggaaa gaaatgcata aacttttgcc attctcaccg gattcagtcg
2760tcactcatgg tgatttctca cttgataacc ttatttttga cgaggggaaa
ttaataggtt 2820gtattgatgt tggacgagtc ggaatcgcag accgatacca
ggatcttgcc atcctatgga 2880actgcctcgg tgagttttct ccttcattac
agaaacggct ttttcaaaaa tatggtattg 2940ataatcctga tatgaataaa
ttgcagtttc atttgatgct cgatgagttt ttctaagaat 3000taattcatga
ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
3060gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
ctgcttgcaa 3120acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
atcaagagct accaactctt 3180tttccgaagg taactggctt cagcagagcg
cagataccaa atactgtcct tctagtgtag 3240ccgtagttag gccaccactt
caagaactct gtagcaccgc ctacatacct cgctctgcta 3300atcctgttac
cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
3360agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
gtgcacacag 3420cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
tacagcgtga gctatgagaa 3480agcgccacgc ttcccgaagg gagaaaggcg
gacaggtatc cggtaagcgg cagggtcgga 3540acaggagagc gcacgaggga
gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc 3600gggtttcgcc
acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
3660ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
ctggcctttt 3720gctcacatgt tctttcctgc gttatcccct gattctgtgg
ataaccgtat taccgccttt 3780gagtgagctg ataccgctcg ccgcagccga
acgaccgagc gcagcgagtc agtgagcgag 3840gaagcggaag a
3851292961DNAartificialvector pBluescript II SK (+) 29ctaaattgta
agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc 60attttttaac
caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
120gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga
acgtggactc 180caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc
ccactacgtg aaccatcacc 240ctaatcaagt tttttggggt cgaggtgccg
taaagcacta aatcggaacc ctaaagggag 300cccccgattt agagcttgac
ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa 360agcgaaagga
gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
420cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat
tcaggctgcg 480caactgttgg gaagggcgat cggtgcgggc ctcttcgcta
ttacgccagc tggcgaaagg 540gggatgtgct gcaaggcgat taagttgggt
aacgccaggg ttttcccagt cacgacgttg 600taaaacgacg gccagtgagc
gcgcgtaata cgactcacta tagggcgaat tgggtaccgg 660gccccccctc
gaggtcgacg gtatcgataa gcttgatatc gaattcctgc agcccggggg
720atccactagt tctagagcgg ccgccaccgc ggtggagctc cagcttttgt
tccctttagt 780gagggttaat tgcgcgcttg gcgtaatcat ggtcatagct
gtttcctgtg tgaaattgtt 840atccgctcac aattccacac aacatacgag
ccggaagcat aaagtgtaaa gcctggggtg 900cctaatgagt gagctaactc
acattaattg cgttgcgctc actgcccgct ttccagtcgg 960gaaacctgtc
gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
1020gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
gttcggctgc 1080ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
atccacagaa tcaggggata 1140acgcaggaaa gaacatgtga gcaaaaggcc
agcaaaaggc caggaaccgt aaaaaggccg 1200cgttgctggc gtttttccat
aggctccgcc cccctgacga gcatcacaaa aatcgacgct 1260caagtcagag
gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
1320gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
tccgcctttc 1380tcccttcggg aagcgtggcg ctttctcata gctcacgctg
taggtatctc agttcggtgt 1440aggtcgttcg ctccaagctg ggctgtgtgc
acgaaccccc cgttcagccc gaccgctgcg 1500ccttatccgg taactatcgt
cttgagtcca acccggtaag acacgactta tcgccactgg 1560cagcagccac
tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
1620tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc
tgcgctctgc 1680tgaagccagt taccttcgga aaaagagttg gtagctcttg
atccggcaaa caaaccaccg 1740ctggtagcgg tggttttttt gtttgcaagc
agcagattac gcgcagaaaa aaaggatctc 1800aagaagatcc tttgatcttt
tctacggggt ctgacgctca gtggaacgaa aactcacgtt 1860aagggatttt
ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
1920aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
agttaccaat 1980gcttaatcag tgaggcacct atctcagcga tctgtctatt
tcgttcatcc atagttgcct 2040gactccccgt cgtgtagata actacgatac
gggagggctt accatctggc cccagtgctg 2100caatgatacc gcgagaccca
cgctcaccgg ctccagattt atcagcaata aaccagccag 2160ccggaagggc
cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
2220attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
aacgttgttg 2280ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
tatggcttca ttcagctccg 2340gttcccaacg atcaaggcga gttacatgat
cccccatgtt gtgcaaaaaa gcggttagct 2400ccttcggtcc tccgatcgtt
gtcagaagta agttggccgc agtgttatca ctcatggtta 2460tggcagcact
gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
2520gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
tgctcttgcc 2580cggcgtcaat acgggataat accgcgccac atagcagaac
tttaaaagtg ctcatcattg 2640gaaaacgttc ttcggggcga aaactctcaa
ggatcttacc gctgttgaga tccagttcga 2700tgtaacccac tcgtgcaccc
aactgatctt cagcatcttt tactttcacc agcgtttctg 2760ggtgagcaaa
aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
2820gttgaatact catactcttc ctttttcaat attattgaag catttatcag
ggttattgtc 2880tcatgagcgg atacatattt gaatgtattt agaaaaataa
acaaataggg gttccgcgca 2940catttccccg aaaagtgcca c
2961302686DNAartificialvector pUC 19 30tcgcgcgttt cggtgatgac
ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60cagcttgtct gtaagcggat
gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120ttggcgggtg
tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
180accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc
atcaggcgcc 240attcgccatt caggctgcgc aactgttggg aagggcgatc
ggtgcgggcc tcttcgctat 300tacgccagct ggcgaaaggg ggatgtgctg
caaggcgatt aagttgggta acgccagggt 360tttcccagtc acgacgttgt
aaaacgacgg ccagtgaatt cgagctcggt acccggggat 420cctctagagt
cgacctgcag gcatgcaagc ttggcgtaat catggtcata gctgtttcct
480gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag
cataaagtgt 540aaagcctggg gtgcctaatg agtgagctaa ctcacattaa
ttgcgttgcg ctcactgccc 600gctttccagt cgggaaacct gtcgtgccag
ctgcattaat gaatcggcca acgcgcgggg 660agaggcggtt tgcgtattgg
gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg 720gtcgttcggc
tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
780gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
ggccaggaac 840cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
gcccccctga cgagcatcac 900aaaaatcgac gctcaagtca gaggtggcga
aacccgacag gactataaag ataccaggcg 960tttccccctg gaagctccct
cgtgcgctct cctgttccga ccctgccgct taccggatac 1020ctgtccgcct
ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
1080ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
ccccgttcag 1140cccgaccgct gcgccttatc cggtaactat cgtcttgagt
ccaacccggt aagacacgac 1200ttatcgccac tggcagcagc cactggtaac
aggattagca gagcgaggta tgtaggcggt 1260gctacagagt tcttgaagtg
gtggcctaac tacggctaca ctagaaggac agtatttggt 1320atctgcgctc
tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
1380aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
tacgcgcaga 1440aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
ggtctgacgc tcagtggaac 1500gaaaactcac gttaagggat tttggtcatg
agattatcaa aaaggatctt cacctagatc 1560cttttaaatt aaaaatgaag
ttttaaatca atctaaagta tatatgagta aacttggtct 1620gacagttacc
aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
1680tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
cttaccatct 1740ggccccagtg ctgcaatgat accgcgagac ccacgctcac
cggctccaga tttatcagca 1800ataaaccagc cagccggaag ggccgagcgc
agaagtggtc ctgcaacttt atccgcctcc 1860atccagtcta ttaattgttg
ccgggaagct agagtaagta gttcgccagt taatagtttg 1920cgcaacgttg
ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
1980tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
gttgtgcaaa 2040aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
gtaagttggc cgcagtgtta 2100tcactcatgg ttatggcagc actgcataat
tctcttactg tcatgccatc cgtaagatgc 2160ttttctgtga ctggtgagta
ctcaaccaag tcattctgag aatagtgtat gcggcgaccg 2220agttgctctt
gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
2280gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
accgctgttg 2340agatccagtt cgatgtaacc cactcgtgca cccaactgat
cttcagcatc ttttactttc 2400accagcgttt ctgggtgagc aaaaacagga
aggcaaaatg ccgcaaaaaa gggaataagg 2460gcgacacgga aatgttgaat
actcatactc ttcctttttc aatattattg aagcatttat 2520cagggttatt
gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
2580ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac
cattattatc 2640atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtc
2686316267DNAartificialvector pSK9 31cacctaaatt gtaagcgtta
atattttgtt aaaattcgcg ttaaattttt gttaaatcag 60ctcatttttt aaccaatagg
ccgaaatcgg caaaatccct tataaatcaa aagaatagac 120cgagataggg
ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga
180ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac
gtgaaccatc 240accctaatca agttttttgg ggtcgaggtg ccgtaaagca
ctaaatcgga accctaaagg 300gagcccccga tttagagctt gacggggaaa
gccggcgaac gtggcgagaa aggaagggaa 360gaaagcgaaa ggagcgggcg
ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac 420caccacaccc
gccgcgctta atgcgccgct acagggcgcg tcccattcgc cattcaggct
480gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc
agctggcgaa 540agggggatgt gctgcaaggc gattaagttg ggtaacgcca
gggttttccc agtcacgacg 600ttgtaaaacg acggccagtg aattgtaata
cgactcacta tagggcgaat tggaggccag 660tgctggagga atatgatttt
gtcatcctcg actgtgcccc tggttataat ctgttgaccc 720gcagtggcat
tgcggccagc gacttttatc tgttgccggc tcgtcctgaa cccctatcgg
780tggtggggat gcagttactg gaaagaagaa ttgagaaact gaaggaaagc
cataaggcct 840ccgatgatcc cctgaatatc aatctgatcg gagtggtgtt
tattctgtcc ggcggcggtt 900tgatgagtcg ctactataac caggtaatgc
ggcgggtaca aacggatttc accccgggac 960aactttttca gcagtccatt
cccatggatg tcaatgtggc taaggcagtg gatagcttta 1020tgccggtggt
tacctccatg cccaatacgg cgggttcaaa agcttttatt aaattaaccc
1080aggaattttt acagaaagta gaagcttttg gctaaagcaa agcccccatt
gattaacaac 1140gggaggggta ccgaggtgct gctgaagttg cccgcaacag
agagtggaac caaccggtga 1200taccacgata ctatgactga gagtcaacgc
catgagcggc ctcatttctt attctgagtt 1260acaacagtcc gcaccgctgt
ccggtagctc cttccggtgg gcgcggggca tgactatcgt 1320cgccgcactt
atgactgtct tctttatcat gcaactcgta ggacaggtgc cggcagcgcc
1380caacagtccc ccggccacgg ggcctgccac catacccacg ccgaaacaag
cgccctgcac 1440cattatgttc cggatctgca tcgcaggatg ctgctggcta
ccctgtggaa cacctacatc 1500tgtattaacg aagcgctaac cgtttttatc
aggctctggg aggcagaata aatgatcata 1560tcgtcaatta ttacctccac
ggggagagcc tgagcaaact ggcctcaggc atttgagaag 1620cacacggtca
cactgcttcc ggtagtcaat aaaccggtaa accagcaata gacataagcg
1680gctatttaac gaccctgccc tgaaccgacg accgggtcga atttgctttc
gaatttctgc 1740cattcatccg cttattatca cttattcagg cgtagcacca
ggcgtttaag ggcaccaata 1800actgccttaa aaaaattacg ccccgccctg
ccactcatcg cagtactgtt gtaattcatt 1860aagcattctg ccgacatgga
agccatcaca gacggcatga tgaacctgaa tcgccagcgg 1920catcagcacc
ttgtcgcctt gcgtataata tttgcccatg gtgaaaacgg gggcgaagaa
1980gttgtccata ttggccacgt ttaaatcaaa actggtgaaa ctcacccagg
gattggctga 2040gacgaaaaac atattctcaa taaacccttt agggaaatag
gccaggtttt caccgtaaca 2100cgccacatct tgcgaatata tgtgtagaaa
ctgccggaaa tcgtcgtggt attcactcca 2160gagcgatgaa aacgtttcag
tttgctcatg gaaaacggtg taacaagggt gaacactatc 2220ccatatcacc
agctcaccgt ctttcattgc catacggaat tccggatgag cattcatcag
2280gcgggcaaga atgtgaataa aggccggata aaacttgtgc ttatttttct
ttacggtctt 2340taaaaaggcc gtaatatcca gctgaacggt ctggttatag
gtacattgag caactgactg 2400aaatgcctca aaatgttctt tacgatgcca
ttgggatata tcaacggtgg tatatccagt 2460gatttttttc tccattttag
cttccttagc tcctgaaaat ctcgataact caaaaaatac 2520gcccggtagt
gatcttattt cattatggtg aaagttggaa cctcttacct cggtacccct
2580catcgggggc tgtgttggcc gagacggcac tgaggatttt actctccatg
gcattccaag 2640gaatatctac ccaactcacc tgctccggcg gattgttccg
ctcaaaagta ctaatcaagt 2700cgtcaaaata cttattaaat tttggctgca
attgcatagt ccaaaagctg actttcccct 2760ccatgctctg gggggaattg
ctctggcaac tgattaatcc actgagcaac agcccaagac 2820acgcaaacaa
aaaccaacgt cttggcgatc gccatcggca ccatgaaacc atcgtaaaag
2880ctggggaaag aataaaaaac agtggttcag gaattgcatt gccatggcca
cttcacaaac 2940ctagccaatt ttagcttgac cgcaactttg acagattgtc
ttttgacttt gcctggaccg 3000cctcccataa taccttcgcg tcttgaagac
tttatccttg aaaggagaac atatgtttct 3060cggcaaaaat taattatcga
ttggctggaa cctggtcaaa ccagggcttt tcatccattg 3120gaaaagcgat
tttgatcatc tagggtcagg agcaaagatc tgatcaaata ttgatcattt
3180attaggaaag ctgaactttc accactttat ttttggcttc ctctactttg
ggcaaagtca 3240aagttaggat accggcatcg taattagctt taacttctgt
gttttggatt gctccaggta 3300caggaataac ccggcggaaa ctgccatagc
ggaactctgt gcgccgcacc ccatcttttt 3360cggtgctatg ggtatcctgg
cgatcgccgc tgacggtcac cgcatccctg gcggcttgga 3420tgtccaaatt
atcggggtcc atgccaggta attctagttt gagcacatag gcttcttcag
3480tttcagttag ttctgcttta ggattaaacc cttggcgatc gccgtggcgg
tccgtaggga 3540caaaaacttc ttcaaacagt tggttcatct gctgctggaa
attatccatt tcccgcaggg 3600gattgtaaag aatgagagac ataatgttaa
ctcctgatgt gtggaaggaa ttgattaccc 3660ttgaatggtt ctatcttaaa
atttcccctt ccaggttaga ttcggttttc aggaaagaag 3720gtggggggat
tgccgaaatt acatttctag ccgcaatttt tagtaaaaaa aagatgagtt
3780tttacctcac cttaagtaaa tatttgagtg gcaaaacaaa atggtaaaaa
tagctaagct 3840tccaccgccc tatggatttt tggaaggaag tcttaggttg
tgaaaaacta taaaaaccaa 3900ccataggaat ggagaccttt acccaacaag
ttgaccccta ggtaacaaat ccaaaccacc 3960gtaaaaccgc tggcggccaa
aatagcgggc ttgcggcctt gccaaccttt ggtaatgcgg 4020gcatggagat
aggcggcaaa tactagccag gtgattaggg cccggtaccc agcttttgtt
4080ccctttagtg agggttaatt tcgagcttgg cgtaatcatg gtcatagctg
tttcctgtgt 4140gaaattgtta tccgctcaca attccacaca acatacgagc
cggaagcata aagtgtaaag 4200cctggggtgc ctaatgagtg agctaactca
cattaattgc gttgcgctca ctgcccgctt 4260tccagtcggg aaacctgtcg
tgccagctgc attaatgaat cggccaacgc gcggggagag 4320gcggtttgcg
tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
4380ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
tccacagaat 4440caggggataa cgcaggaaag aacatgtgag caaaaggcca
gcaaaaggcc aggaaccgta 4500aaaaggccgc gttgctggcg tttttccata
ggctccgccc ccctgacgag catcacaaaa 4560atcgacgctc aagtcagagg
tggcgaaacc cgacaggact ataaagatac caggcgtttc 4620cccctggaag
ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
4680ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
aggtatctca 4740gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
cgaacccccc gttcagcccg 4800accgctgcgc cttatccggt aactatcgtc
ttgagtccaa cccggtaaga cacgacttat 4860cgccactggc agcagccact
ggtaacagga ttagcagagc gaggtatgta ggcggtgcta 4920cagagttctt
gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct
4980gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
tccggcaaac 5040aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
gcagattacg cgcagaaaaa 5100aaggatctca agaagatcct ttgatctttt
ctacggggtc tgacgctcag tggaacgaaa 5160actcacgtta agggattttg
gtcatgagat tatcaaaaag gatcttcacc tagatccttt 5220taaattaaaa
atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
5280gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
cgttcatcca 5340tagttgcctg actccccgtc gtgtagataa ctacgatacg
ggagggctta ccatctggcc 5400ccagtgctgc aatgataccg cgagacccac
gctcaccggc tccagattta tcagcaataa 5460accagccagc cggaagggcc
gagcgcagaa gtggtcctgc aactttatcc gcctccatcc 5520agtctattaa
ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
5580acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt
atggcttcat 5640tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
ccccatgttg tgcaaaaaag 5700cggttagctc cttcggtcct ccgatcgttg
tcagaagtaa gttggccgca gtgttatcac 5760tcatggttat ggcagcactg
cataattctc ttactgtcat gccatccgta agatgctttt 5820ctgtgactgg
tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
5880gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
ttaaaagtgc 5940tcatcattgg aaaacgttct tcggggcgaa aactctcaag
gatcttaccg ctgttgagat 6000ccagttcgat gtaacccact cgtgcaccca
actgatcttc agcatctttt actttcacca 6060gcgtttctgg gtgagcaaaa
acaggaaggc aaaatgccgc aaaaaaggga ataagggcga 6120cacggaaatg
ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
6180gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
caaatagggg 6240ttccgcgcac atttccccga aaagtgc
626732463PRTSynechocystis sp. strain PCC6803 32Met Val Ser Leu Thr
Pro Asn Pro Ser Tyr Ser Val Ser Leu Leu Leu 1 5 10 15 Glu Leu Pro
Asn His Ala Gly Thr Leu Ala Ser Val Thr Gln Ala Ile 20 25 30 Ala
Asp Ala Gly Gly Ser Phe Gly Gln Ile Ser Leu Ile Glu Ser Asn 35 40
45 Leu Lys Leu Thr Arg Arg Glu Ile Ala Val Asp Ala Ser Ser Ser Glu
50 55 60 His Ala Glu Lys Ile Ile Gly Ala Val Lys Ala Leu Asp Asn
Val Lys 65 70 75 80 Leu Leu Lys Val Ser Asp Arg Thr Phe Asp Leu His
Arg Gln Gly Lys 85 90 95 Ile Ser Val Val Ser Arg Ile Pro Leu Thr
Ser Gln Ser Asp Leu Ala 100 105 110 Met Ala Tyr Thr Pro Gly Val Gly
Arg Ile Cys Arg Ala Ile Ala Glu 115 120 125 Asp Pro Glu Lys Val Tyr
Ser Leu Thr Ile Lys Ser Asn Thr Val Ala 130 135 140 Val Val Thr Asp
Gly Ser Ala Val Leu Gly Leu Gly Asn Leu Gly Pro 145 150 155 160 Glu
Ala Ala Leu Pro Val Met Glu Gly Lys Ala Met Leu Phe Lys Glu 165 170
175 Phe Ala Gln Leu Asp Ala Phe Pro Ile Cys Leu Asp Thr Gln Asp Thr
180 185 190 Glu Glu Ile Ile Arg Thr Val Lys Ala Ile Ala Pro Val Phe
Gly Gly 195 200 205 Val Asn Leu Glu Asp Ile Ala Ala Pro Arg Cys Phe
Glu Ile Glu Ala 210 215 220 Arg Leu Lys Lys Glu Leu Asn Ile Pro Val
Phe His Asp Asp Gln His 225 230 235 240 Gly Thr Ala Ile Val Thr Leu
Ala Ala Leu Leu Asn Ala Leu Lys Phe 245 250 255 Val Gly Lys Ala Met
Ala Ala Val Arg Ile Val Ile Asn Gly Ala Gly 260 265 270 Ala Ala Gly
Leu Ala Ile Ala Glu Leu Leu Lys Glu Ser Gly Ala Thr 275 280 285 Asp
Ile Trp Ile Cys Asp Ser Lys Gly Ile Val Gly Lys His Arg Thr 290 295
300 Asp Leu Asn Ser Lys Lys Gln Ser Phe Ala Val Asp Ala Glu Gly Thr
305 310 315 320 Leu Ala Asp Ala Met Ala Gly Ala Asp Val Phe Leu Gly
Val Ser Ala 325 330 335 Pro Gly Val Val Thr Lys Glu Met Val Gln Ser
Met Ala Lys Asp Pro 340 345 350 Ile Val Phe Ala Met Ala Asn Pro Ile
Pro Glu Ile Gln Pro Glu Leu 355 360 365 Ile Gln Glu Asp Ala Ala Val
Ile Ala Thr Gly Arg Ser Asp Tyr Pro 370 375 380 Asn Gln Ile Asn Asn
Val Leu Ala Phe Pro Gly Val Phe Arg Gly Ala 385 390 395 400 Ile Asp
Cys Arg Ala Ser Ile Ile Thr Thr Thr Met Cys Ile Glu Ala 405 410 415
Ala Lys Ala Ile Ala Ser Leu Val His Ser Asn Thr Leu Asp Ser Glu 420
425 430 His Ile Ile Pro Ser Val Phe Asp Asn Arg Val Ala Thr Thr Val
Ala 435 440 445 Ser Ala Val Gln Leu Ala Ala Arg Asn Glu Gly Val Ala
Gly Gln 450 455 460 331545DNAartificialinsert of construct
pSK9/me-long 33tatggttagc ctcaccccca atccgagtta tagcgtcagc
ctactgttgg aactccccaa 60ccacgccgga actttggcca gcgttaccca ggcgatcgcc
gatgcggggg gcagttttgg 120gcaaatttcc ctgattgaga gtaacttaaa
actcacccgg cgggaaattg cggtggatgc 180ttccagcagt gagcacgccg
aaaaaattat tggggcagtg aaagctctgg ataatgtcaa 240attgctgaag
gtgtccgatc gcacctttga tttacaccgt cagggcaaaa ttagcgtggt
300tagtcgcatt cccctcacct cccaatcgga tttggccatg gcctataccc
caggggtggg 360gcgcatctgt cgggcgatcg ccgaagatcc ggaaaaggtt
tattccctga ccattaaaag 420caatacggtg gcggtggtga ccgatggcag
tgcggtgttg gggttgggta acctggggcc 480ggaagcggct ttaccagtga
tggaaggcaa ggccatgtta ttcaaggaat ttgcccaact 540ggacgctttt
cccatctgtt tggataccca ggatacggag gaaattattc gcaccgtcaa
600ggcgatcgcc ccggtgtttg gcggcgtaaa tttggaagac attgccgctc
cccggtgttt 660tgaaattgaa gcccggctga aaaaagaatt aaatattcct
gtatttcacg atgatcagca 720cggcaccgcc attgttaccc tggccgcttt
gttaaatgcc ctcaaatttg ttggtaaagc 780catggccgct gtccgcattg
tcatcaacgg cgctggggct gctgggttgg cgatcgccga 840attgctcaag
gaatccggag ccaccgatat ttggatttgc gactccaagg gcattgtggg
900caaacatcgc accgatttaa acagcaaaaa acagagcttt gcggtggatg
cggaagggac 960tttagccgat gctatggctg gagctgatgt gtttttaggg
gtgagtgcgc cgggggtagt 1020gaccaaggaa atggtgcaat ccatggccaa
ggacccgatt gtgtttgcca tggccaaccc 1080tatccccgaa attcagccgg
aattaatcca agaggatgcg gcggttattg ccacggggcg 1140cagtgattac
cccaaccaaa ttaacaatgt gcttgccttt ccgggggttt tccggggagc
1200cattgactgt agagctagca ttattaccac caccatgtgc atcgaagcgg
ccaaggcgat 1260cgcctctttg gtgcacagca acaccctaga tagtgagcat
attattcctt cggtttttga 1320caatcgggtc gccactaccg tagccagtgc
agtgcagttg gccgcccgca atgaaggggt 1380ggccggtcaa tagttaatcg
ggaattgtta aacctttact ggtcaaccat tcctgattgt 1440aaagacggga
ttggtaacgg gctcctccgt cacaaagtac ggtaacaatg gtatgccccg
1500gcccaagttt tttggccaat tggtaagccg ctcccacatt aatat
154534324PRTSynechocystis sp. strain PCC6803 34Met Asn Ile Leu Glu
Tyr Ala Pro Ile Ala Cys Gln Ser Trp Gln Val 1 5 10 15 Thr Val Val
Gly Ala Gly Asn Val Gly Arg Thr Leu Ala Gln Arg Leu 20 25 30 Val
Gln Gln Asn Val Ala Asn Val Val Leu Leu Asp Ile Val Pro Gly 35 40
45 Leu Pro Gln Gly Ile Ala Leu Asp Leu Met Ala Ala Gln Ser Val Glu
50 55 60 Glu Tyr Asp Ser Lys Ile Ile Gly Thr Asn Glu Tyr Glu Ala
Thr Ala 65 70 75 80 Gly Ser Asp Val Val Val Ile Thr Ala Gly Leu Pro
Arg Arg Pro Gly 85 90 95 Met Ser Arg Asp Asp Leu Leu Gly Lys Asn
Ala Asn Ile Val Ala Gln 100 105 110 Gly Ala Arg Glu Ala Leu Arg Tyr
Ser Pro Asn Ala Ile Leu Ile Val 115 120 125 Val Thr Asn Pro Leu Asp
Val Met Thr Tyr Leu Ala Trp Lys Val Thr 130 135 140 Gly Leu Pro Ser
Gln Arg Val Met Gly Met Ala Gly Val Leu Asp Ser 145 150 155 160 Ala
Arg Leu Lys Ala Phe Ile Ala Met Lys Leu Gly Ala Cys Pro Ser 165 170
175 Asp Ile Asn Thr Leu Val Leu Gly Gly His Gly Asp Leu Met Leu Pro
180 185 190 Leu Pro Arg Tyr Cys Thr Val Ser Gly Val Pro Ile Thr Glu
Leu Ile 195 200 205 Pro Pro Gln Thr Ile Glu Glu Leu Val Glu Arg Thr
Arg Asn Gly Gly 210 215 220 Ala Glu Ile Ala Ala Leu Leu Gln Thr Gly
Thr Ala Tyr Tyr Ala Pro 225 230 235 240 Ala Ser Ser Ala Ala Val Met
Val Glu Ser Ile Leu Arg Asn Gln Ser 245 250 255 Arg Ile Leu Pro Ala
Ala Thr Tyr Leu Asp Gly Ala Tyr Gly Leu Lys 260
265 270 Asp Ile Phe Leu Gly Val Pro Cys Arg Leu Gly Cys Arg Gly Val
Glu 275 280 285 Asp Ile Leu Glu Val Gln Leu Thr Pro Glu Glu Lys Ala
Ala Leu His 290 295 300 Leu Ser Ala Glu Ala Val Arg Leu Asn Ile Asp
Val Ala Leu Ala Met 305 310 315 320 Val Ser Asp Gly
351027DNAartificialinsert of construct pSK9-mdh 35tatgaatatt
ttggagtatg ctccgatcgc ctgtcagtcc tggcaggtta ccgtggtcgg 60cgctggcaat
gtggggcgga cccttgccca gaggttagtg cagcaaaatg tcgccaacgt
120agttttgttg gacattgtgc caggcttacc ccagggcatt gccttggatt
tgatggccgc 180ccagagcgtg gaggaatacg acagcaaaat cattggcacc
aatgaatacg aggccaccgc 240cggctccgat gtggtggtaa ttaccgctgg
tctaccccgc aggcccggca tgagtcggga 300tgatttgttg ggcaaaaacg
ccaacattgt ggcccagggg gcccgggaag cattgcgtta 360ttcccccaac
gccattttga ttgtggtcac caatcccctg gatgtaatga cctatttggc
420ctggaaagta actggtttac cttcccaacg ggttatgggc atggcggggg
tgttggactc 480ggctcggctc aaggccttca ttgcgatgaa attaggggcc
tgtccttctg atatcaacac 540cttagtgctg ggcgggcacg gagatttgat
gctgcccttg ccacgatact gcaccgtcag 600cggggttccc attaccgaat
taataccccc ccaaaccatt gaagagttgg tggagcgtac 660ccgtaacggt
ggggctgaaa ttgccgcctt actacaaacg ggcacagcct attatgcgcc
720ggcctcttcc gctgcggtga tggtggagtc cattttacgc aatcagtcta
gaattctccc 780cgccgccacc taccttgatg gtgcctatgg attgaaggac
attttccttg gagtgccctg 840ccgtttgggg tgtcgaggag tggaagatat
tctcgaagtg caattaaccc ctgaagaaaa 900agctgccctc catctttctg
cagaagcagt tcgccttaat attgatgtgg cgttggccat 960ggttagcgac
ggttaacacg ataacggaca gtgccaatac cgttttttca ccgaggttag 1020ggcttat
1027362606DNAartificialinsert of construct pSK9/me-mdh 36tatggttagc
ctcaccccca atccgagtta tagcgtcagc ctactgttgg aactccccaa 60ccacgccgga
actttggcca gcgttaccca ggcgatcgcc gatgcggggg gcagttttgg
120gcaaatttcc ctgattgaga gtaacttaaa actcacccgg cgggaaattg
cggtggatgc 180ttccagcagt gagcacgccg aaaaaattat tggggcagtg
aaagctctgg ataatgtcaa 240attgctgaag gtgtccgatc gcacctttga
tttacaccgt cagggcaaaa ttagcgtggt 300tagtcgcatt cccctcacct
cccaatcgga tttggccatg gcctataccc caggggtggg 360gcgcatctgt
cgggcgatcg ccgaagatcc ggaaaaggtt tattccctga ccattaaaag
420caatacggtg gcggtggtga ccgatggcag tgcggtgttg gggttgggta
acctggggcc 480ggaagcggct ttaccagtga tggaaggcaa ggccatgtta
ttcaaggaat ttgcccaact 540ggacgctttt cccatctgtt tggataccca
ggatacggag gaaattattc gcaccgtcaa 600ggcgatcgcc ccggtgtttg
gcggcgtaaa tttggaagac attgccgctc cccggtgttt 660tgaaattgaa
gcccggctga aaaaagaatt aaatattcct gtatttcacg atgatcagca
720cggcaccgcc attgttaccc tggccgcttt gttaaatgcc ctcaaatttg
ttggtaaagc 780catggccgct gtccgcattg tcatcaacgg cgctggggct
gctgggttgg cgatcgccga 840attgctcaag gaatccggag ccaccgatat
ttggatttgc gactccaagg gcattgtggg 900caaacatcgc accgatttaa
acagcaaaaa acagagcttt gcggtggatg cggaagggac 960tttagccgat
gctatggctg gagctgatgt gtttttaggg gtgagtgcgc cgggggtagt
1020gaccaaggaa atggtgcaat ccatggccaa ggacccgatt gtgtttgcca
tggccaaccc 1080tatccccgaa attcagccgg aattaatcca agaggatgcg
gcggttattg ccacggggcg 1140cagtgattac cccaaccaaa ttaacaatgt
gcttgccttt ccgggggttt tccggggagc 1200cattgactgt agagctagca
ttattaccac caccatgtgc atcgaagcgg ccaaggcgat 1260cgcctctttg
gtgcacagca acaccctaga tagtgagcat attattcctt cggtttttga
1320caatcgggtc gccactaccg tagccagtgc agtgcagttg gccgcccgca
atgaaggggt 1380ggccggtcaa tagttaatcg ggaattgtta aacctttact
ggtcaaccat tcctgattgt 1440aaagacggga ttggtaacgg gctcctccgt
cacaaagtac ggtaacaatg gtatgccccg 1500gcccaagttt tttggccaat
tggtaagccg ctcccacatt aatatcgatt tttctccacc 1560atcaacaccc
cggagggtgc catgaatatt ttggagtatg ctccgatcgc ctgtcagtcc
1620tggcaggtta ccgtggtcgg cgctggcaat gtggggcgga cccttgccca
gaggttagtg 1680cagcaaaatg tcgccaacgt agttttgttg gacattgtgc
caggcttacc ccagggcatt 1740gccttggatt tgatggccgc ccagagcgtg
gaggaatacg acagcaaaat cattggcacc 1800aatgaatacg aggccaccgc
cggctccgat gtggtggtaa ttaccgctgg tctaccccgc 1860aggcccggca
tgagtcggga tgatttgttg ggcaaaaacg ccaacattgt ggcccagggg
1920gcccgggaag cattgcgtta ttcccccaac gccattttga ttgtggtcac
caatcccctg 1980gatgtaatga cctatttggc ctggaaagta actggtttac
cttcccaacg ggttatgggc 2040atggcggggg tgttggactc ggctcggctc
aaggccttca ttgcgatgaa attaggggcc 2100tgtccttctg atatcaacac
cttagtgctg ggcgggcacg gagatttgat gctgcccttg 2160ccacgatact
gcaccgtcag cggggttccc attaccgaat taataccccc ccaaaccatt
2220gaagagttgg tggagcgtac ccgtaacggt ggggctgaaa ttgccgcctt
actacaaacg 2280ggcacagcct attatgcgcc ggcctcttcc gctgcggtga
tggtggagtc cattttacgc 2340aatcagtcta gaattctccc cgccgccacc
taccttgatg gtgcctatgg attgaaggac 2400attttccttg gagtgccctg
ccgtttgggg tgtcgaggag tggaagatat tctcgaagtg 2460caattaaccc
ctgaagaaaa agctgccctc catctttctg cagaagcagt tcgccttaat
2520attgatgtgg cgttggccat ggttagcgac ggttaacacg ataacggaca
gtgccaatac 2580cgttttttca ccgaggttag ggctta
260637473PRTSynechocystis sp. strain PCC6803 37Met Arg Pro Leu Ser
His Arg Thr Lys Ile Val Ala Thr Ile Gly Pro 1 5 10 15 Ala Ser Ser
Ser Val Glu Val Ile Arg Gln Met Val Asp Ala Gly Met 20 25 30 Asn
Val Ala Arg Leu Asn Phe Ser His Gly Ser Tyr Glu Asp His Ala 35 40
45 Thr Met Val Arg Leu Leu Arg Ser Val Glu Gln Glu Met Asp Thr Pro
50 55 60 Ile Thr Leu Leu Gln Asp Leu Gln Gly Pro Lys Ile Arg Ile
Gly Gln 65 70 75 80 Leu Pro Gly Gly Glu Lys Gln Leu Arg Glu Gly Glu
Lys Val Ser Leu 85 90 95 Val Pro Val Glu Ile Gly Asp Arg His Pro
Gly Ala Val Gly Ile Asp 100 105 110 Tyr Pro His Leu Ala Thr Glu Ala
Lys Val Gly Glu Arg Ile Leu Leu 115 120 125 Asp Asp Gly Leu Leu Glu
Met Lys Val Val Ser Ile Gln Asp Pro Glu 130 135 140 Val Ile Cys Glu
Val Val Thr Gly Gly Ile Leu Lys Ser Arg Lys Gly 145 150 155 160 Val
Asn Leu Pro Gly Leu Val Leu Thr Leu Pro Ser Met Thr Thr Lys 165 170
175 Asp Lys Gln Asp Leu Glu Phe Gly Leu Ser Gln Gly Ile Asp Trp Val
180 185 190 Ser Leu Ser Phe Val Arg Lys Gly Glu Asp Ile His Thr Leu
Lys Gln 195 200 205 Phe Leu Ala Glu Arg Gly His Pro Asp Leu Pro Val
Ile Ala Lys Ile 210 215 220 Glu Lys Pro Gln Ala Ile Asp Asn Leu Glu
Glu Ile Val Ala Val Ser 225 230 235 240 Asn Gly Ile Met Val Ala Arg
Gly Asp Leu Gly Val Glu Val Asn Pro 245 250 255 Glu Lys Val Pro Arg
Leu Gln Lys Glu Ile Ile Arg Arg Cys Asn Val 260 265 270 Arg Ala Ile
Pro Val Ile Thr Ala Thr Gln Met Leu Asp Ser Met Ile 275 280 285 Gln
Asn Ser Arg Pro Thr Arg Ala Glu Ala Ser Asp Val Ala Asn Ala 290 295
300 Ile Leu Asp Gly Thr Asp Ala Val Met Leu Ser Gly Glu Ser Ala Val
305 310 315 320 Gly Gln Tyr Pro Val Lys Ser Val Gln Met Leu Arg Lys
Ile Ala Glu 325 330 335 Glu Thr Glu Val Gly Leu His Leu Val Asn Asn
Pro Pro Ile Glu Asn 340 345 350 Thr Glu Thr His Ala Leu Ser Glu Ala
Leu Val Val Ile Asp Gly Ile 355 360 365 Leu Asp Leu Lys Tyr Ile Val
Thr Phe Thr Thr Ser Gly Phe Thr Ser 370 375 380 Leu Leu Ala Ser Asn
Gln Arg Pro Ser Val Pro Val Ile Ala Phe Thr 385 390 395 400 Pro Ser
Glu Lys Val Tyr His Ser Leu Asn Leu Val Trp Gly Ile Ile 405 410 415
Pro Phe Leu Ile Asn Glu Glu Phe Asp Thr Phe Glu Asp Leu Ile Gln 420
425 430 Gln Ala Glu Val Leu Leu Arg Asp Arg Lys Met Val Glu Lys Gly
Asp 435 440 445 Gln Leu Leu Ile Met Ala Gly Ile Pro Thr Lys Ile Pro
Arg Gly Thr 450 455 460 Asn Phe Leu Lys Ile His Arg Ile Ser 465 470
382350DNAartificialinsert of construct pVZ321-pyk1 38tcgactaccc
attgggggct gagcggccca gcggtgttaa agctatctgc ctggggagca 60agacaattac
aggcatcggg ttatgaccat tccctctgga tcaactggct gcctaaagtg
120actcccctgg aatccatggc ccaatggcga gcaacaaagc aaagccatcc
ccgcaaacaa 180atcgctaact ttaccgccac tgctttacct aaacgacttt
ggcagaggtt gacgacccag 240gctggtatta aaccggggca atgttgggcc
gatttttcca aggttcagga gcgacaacta 300acagagaata tccaccgcta
ccactgtcag atcaaaggta aaggggtgtt caaagaagaa 360tttgtcacct
gtgggggcat taccctcaag gaagtggatt ttaagaccat ggctagccgt
420tgttgccctg gattatattt tgccggggaa atcctcgacg tagatggtat
taccggcggt 480tttaattttc aaaatgcctg gactacggct tggttggcgg
cccaggggat ggcggctcca 540taacattccc ttaattcctc ctcagtaagt
tggactaaaa ttccagtgcc agccctgatt 600aacccggtga agtttatgag
acccctaagt catcgcacca aaattgtggc caccattggc 660cccgccagta
gttcggtaga ggtgatacgt cagatggtgg atgcgggcat gaatgtggcc
720cggctgaatt tttcccacgg tagttatgag gaccatgcca ccatggttcg
cctactacgg 780tcagtggagc aggaaatgga cacccccatt acccttttgc
aagatttaca ggggcccaaa 840attcggattg gtcagttgcc ggggggagaa
aagcaactgc gggaagggga aaaagtttct 900ttggtgccgg tggaaattgg
cgatcgccat cctggagcgg tgggcattga ctatccccat 960ttggcgacgg
aggcaaaagt aggggaaaga attttattgg acgatggttt actggaaatg
1020aaagttgtgt ccattcaaga tccagaggta atttgtgaag tggtgaccgg
gggcatcctc 1080aaaagtcgca aaggggtcaa tctaccgggt ttagtcctaa
ccctaccttc catgacgacc 1140aaagacaagc aagacttaga atttggtttg
agccaaggca ttgactgggt ttcccttagt 1200tttgtccgca aaggggaaga
tatccacacc cttaaacaat ttctcgctga acggggccat 1260cctgatctgc
cggtcattgc caaaattgaa aaaccccagg cgatcgataa tctagaagaa
1320atcgtggcag tttccaacgg cattatggtg gccagggggg atctgggggt
ggaagtaaac 1380ccagaaaagg ttccccgttt gcaaaaggaa attattcggc
gctgtaacgt gcgggccatt 1440ccggtcatca ccgctaccca aatgctagat
agcatgattc aaaattcccg acccaccagg 1500gcggaagcca gtgacgtggc
caacgctatt ttggacggca ccgatgcggt gatgttatcg 1560ggggaatcgg
cagtgggaca atatcccgtt aaatcagtgc aaatgttgcg aaaaattgcc
1620gaagagacgg aagtgggtct acatcttgtt aataatcccc caatagaaaa
tacggaaacc 1680catgccctaa gtgaagcgtt agtggtaatt gacggaattc
tagatttaaa atacattgtc 1740acgttcacca cctcaggttt tacttctctc
ctcgcttcca accaaagacc gtcggtgccg 1800gtgattgctt tcactccctc
agaaaaggtt taccatagcc tcaacttagt ttggggtatt 1860attccctttt
taattaacga agaatttgac acctttgagg atttaatcca acaggcggag
1920gtactactgc gggatagaaa aatggtggaa aagggcgatc agttgctaat
catggcggga 1980attcccacta aaatacccag gggcactaat ttcctcaaga
ttcaccgtat ttcttaaaac 2040ctatgcctca aatccatagt tgctggacaa
ggattctaca ctactgtttt caggaagtat 2100acttggaata atttctagaa
aatagcaaaa ttatgtcatt attccctctt ttaaccgctt 2160tgttagggat
tatgcccgca aacaccattg aacaagttcc tgctgttgtt gaagctgaag
2220ctcgtccttt tctggttagc caagctaaca gcgcagatat tttggtgaaa
ctcccccgac 2280cccagggtag tcccaagaat gtaggtagca tgttcatggc
caatgcctat ggacaacagg 2340gcctaaattc 2350392236DNAartificialinsert
found in construct pVZ321 PpetJ pyk1 39tcgactctag aggatccccg
ggtacccctc atcgggggct gtgttggccg agacggcact 60gaggatttta ctctccatgg
cattccaagg aatatctacc caactcacct gctccggcgg 120attgttccgc
tcaaaagtac taatcaagtc gtcaaaatac ttattaaatt ttggctgcaa
180ttgcatagtc caaaagctga ctttcccctc catgctctgg ggggaattgc
tctggcaact 240gattaatcca ctgagcaaca gcccaagaca cgcaaacaaa
aaccaacgtc ttggcgatcg 300ccatcggcac catgaaacca tcgtaaaagc
tggggaaaga ataaaaaaca gtggttcagg 360aattgcattg ccatggccac
ttcacaaacc tagccaattt tagcttgacc gcaactttga 420cagattgtct
tttgactttg cctggaccgc ctcccataat accttcgcgt cttgaagact
480ttatccttga aaggagaaca tatgagaccc ctaagtcatc gcaccaaaat
tgtggccacc 540attggccccg ccagtagttc ggtagaggtg atacgtcaga
tggtggatgc gggcatgaat 600gtggcccggc tgaatttttc ccacggtagt
tatgaggacc atgccaccat ggttcgccta 660ctacggtcag tggagcagga
aatggacacc cccattaccc ttttgcaaga tttacagggg 720cccaaaattc
ggattggtca gttgccgggg ggagaaaagc aactgcggga aggggaaaaa
780gtttctttgg tgccggtgga aattggcgat cgccatcctg gagcggtggg
cattgactat 840ccccatttgg cgacggaggc aaaagtaggg gaaagaattt
tattggacga tggtttactg 900gaaatgaaag ttgtgtccat tcaagatcca
gaggtaattt gtgaagtggt gaccgggggc 960atcctcaaaa gtcgcaaagg
ggtcaatcta ccgggtttag tcctaaccct accttccatg 1020acgaccaaag
acaagcaaga cttagaattt ggtttgagcc aaggcattga ctgggtttcc
1080cttagttttg tccgcaaagg ggaagatatc cacaccctta aacaatttct
cgctgaacgg 1140ggccatcctg atctgccggt cattgccaaa attgaaaaac
cccaggcgat cgataatcta 1200gaagaaatcg tggcagtttc caacggcatt
atggtggcca ggggggatct gggggtggaa 1260gtaaacccag aaaaggttcc
ccgtttgcaa aaggaaatta ttcggcgctg taacgtgcgg 1320gccattccgg
tcatcaccgc tacccaaatg ctagatagca tgattcaaaa ttcccgaccc
1380accagggcgg aagccagtga cgtggccaac gctattttgg acggcaccga
tgcggtgatg 1440ttatcggggg aatcggcagt gggacaatat cccgttaaat
cagtgcaaat gttgcgaaaa 1500attgccgaag agacggaagt gggtctacat
cttgttaata atcccccaat agaaaatacg 1560gaaacccatg ccctaagtga
agcgttagtg gtaattgacg gaattctaga tttaaaatac 1620attgtcacgt
tcaccacctc aggttttact tctctcctcg cttccaacca aagaccgtcg
1680gtgccggtga ttgctttcac tccctcagaa aaggtttacc atagcctcaa
cttagtttgg 1740ggtattattc cctttttaat taacgaagaa tttgacacct
ttgaggattt aatccaacag 1800gcggaggtac tactgcggga tagaaaaatg
gtggaaaagg gcgatcagtt gctaatcatg 1860gcgggaattc ccactaaaat
acccaggggc actaatttcc tcaagattca ccgtatttct 1920taaaacctat
gcctcaaatc catagttgct ggacaaggat tctacactac tgttttcagg
1980aagtatactt ggaataattt ctagaaaata gcaaaattat gtcattattc
cctcttttaa 2040ccgctttgtt agggattatg cccgcaaaca ccattgaaca
agttcctgct gttgttgaag 2100ctgaagctcg tccttttctg gttagccaag
ctaacagcgc agatattttg gtgaaactcc 2160cccgacccca gggtagtccc
aagaatgtag gtagcatgtt catggccaat gcctatggac 2220aacagggcct aaattc
223640591PRTSynechocystis sp. strain PCC6803 40Met Gln Thr Ser Pro
Leu Pro Arg Arg Thr Lys Ile Val Ala Thr Ile 1 5 10 15 Gly Pro Ala
Thr Gln Ser Lys Glu Val Leu Arg Gln Leu Ile Gln Ala 20 25 30 Gly
Ala Thr Thr Phe Arg Leu Asn Phe Ser His Gly Asp His Ala Tyr 35 40
45 His Gln Gln Ser Ile Arg Leu Ile Arg Gln Ile Ala Phe Glu Leu Asn
50 55 60 Gln Pro Val Gly Ile Leu Gln Asp Leu Gln Gly Pro Lys Ile
Arg Val 65 70 75 80 Gly Lys Phe Leu Asn Asp Ala Gly Ser Val Gln Leu
Lys Asn Gly Asp 85 90 95 Pro Tyr Thr Leu Thr Ser Arg Pro Val Glu
Cys Thr Glu Thr Ile Ser 100 105 110 Ser Ile Ser Tyr Glu Tyr Leu Ala
Asp Glu Val Pro Ser Gly Ala Arg 115 120 125 Ile Leu Leu Asp Asp Gly
Lys Leu Glu Met Leu Val Glu Glu Val Asp 130 135 140 Thr Val Ala Arg
Asp Leu His Cys Arg Val Ile Val Gly Gly Thr Leu 145 150 155 160 Ser
Ser Asn Lys Gly Val Asn Phe Pro Gly Val Cys Leu Ser Val Lys 165 170
175 Ala Met Thr Asp Lys Asp Lys Glu Asp Leu Met Phe Gly Leu Asp Gln
180 185 190 Gly Val Asp Trp Val Ala Leu Ser Phe Val Arg Asn Pro Gln
Asp Ile 195 200 205 Asp Glu Ile Lys Gly Leu Ile Ala Ala Ala Gly Lys
Ser Val Pro Val 210 215 220 Ile Ala Lys Ile Glu Lys His Glu Ala Ile
Lys Asp Met Gln Ala Val 225 230 235 240 Leu Glu Lys Cys Asp Gly Val
Met Val Ala Arg Gly Asp Leu Gly Val 245 250 255 Glu Leu Pro Ala Glu
Asp Val Pro Ile Leu Gln Lys Lys Leu Ile Ala 260 265 270 Thr Ala Asn
Arg Leu Gly Ile Pro Val Ile Thr Ala Thr Gln Met Leu 275 280 285 Asp
Ser Met Val Asn Ser Pro Arg Pro Thr Arg Ala Glu Val Ser Asp 290 295
300 Val Ala Asn Ala Ile Leu Asp Gly Thr Asp Ala Val Met Leu Ser Asn
305 310 315 320 Glu Thr Ala Ile Gly Lys Phe Pro Val Glu Ala Val Ala
Ile Met Ala 325 330 335 Lys Ile Ala Glu Arg Ile Glu Gln Glu Asp Ile
Asn Pro Ser Gln Ala 340 345 350 Glu Ala Ser Arg Thr Ser Ile Pro Asn
Ala Ile Ser Ser Ala Val Ser 355 360 365 Gln Ile Ala Glu Thr Leu Asn
Ala Ala Ala Ile Met Ser Leu Thr Lys 370 375 380 Thr Gly Ser Thr Ala
Arg His Val Ser Lys Phe Arg Pro Lys Thr Pro 385 390 395 400 Ile Leu
Ala Val Thr Pro His Val Asp Val Ser Arg Gln Leu Gln Leu 405 410 415
Val Trp Gly Val Lys Pro Leu Leu Val Leu Asp Leu Pro Ser Thr Ser 420
425 430 Gln Thr Phe Gln Ala Ala Ile Asn Val Ala Gln Glu Asn His Phe
Leu 435 440 445 Arg Asp Gly Asp Leu Val Val Met Thr Ala Gly Thr
Leu Gln Gly Val 450 455 460 Ala Gly Ser Thr Asp Leu Ile Lys Val Glu
Val Val Lys Ala Ile Leu 465 470 475 480 Gly Arg Gly Val Gly Ile Gly
Gln Gly Ala Val Ser Gly Arg Ala Arg 485 490 495 Val Ala Ser Arg Pro
Gln Ala Ile Ala Gln Phe Thr Gln Gly Glu Ile 500 505 510 Leu Val Val
Pro Ser Thr Asn Ala Asp Cys Val Asp Met Met Arg Arg 515 520 525 Ala
Ala Gly Ile Ile Thr Glu Glu Glu Ser Leu Thr Ser His Ala Ala 530 535
540 Ile Ile Gly Leu Arg Leu Gly Val Pro Val Ile Val Gly Phe Lys Gly
545 550 555 560 Ala Thr Gln Lys Ile Arg Asp Gly Ala Ile Val Thr Ile
Asp Ala Gln 565 570 575 Lys Gly Leu Ile Tyr Ser Gly Ala Leu Pro Pro
Val Ser Lys Gly 580 585 590 412620DNAartificialinsert of pVZ321pyk2
41tcgaccggca aacaaatcca gcggaaaata accgtaggtt cccgctaagg tatcatcctc
60cgcctgacag gaaattaata ctccccgata atccccgtca taactgtcga gataaaagcg
120cagggattgg ctctggcatt tcaaatacac tggcccttca atggttttac
ctggttccat 180ggcgatcgcc tctccgtaat ccaacccctg tagatattgc
cccaaagcat tttccgcact 240agcttggtcg tcggcacaaa tgccaaaatt
ttccgcttca ctctggccac aaatccagac 300aatggcttgg cgtaaatctt
ccctttcctg gtcattctgg ggaatacgga tttctaaacg 360actgtaatcc
tttaacaacg tcaactgagc ggaagcattc atagcaaacc catcaaactg
420tgcagactat tgggaaaaat tgcccaaagc ccagcctacc atcgttggcc
ctgtaagccg 480atgcaactga aacggcgatc gtggcggata atgagaatat
ttccctaaca ccgcttaaaa 540gcagacggcc ttaactatta tatttaactg
cccctaattt cagcccatta tgcaaacgtc 600tccccttccc cgtcgtacca
aaatcgtcgc taccattggc cccgcgaccc aaagcaagga 660agtgctgaga
caactgatcc aagccggtgc taccactttc cgcctcaatt tttcccacgg
720agaccacgct taccaccaac aaagtatccg tttgattcgc cagattgcct
ttgaactgaa 780ccaaccagtg ggcattctcc aggatttaca ggggccaaag
attcgggtgg gcaaatttct 840caatgatgcc ggctctgtgc aactcaaaaa
cggtgatccc tataccctca ccagtcgccc 900ggtggaatgt acggaaacca
ttagttccat tagctacgaa tatttagccg acgaagtacc 960ttctggggca
agaattttgc tcgacgacgg caaactggaa atgttggtgg aggaagtgga
1020cactgttgcc cgggatctcc actgtcgggt gattgtgggg ggaacccttt
ccagcaataa 1080aggggttaat tttcccgggg tctgcctttc cgttaaggcc
atgaccgata aagataagga 1140agatttgatg ttcgggctgg accaaggggt
ggactgggtg gccctgagtt ttgttcgtaa 1200tccccaggat attgatgaga
ttaaggggtt aattgcggcg gcagggaaat ccgtgccggt 1260aatcgccaaa
attgagaagc acgaagcgat taaggatatg caggcggtgc tggaaaaatg
1320tgacggtgtc atggtggccc ggggggactt gggggtagaa ctacccgcag
aagatgtacc 1380tattttgcaa aagaaactca ttgccactgc taaccggttg
ggcattcctg tcattactgc 1440tacccaaatg ttggacagta tggtcaacag
cccccgacct actagggctg aagtgtccga 1500cgtggccaat gccatcctcg
atggtaccga tgcggtgatg ctctccaacg aaacggcgat 1560cggtaaattt
cccgtggaag cagtggctat tatggccaaa attgcggagc gcattgaaca
1620ggaagatatc aatccttccc aagcggaagc cagtcgcact tctattccca
atgctatttc 1680cagcgccgtt agccagattg cggaaaccct caacgcggcg
gctattatgt ctttgactaa 1740gaccggatcc accgcccgcc atgtgtcaaa
gttccgcccc aaaaccccca ttctggccgt 1800tacgccccat gtggatgtgt
cccgtcagtt gcagttggtg tggggagtta agcccctgtt 1860ggtattggat
ttaccttcca ccagccaaac gttccaagcc gccattaacg tggcccagga
1920aaaccatttt ctccgggatg gagatttggt ggtgatgacc gccgggacat
tgcagggagt 1980tgccggttcg acggatttaa tcaaagtgga agtggtcaag
gccattcttg gtcggggtgt 2040aggtattggc caaggagctg taagtggccg
ggccagggtt gccagtcgtc cccaggcgat 2100cgcccaattt acccagggag
aaattttagt agttccctct accaacgctg attgtgtgga 2160catgatgcga
cgggcggcgg gcattatcac cgaagaagaa agcctgacta gccatgcggc
2220cattattggt ttgcggctgg gggtgccggt cattgtgggt tttaaaggcg
ctacccaaaa 2280gattcgagat ggagccattg tcaccatcga tgcccaaaaa
ggactgattt attccggtgc 2340attacccccg gtgtccaaag gatagggaat
aattagggac ccagtaacag ggcgagaaca 2400ggaagcatca tcccaatgga
gttcccattc cccaccccct ggaacagtaa cattttggta 2460gaatcaacgg
ggttcttttc ccgacgtaat ttagtccttt gcagaaaaaa tcttccctta
2520aatcccgcta ctggctgtta atcccctata ttcgtcccca tcggcggact
attgggctgg 2580ctttcctctg tactctgcta ttcactgttt tttggcccac
2620422532DNAartificialinsert of the construct pVZ321 PpetJ pyk2
42tcgactctag aggatccccg ggtacccctc atcgggggct gtgttggccg agacggcact
60gaggatttta ctctccatgg cattccaagg aatatctacc caactcacct gctccggcgg
120attgttccgc tcaaaagtac taatcaagtc gtcaaaatac ttattaaatt
ttggctgcaa 180ttgcatagtc caaaagctga ctttcccctc catgctctgg
ggggaattgc tctggcaact 240gattaatcca ctgagcaaca gcccaagaca
cgcaaacaaa aaccaacgtc ttggcgatcg 300ccatcggcac catgaaacca
tcgtaaaagc tggggaaaga ataaaaaaca gtggttcagg 360aattgcattg
ccatggccac ttcacaaacc tagccaattt tagcttgacc gcaactttga
420cagattgtct tttgactttg cctggaccgc ctcccataat accttcgcgt
cttgaagact 480ttatccttga aaggagaaca tatgcaaacg tctccccttc
cccgtcgtac caaaatcgtc 540gctaccattg gccccgcgac ccaaagcaag
gaagtgctga gacaactgat ccaagccggt 600gctaccactt tccgcctcaa
tttttcccac ggagaccacg cttaccacca acaaagtatc 660cgtttgattc
gccagattgc ctttgaactg aaccaaccag tgggcattct ccaggattta
720caggggccaa agattcgggt gggcaaattt ctcaatgatg ccggctctgt
gcaactcaaa 780aacggtgatc cctataccct caccagtcgc ccggtggaat
gtacggaaac cattagttcc 840attagctacg aatatttagc cgacgaagta
ccttctgggg caagaatttt gctcgacgac 900ggcaaactgg aaatgttggt
ggaggaagtg gacactgttg cccgggatct ccactgtcgg 960gtgattgtgg
ggggaaccct ttccagcaat aaaggggtta attttcccgg ggtctgcctt
1020tccgttaagg ccatgaccga taaagataag gaagatttga tgttcgggct
ggaccaaggg 1080gtggactggg tggccctgag ttttgttcgt aatccccagg
atattgatga gattaagggg 1140ttaattgcgg cggcagggaa atccgtgccg
gtaatcgcca aaattgagaa gcacgaagcg 1200attaaggata tgcaggcggt
gctggaaaaa tgtgacggtg tcatggtggc ccggggggac 1260ttgggggtag
aactacccgc agaagatgta cctattttgc aaaagaaact cattgccact
1320gctaaccggt tgggcattcc tgtcattact gctacccaaa tgttggacag
tatggtcaac 1380agcccccgac ctactagggc tgaagtgtcc gacgtggcca
atgccatcct cgatggtacc 1440gatgcggtga tgctctccaa cgaaacggcg
atcggtaaat ttcccgtgga agcagtggct 1500attatggcca aaattgcgga
gcgcattgaa caggaagata tcaatccttc ccaagcggaa 1560gccagtcgca
cttctattcc caatgctatt tccagcgccg ttagccagat tgcggaaacc
1620ctcaacgcgg cggctattat gtctttgact aagaccggat ccaccgcccg
ccatgtgtca 1680aagttccgcc ccaaaacccc cattctggcc gttacgcccc
atgtggatgt gtcccgtcag 1740ttgcagttgg tgtggggagt taagcccctg
ttggtattgg atttaccttc caccagccaa 1800acgttccaag ccgccattaa
cgtggcccag gaaaaccatt ttctccggga tggagatttg 1860gtggtgatga
ccgccgggac attgcaggga gttgccggtt cgacggattt aatcaaagtg
1920gaagtggtca aggccattct tggtcggggt gtaggtattg gccaaggagc
tgtaagtggc 1980cgggccaggg ttgccagtcg tccccaggcg atcgcccaat
ttacccaggg agaaatttta 2040gtagttccct ctaccaacgc tgattgtgtg
gacatgatgc gacgggcggc gggcattatc 2100accgaagaag aaagcctgac
tagccatgcg gccattattg gtttgcggct gggggtgccg 2160gtcattgtgg
gttttaaagg cgctacccaa aagattcgag atggagccat tgtcaccatc
2220gatgcccaaa aaggactgat ttattccggt gcattacccc cggtgtccaa
aggataggga 2280ataattaggg acccagtaac agggcgagaa caggaagcat
catcccaatg gagttcccat 2340tccccacccc ctggaacagt aacattttgg
tagaatcaac ggggttcttt tcccgacgta 2400atttagtcct ttgcagaaaa
aatcttccct taaatcccgc tactggctgt taatccccta 2460tattcgtccc
catcggcgga ctattgggct ggctttcctc tgtactctgc tattcactgt
2520tttttggccc ac 253243470PRTE. coli K12 43Met Lys Lys Thr Lys Ile
Val Cys Thr Ile Gly Pro Lys Thr Glu Ser 1 5 10 15 Glu Glu Met Leu
Ala Lys Met Leu Asp Ala Gly Met Asn Val Met Arg 20 25 30 Leu Asn
Phe Ser His Gly Asp Tyr Ala Glu His Gly Gln Arg Ile Gln 35 40 45
Asn Leu Arg Asn Val Met Ser Lys Thr Gly Lys Thr Ala Ala Ile Leu 50
55 60 Leu Asp Thr Lys Gly Pro Glu Ile Arg Thr Met Lys Leu Glu Gly
Gly 65 70 75 80 Asn Asp Val Ser Leu Lys Ala Gly Gln Thr Phe Thr Phe
Thr Thr Asp 85 90 95 Lys Ser Val Ile Gly Asn Ser Glu Met Val Ala
Val Thr Tyr Glu Gly 100 105 110 Phe Thr Thr Asp Leu Ser Val Gly Asn
Thr Val Leu Val Asp Asp Gly 115 120 125 Leu Ile Gly Met Glu Val Thr
Ala Ile Glu Gly Asn Lys Val Ile Cys 130 135 140 Lys Val Leu Asn Asn
Gly Asp Leu Asp Glu Asn Lys Gly Val Asn Leu 145 150 155 160 Pro Gly
Val Ser Ile Ala Leu Pro Ala Leu Ala Glu Lys Asp Lys Gln 165 170 175
Asp Leu Ile Phe Gly Cys Glu Gln Gly Val Asp Phe Val Ala Ala Ser 180
185 190 Phe Ile Arg Lys Arg Ser Asp Val Ile Glu Ile Arg Glu His Leu
Lys 195 200 205 Ala His Gly Gly Glu Asn Ile His Ile Ile Ser Lys Ile
Glu Asn Gln 210 215 220 Glu Gly Leu Asn Asn Phe Asp Glu Ile Leu Glu
Ala Ser Asp Gly Ile 225 230 235 240 Met Val Ala Arg Gly Asp Leu Gly
Val Glu Ile Pro Val Glu Glu Val 245 250 255 Ile Phe Ala Gln Lys Met
Met Ile Glu Lys Cys Ile Arg Ala Arg Lys 260 265 270 Val Val Ile Thr
Ala Thr Gln Met Leu Asp Ser Met Ile Lys Asn Pro 275 280 285 Arg Pro
Thr Arg Ala Glu Ala Gly Asp Val Ala Asn Ala Ile Leu Asp 290 295 300
Gly Thr Asp Ala Val Met Leu Ser Gly Glu Ser Ala Lys Gly Lys Tyr 305
310 315 320 Pro Leu Glu Ala Val Ser Ile Met Ala Thr Ile Cys Glu Arg
Thr Asp 325 330 335 Arg Val Met Asn Ser Arg Leu Glu Phe Asn Asn Asp
Asn Arg Lys Leu 340 345 350 Arg Ile Thr Glu Ala Val Cys Arg Gly Ala
Val Glu Thr Ala Glu Lys 355 360 365 Leu Asp Ala Pro Leu Ile Val Val
Ala Thr Gln Gly Gly Lys Ser Ala 370 375 380 Arg Ala Val Arg Lys Tyr
Phe Pro Asp Ala Thr Ile Leu Ala Leu Thr 385 390 395 400 Thr Asn Glu
Lys Thr Ala His Gln Leu Val Leu Ser Lys Gly Val Val 405 410 415 Pro
Gln Leu Val Lys Glu Ile Thr Ser Thr Asp Asp Phe Tyr Arg Leu 420 425
430 Gly Lys Glu Leu Ala Leu Gln Ser Gly Leu Ala His Lys Gly Asp Val
435 440 445 Val Val Met Val Ser Gly Ala Leu Val Pro Ser Gly Thr Thr
Asn Thr 450 455 460 Ala Ser Val His Val Leu 465 470
44429PRTZymomonas mobilis 44Met Thr Ala Ile Val Ser Ile His Gly Arg
Gln Val Val Asp Ser Arg 1 5 10 15 Gly Asn Pro Thr Val Glu Val Asp
Val Thr Leu Glu Asp Gly Ser Phe 20 25 30 Gly Arg Ala Ala Val Pro
Ser Gly Ala Ser Thr Gly Val His Glu Ala 35 40 45 Val Glu Leu Arg
Asp Gly Asp Lys Thr Arg Trp Gly Gly Lys Gly Val 50 55 60 Thr Lys
Ala Val His Ala Val Asn Asn Glu Ile Ala Asn Ala Ile Ile 65 70 75 80
Gly Leu Glu Ala Glu Asp Gln Glu Leu Ile Asp Gln Thr Met Ile Lys 85
90 95 Leu Asp Gly Thr Pro Asn Lys Gly Lys Phe Gly Ala Asn Ala Ile
Leu 100 105 110 Gly Val Ser Leu Ala Val Ala Lys Ala Ala Ala Glu Ala
Arg Gly Leu 115 120 125 Pro Leu Tyr Arg Tyr Val Gly Gly Thr Ala Ala
His Val Leu Pro Val 130 135 140 Pro Met Met Asn Ile Val Asn Gly Gly
Met His Ala Asp Asn Pro Ile 145 150 155 160 Asp Phe Gln Glu Phe Met
Ile Ala Pro Val Gly Ala Ser Ser Ile Asn 165 170 175 Glu Ala Val Arg
Ile Gly Thr Glu Val Phe His Thr Leu Lys Lys Glu 180 185 190 Leu Ser
Ala Lys Gly Met Asn Thr Asn Val Gly Asp Glu Gly Gly Phe 195 200 205
Ala Pro Ser Leu Asp Ser Ala Ser Ser Ala Leu Asp Phe Ile Val Asp 210
215 220 Ser Ile Ser Lys Ala Gly Tyr Lys Pro Gly Glu Asp Val Phe Ile
Ala 225 230 235 240 Leu Asp Ala Ala Ser Ser Glu Phe Tyr Asn Lys Asp
Gln Asn Ile Tyr 245 250 255 Asp Leu Lys Gly Glu Gly Arg Lys Leu Thr
Ser Ala Gln Leu Val Asp 260 265 270 Tyr Tyr Val Glu Leu Cys Gly Lys
Tyr Pro Ile Tyr Ser Ile Glu Asp 275 280 285 Gly Leu Ala Glu Asp Asp
Phe Glu Gly Trp Lys Ile Leu Thr Glu Lys 290 295 300 Leu Gly Asp Lys
Val Gln Leu Val Gly Asp Asp Leu Phe Val Thr Asn 305 310 315 320 Val
Lys Arg Leu Ser Asp Gly Ile Glu Arg Gly Ile Ala Asn Ser Leu 325 330
335 Leu Val Lys Phe Asn Gln Ile Gly Ser Leu Ser Glu Thr Leu Ala Ala
340 345 350 Val Asn Met Ala Asn Asp Ala Ser Tyr Thr Ala Val Met Ser
His Arg 355 360 365 Ser Gly Glu Thr Glu Asp Thr Thr Ile Ala Asp Leu
Ala Val Ala Thr 370 375 380 Asn Cys Gly Gln Ile Lys Thr Gly Ser Leu
Cys Arg Ser Glu Arg Ile 385 390 395 400 Ala Lys Tyr Asn Gln Leu Met
Arg Ile Glu Glu Glu Leu Gly Ser Val 405 410 415 Ala Lys Tyr Ala Gly
Arg Ser Val Leu Arg Lys Ala Lys 420 425 45228PRTZymomonas mobilis
45Met Pro Thr Leu Val Leu Ser Arg His Gly Gln Ser Glu Trp Asn Leu 1
5 10 15 Glu Asn Arg Phe Thr Gly Trp Trp Asp Val Asn Leu Thr Glu Gln
Gly 20 25 30 Val Gln Glu Ala Thr Ala Gly Gly Lys Ala Leu Ala Glu
Lys Gly Phe 35 40 45 Glu Phe Asp Ile Ala Phe Thr Ser Val Leu Thr
Arg Ala Ile Lys Thr 50 55 60 Thr Asn Leu Ile Leu Glu Ala Gly Lys
Thr Leu Trp Val Pro Thr Glu 65 70 75 80 Lys Asp Trp Arg Leu Asn Glu
Arg His Tyr Gly Gly Leu Thr Gly Leu 85 90 95 Asn Lys Ala Glu Thr
Ala Ala Lys His Gly Glu Glu Gln Val His Ile 100 105 110 Trp Arg Arg
Ser Tyr Gly Val Pro Pro Pro Pro Met Glu Lys Gly Ser 115 120 125 Lys
Phe Asp Leu Ser Gly Asp Arg Arg Tyr Asp Gly Val Lys Ile Pro 130 135
140 Glu Thr Glu Ser Leu Lys Asp Thr Val Ala Arg Val Leu Pro Tyr Trp
145 150 155 160 Glu Glu Arg Ile Ala Pro Glu Leu Lys Ala Gly Lys Arg
Val Leu Ile 165 170 175 Gly Ala His Gly Asn Ser Leu Arg Ala Leu Val
Lys His Leu Ser Lys 180 185 190 Leu Ser Asp Glu Glu Ile Val Lys Phe
Glu Leu Pro Thr Gly Gln Pro 195 200 205 Leu Val Tyr Glu Leu Asn Asp
Asp Leu Thr Pro Lys Asp Arg Tyr Phe 210 215 220 Leu Asn Glu Arg 225
464274DNAartificialinsert of plasmid #67 46cccgggatct ctagaaagtt
tcggactcag tagacctaag tacagagtga tgtcaacgcc 60ttcaagctag acgggaggcg
gcttttgcca tggttcagcg atcgctcctc atcttcaata 120agcagggcat
gagccagcgt taagcaaatc aaatcaaatc tcgcttctgg gcttcaataa
180atggttccga ttgatgatag gttgattcat gaggaatcta aggcttaatt
ctccacaaaa 240gaattaagcg tccgtcgcaa cggaatgctc cgctggactt
gcgctgtggg actgcagctt 300tacaggctcc ccctgccaga aatcctgaat
cgtcgagcat atctgacata tctctaggga 360gagacgacat gtcgacgatt
aatttcagcg tataatgcgc gccaattgac tcttgaatgg 420tttcagcact
ttggactgta gaactcaacg actcaaaaac aggcactcac gttgggctga
480gacacaagca cacattcctc tgcacgcttt ttcgatgtca cctatcctta
gagcgaggca 540ccaccacttt cgtaataccg gattcgcttt ccggcagtgc
gcccagaaag caagtttctc 600ccatccttct caacttaaag actaagactg
tcatgaaaaa gaccaaaatt gtttgcacca 660tcggaccgaa aaccgaatct
gaagagatgt tagctaaaat gctggacgct ggcatgaacg 720ttatgcgtct
gaacttctct catggtgact atgcagaaca cggtcagcgc attcagaatc
780tgcgcaacgt gatgagcaaa actggtaaaa ccgccgctat cctgcttgat
accaaaggtc 840cggaaatccg caccatgaaa ctggaaggcg gtaacgacgt
ttctctgaaa gctggtcaga 900cctttacttt caccactgat aaatctgtta
tcggcaacag cgaaatggtt gcggtaacgt 960atgaaggttt cactactgac
ctgtctgttg gcaacaccgt actggttgac gatggtctga 1020tcggtatgga
agttaccgcc attgaaggta acaaagttat ctgtaaagtg ctgaacaacg
1080gtgacctgga cgaaaacaaa ggtgtgaacc tgcctggcgt ttccattgct
ctgccagcac 1140tggctgaaaa agacaaacag gacctgatct ttggttgcga
acaaggcgta gactttgttg 1200ctgcttcctt tattcgtaag cgttctgacg
ttatcgaaat ccgtgagcac ctgaaagcgc 1260acggcggcga aaacatccac
atcatctcca aaatcgaaaa ccaggaaggc ctcaacaact 1320tcgacgaaat
cctcgaagcc
tctgacggca tcatggttgc gcgtggcgac ctgggtgtag 1380aaatcccggt
agaagaagtt atcttcgccc agaagatgat gatcgaaaaa tgtatccgtg
1440cacgtaaagt cgttatcact gcgacccaga tgctggattc catgatcaaa
aacccacgcc 1500cgactcgcgc agaagccggt gacgttgcaa acgccatcct
cgacggtact gacgcagtga 1560tgctgtctgg tgaatccgca aaaggtaaat
acccgctgga agcggtttct atcatggcga 1620ccatctgcga acgtaccgac
cgcgtgatga acagccgtct cgagttcaac aatgacaacc 1680gtaaactgcg
cattaccgaa gcggtatgcc gtggtgccgt cgaaactgct gaaaaactgg
1740atgctccgct gatcgtggtt gctactcagg gcggtaaatc tgctcgcgca
gtacgtaaat 1800acttcccgga tgccaccatc ctggcactga ccactaacga
aaaaacggct catcagttgg 1860tactgagcaa aggcgttgtg ccgcagcttg
ttaaagagat cacttctact gatgatttct 1920accgtctggg taaagaactg
gctctgcaga gcggtctggc acacaaaggt gacgttgtag 1980ttatggtttc
tggtgcactg gtaccgagcg gcactactaa caccgcatct gttcacgtcc
2040tgtaataagc ttcattgacg gactgagttc aaaaagagac tcgtctaaaa
gattttaaga 2100aaggtttcga tatgacagct attgtcagta tccatggccg
tcaggttgtc gacagccgcg 2160gtaacccgac cgttgaagtt gatgttacgc
ttgaagatgg cagcttcggc cgcgccgcag 2220tgccgtcagg tgcttctacc
ggcgttcatg aagctgttga acttcgtgat ggcgacaaaa 2280cccgttgggg
tggtaaaggc gttaccaaag ctgttcacgc tgtaaacaac gaaattgcta
2340acgcaattat tggtctggaa gccgaagatc aggaactgat cgaccagacg
atgatcaagc 2400tcgatggcac cccgaacaag ggtaaattcg gtgctaacgc
tatcctcggt gtcagcttgg 2460ctgttgctaa agctgctgct gaagctcgcg
gtctcccgct ttaccgttat gttggtggta 2520cggcagctca cgttcttccg
gttccgatga tgaacatcgt taacggtggt atgcacgctg 2580acaaccccat
cgatttccag gaattcatga ttgctccggt tggcgccagc tctatcaatg
2640aagctgtccg catcggtacc gaagttttcc ataccctgaa aaaagaactg
tctgctaaag 2700gcatgaacac caacgtcggt gacgaaggtg gtttcgctcc
tagccttgac agtgcttctt 2760ctgctctgga cttcatcgtc gattccatct
ccaaagccgg ttataagccg ggcgaagatg 2820tgttcatcgc tctcgatgca
gcttcctccg agttctacaa caaagatcag aacatctacg 2880atcttaaggg
tgaaggccgt aaactgacct ccgctcagct cgttgattac tatgtcgaac
2940tctgcggcaa atatccgatc tattccatcg aagatggtct ggccgaagat
gacttcgaag 3000gctggaagat ccttaccgaa aagctcggtg acaaagttca
gttggtcggt gacgatctgt 3060tcgtgaccaa cgtgaagcgt ctttctgatg
gtatcgaacg cggtatcgcc aactcgctgc 3120tcgtgaagtt taaccagatc
ggttctttgt ctgaaacgct cgcagccgtt aacatggcta 3180acgacgcttc
ttacacggct gttatgtctc accgttccgg tgaaaccgaa gacaccacga
3240ttgctgacct cgctgttgcc accaactgcg gtcagatcaa gaccggtagc
ctttgccgtt 3300ccgaacgtat cgctaaatac aatcagctga tgcgcatcga
agaagaactg ggttcggttg 3360ctaaatatgc tggccgttcg gttcttagaa
aagccaaata agaatcacag ctagaaccga 3420gctcacataa cgaagagata
ttgaaaagga gtggaatatg cccacgctcg ttttgtcccg 3480tcacggacag
tccgaatgga accttgaaaa ccgtttcacc ggttggtggg atgttaacct
3540gactgaacag ggtgttcagg aagcaacggc cggtggtaaa gctctggctg
aaaagggttt 3600tgaattcgat atcgctttca ccagcgttct gacccgcgcc
atcaaaacca ccaatcttat 3660tctcgaagcc ggtaaaaccc tttgggttcc
gaccgaaaaa gattggcgtt tgaatgaacg 3720tcactatggt ggtctgaccg
gtctgaacaa ggctgaaacc gccgctaaac atggtgaaga 3780acaggttcat
atttggcgcc gttcttatgg cgttccgccg cccccgatgg aaaaaggcag
3840caagttcgat ctgtctggcg atcgccgtta tgatggtgtc aagattcctg
aaacggaaag 3900cctgaaagac accgttgctc gcgtgctgcc ttattgggaa
gaacgcattg cccctgaact 3960gaaggctggc aagcgcgtcc tgatcggtgc
gcatggtaac tcactgcgcg ctctcgttaa 4020gcatctgtcg aaattgtcgg
acgaagaaat cgtcaaattc gaattgccca ccggtcagcc 4080gttggtctac
gaattgaatg atgatctgac tccgaaagat cgttacttcc ttaacgaacg
4140ttaatagcct tgggctttta aagccttttg gtttgttaac cgttttttcg
gccagagttt 4200tctctggccg aaaatttatg tctatccctt tgtttttcta
tccccatcac ctcggttttg 4260ttggatccac tagt 427447432PRTSynechocystis
sp. strain PCC6803 47Met Leu Ser Lys Val Pro Ala Thr Ile Glu Glu
Ile Ala Ala Arg Glu 1 5 10 15 Ile Leu Asp Ser Arg Gly Arg Pro Thr
Ile Glu Ala Glu Val Arg Leu 20 25 30 Glu Ser Gly Ala His Gly Ile
Ala Gln Val Pro Ser Gly Ala Ser Thr 35 40 45 Gly Ser Phe Glu Ala
His Glu Leu Arg Asp Gly Asp Pro Lys Arg Tyr 50 55 60 Asp Gly Lys
Gly Val Glu Lys Ala Val Arg Asn Val Thr Glu Lys Ile 65 70 75 80 Ala
Pro Val Val Glu Gly Leu Asp Ala Phe Asp Gln Met Ala Val Asp 85 90
95 Gln Ala Met Ile Asp Arg Asp Gly Thr Asp Asn Lys Lys Glu Leu Gly
100 105 110 Ala Asn Ala Ile Leu Gly Val Ser Leu Ala Thr Ala Lys Ala
Ala Ala 115 120 125 Ala Glu Leu Ala Ile Pro Leu Tyr Arg Tyr Leu Gly
Gly Pro Leu Ala 130 135 140 Asn Val Leu Pro Val Pro Met Met Asn Val
Ile Asn Gly Gly Ala His 145 150 155 160 Ala Asp Asn Asn Val Asp Phe
Gln Glu Phe Met Ile Met Pro Val Gly 165 170 175 Ala Glu Thr Phe Lys
Glu Ala Leu Arg Trp Gly Ala Glu Val Phe Ala 180 185 190 Val Leu Gly
Lys Val Leu Lys Glu Arg Lys Leu Leu Ser Gly Gly Val 195 200 205 Gly
Asp Glu Gly Gly Tyr Ala Pro Asn Leu Thr Ser Asn Gln Gln Ala 210 215
220 Leu Asp Ile Leu Ile Glu Ala Ile Glu Gln Ala Gly Tyr Lys Pro Gly
225 230 235 240 Ser Gln Ile Ala Leu Ala Met Asp Ile Ala Ala Ser Glu
Phe Phe Lys 245 250 255 Asn Gly Gln Tyr Glu Tyr Asp Gly Gly Ser His
Ser Pro Gln Glu Phe 260 265 270 Ile Asp Tyr Gln Ala Lys Leu Val Ser
Gln Tyr Pro Ile Val Ser Ile 275 280 285 Glu Asp Gly Leu His Glu Asp
Asp Trp Glu Ser Trp Lys Gly Leu Thr 290 295 300 Thr Ser Leu Gly Thr
Lys Thr Gln Leu Val Gly Asp Asp Leu Met Val 305 310 315 320 Thr Asn
Pro Val Arg Leu Gln Lys Ser Ile Asp Leu Gly Val Ala Asn 325 330 335
Ala Ile Leu Ile Lys Leu Asn Gln Ile Gly Thr Leu Ser Glu Thr Leu 340
345 350 Glu Thr Ile Ser Leu Ala Thr Arg His Ser Tyr Arg Ser Val Ile
Ser 355 360 365 His Arg Ser Gly Glu Thr Glu Asp Thr Thr Ile Ala Asp
Leu Ala Val 370 375 380 Ala Thr Arg Val Gly Gln Ile Lys Thr Gly Ser
Leu Cys Arg Ser Glu 385 390 395 400 Arg Val Ala Lys Tyr Asn Arg Leu
Leu Arg Ile Glu Asp Glu Leu Gly 405 410 415 Asp Arg Ala Val Tyr Ala
Pro Lys Ile Gly Leu Gly Pro Lys His Ser 420 425 430
482026DNAartificialinsert of construct pVZ321-PpetJ-eno
48gtcgactcta gaggatcccc gggtacccct catcgggggc tgtgttggcc gagacggcac
60tgaggatttt actctccatg gcattccaag gaatatctac ccaactcacc tgctccggcg
120gattgttccg ctcaaaagta ctaatcaagt cgtcaaaata cttattaaat
tttggctgca 180attgcatagt ccaaaagctg actttcccct ccatgctctg
gggggaattg ctctggcaac 240tgattaatcc actgagcaac agcccaagac
acgcaaacaa aaaccaacgt cttggcgatc 300gccatcggca ccatgaaacc
atcgtaaaag ctggggaaag aataaaaaac agtggttcag 360gaattgcatt
gccatggcca cttcacaaac ctagccaatt ttagcttgac cgcaactttg
420acagattgtc ttttgacttt gcctggaccg cctcccataa taccttcgcg
tcttgaagac 480tttatccttg aaaggagaac atatggagct cttaagtaaa
gtccccgcca ccattgaaga 540aatcgccgcc cgggaaattt tagactccag
gggtcgcccc accatcgaag cggaagtccg 600gctggaaagt ggggcccacg
gcattgccca ggtgcccagt ggtgcttcca caggcagttt 660tgaagcccat
gaattgcggg acggagaccc caaacgctat gacgggaaag gggtagaaaa
720agcagtacgg aacgtgacag aaaaaattgc cccggtggtg gaaggcctgg
atgccttcga 780tcaaatggcg gtggatcagg ccatgattga ccgggatgga
acggacaata aaaaagaatt 840gggggccaat gccattttgg gggtttcttt
agccaccgct aaggccgccg ccgctgagct 900agccattccc ctctaccgct
acctgggagg ccccctggct aacgtactgc cggtaccgat 960gatgaacgtg
attaacggtg gggcccatgc cgacaataac gtcgattttc aggaattcat
1020gatcatgccg gtgggggcag aaacctttaa agaagctctg cgctgggggg
cggaagtttt 1080cgctgtgttg ggcaaagtgt tgaaagaacg aaaactgctc
tccggtgggg tgggggacga 1140agggggttat gctcccaatt tgacctcgaa
tcaacaggcc ctagatattc tcatcgaggc 1200gattgaacaa gctggttaca
aacccggtag tcaaattgcc ttggccatgg acattgccgc 1260cagtgaattt
ttcaaaaatg gtcagtatga atacgacggt ggttcccatt ctccccagga
1320attcatcgac tatcaggcca agctagtgag tcaatatccc attgtctcca
ttgaagacgg 1380tttgcacgaa gacgattggg aaagttggaa gggtttaacc
acttccctgg gcaccaaaac 1440ccagttggtg ggggatgact tgatggtgac
caacccggtg cgtctgcaaa aatccattga 1500tttgggagtt gccaacgcca
ttttgatcaa actcaatcaa atcggcactt tgagcgaaac 1560tttagagacc
atttccctag ctactcgcca tagttaccgt tctgttattt cccatcgctc
1620cggtgaaacg gaggacacca cgatcgccga cttggccgtg gccaccaggg
tagggcaaat 1680taaaaccggt tccctttgtc gttctgagcg ggttgctaaa
tataaccgtt tactccgcat 1740tgaagatgaa cttggcgatc gggccgttta
tgcccctaaa attggcctgg gtcccaaaca 1800ttcttaaaaa gtgttttaaa
gttcccctag cccaagggtt ggggttactt cgggagataa 1860tcaaaccatt
gccaacaggt tcttttggtt aggttgttgc ttaggattgt ctaaaattcc
1920tcagtttttt attcaagact taatttttcc catggttacc ctaccggtga
actgtgggaa 1980tttttatgcc taacccgttc taagccaagg aagcaatgac ctcgag
202649443PRTSynechocystis sp. strain PCC6803 49Met Ala Thr Arg Val
Ile Ile Val Arg His Gly Gln Ser Thr Tyr Asn 1 5 10 15 Ala Glu Lys
Arg Ile Gln Gly Arg Ser Asn Leu Ser Val Leu Thr Asp 20 25 30 Lys
Gly Lys Ala Asp Ala Gln Lys Val Gly Gln Thr Leu Asn Ser Leu 35 40
45 Ala Ile Asp Lys Ile Tyr Cys Ser Pro Leu Arg Arg Ala Lys Glu Thr
50 55 60 Ala Gln Ile Ile Gln Ala Ser Phe Ala His Pro Pro Glu Leu
Ile Pro 65 70 75 80 Ser Glu Asn Leu Leu Glu Val Asn Leu Pro Leu Trp
Glu Lys Met Thr 85 90 95 Lys Asp Asp Val Ala His Gln Tyr Pro Glu
Gln Tyr Arg Leu Trp His 100 105 110 Glu Ala Pro Asp Gln Leu Ala Met
Thr Val Asp Gly Ala Glu Tyr Tyr 115 120 125 Pro Val Ala Ala Leu Tyr
Ala Gln Ala Gln Arg Phe Trp Gln Asp Val 130 135 140 Leu Thr Asp Ala
Ala Gly Gln Thr Leu Leu Ile Val Ala His Asn Gly 145 150 155 160 Ile
Asn Arg Cys Leu Leu Met Ser Ala Ile Gly Met Pro Ala Ser His 165 170
175 Tyr Gln Arg Leu Gln Gln Ser Asn Cys Asn Ile Asn Val Leu Asn Phe
180 185 190 Ser Gly Gly Trp Gly Asp Pro Val Gln Leu Glu Ser Leu Asn
Gln Thr 195 200 205 Ala His Met Gly Val Pro Leu Pro Pro Pro Arg Lys
Asp Asn Asn Arg 210 215 220 Leu Arg Leu Leu Leu Ile Arg His Gly Glu
Thr Gln Trp Asn Arg Glu 225 230 235 240 Gly Arg Phe Gln Gly Ile Arg
Asp Ile Pro Leu Asn Asp Asn Gly Arg 245 250 255 His Gln Ala Gln Lys
Ala Ala Glu Phe Leu Lys Asp Val Pro Ile Asn 260 265 270 Leu Gly Ile
Ser Ser Pro Met Ala Arg Pro Lys Glu Thr Ala Glu Ile 275 280 285 Ile
Leu Gln Tyr His Pro Ser Ile Glu Leu Asp Leu Gln Pro Glu Leu 290 295
300 Ala Glu Ile Cys His Gly Leu Trp Glu Gly Lys Leu Glu Thr Glu Ile
305 310 315 320 Glu Ala Glu Tyr Pro Gly Leu Leu Gln Gln Trp Lys Asp
Ala Pro Ala 325 330 335 Thr Val Gln Met Pro Glu Gly Glu Asn Leu Gln
Gln Val Trp Asp Arg 340 345 350 Ala Ile Ala Cys Trp Gln Asp Arg Val
Lys Phe Tyr Ser Gln Gly Asp 355 360 365 Gly Ser Thr Val Gly Ile Val
Val Ala His Asp Ala Ile Asn Lys Val 370 375 380 Ile Leu Ala Tyr Leu
Leu Gly Leu Thr Pro Ala His Phe Trp Gln Val 385 390 395 400 Lys Gln
Gly Asn Gly Gly Val Ser Val Ile Asp Tyr Pro Gln Gly Leu 405 410 415
Asp Lys Pro Pro Val Ile Gln Ala Ile Asn Leu Met Gly His Leu Gly 420
425 430 Thr Val Leu Asp Lys Thr Ala Ala Gly Ala Leu 435 440
501706DNAartificialinsert of construct pVZ321-PpetJ-pgm
50gtcgactcta gaggatcccc gggtacccct catcgggggc tgtgttggcc gagacggcac
60tgaggatttt actctccatg gcattccaag gaatatctac ccaactcacc tgctccggcg
120gattgttccg ctcaaaagta ctaatcaagt cgtcaaaata cttattaaat
tttggctgca 180attgcatagt ccaaaagctg actttcccct ccatgctctg
gggggaattg ctctggcaac 240tgattaatcc actgagcaac agcccaagac
acgcaaacaa aaaccaacgt cttggcgatc 300gccatcggca ccatgaaacc
atcgtaaaag ctggggaaag aataaaaaac agtggttcag 360gaattgcatt
gccatggcca cttcacaaac ctagccaatt ttagcttgac cgcaactttg
420acagattgtc ttttgacttt gcctggaccg cctcccataa taccttcgcg
tcttgaagac 480tttatccttg aaaggagaac atatggagct caccaaagac
gatgtggccc accaatatcc 540cgaacaatat cgtctctggc acgaagcacc
ggatcaattg gccatgaccg tagatggagc 600ggaatattac cccgttgcgg
ctctctatgc ccaggcccaa agattttggc aggatgtgtt 660aaccgatgcg
gcgggacaaa ccctgctgat tgtggcccac aatggcatca atcgttgcct
720gttaatgagc gccattggta tgcccgcttc ccattaccaa cgcctgcaac
agtccaactg 780caatattaat gtgttgaatt ttagtggtgg ctggggcgat
ccggtgcaac tggaatcctt 840gaatcaaacc gcccatatgg gggtacctct
gccacctccc cgcaaggata ataatcgtct 900gcggttactg cttatccgcc
atggggaaac ccaatggaat cgggaaggac ggttccaagg 960tattcgggat
attcccctca atgacaatgg ccgccatcaa gcccaaaaag cggcggaatt
1020cctcaaagat gtgcccatta acctaggcat tagcagtccc atggctcggc
ccaaggaaac 1080ggcggagatt attctgcaat atcacccaag catagagttg
gatttacagc cggaattggc 1140ggaaatttgc catggcctgt gggaaggcaa
gctagaaacg gaaattgaag cggaatatcc 1200cggattattg caacagtgga
aagatgcccc cgccacagtg cagatgccgg aaggggaaaa 1260tttacaacag
gtctgggacc gggcgatcgc ctgttggcag gaccgggtca aattctatag
1320ccagggggat ggttccacag tgggcattgt ggtggcccat gatgccatca
acaaggtgat 1380tttggcttat ttgttgggtc ttactcccgc tcacttttgg
caagttaaac agggtaatgg 1440cggggtgagc gtcattgact atccccaggg
tctagataag cccccagtta ttcaagccat 1500taatttgatg ggccatttgg
gcacagtgtt ggataaaacc gccgccggag ccctatagtc 1560ctgtccatag
ccaattatcc ccccatttgt tccctaactc ttgtttgcta tgactcactt
1620tggtttgctc tgtccagcaa cgacgggtca tctcaatacc atgttgccct
tgggtaagga 1680actgcaacag cggggtcata ctcgag
1706514479DNAartificialinsert of construct
pVZ322-PpetJ-pyk1-eno-pgm 51ctgcaggtcg actctagagg atccccgggt
acccctcatc gggggctgtg ttggccgaga 60cggcactgag gattttactc tccatggcat
tccaaggaat atctacccaa ctcacctgct 120ccggcggatt gttccgctca
aaagtactaa tcaagtcgtc aaaatactta ttaaattttg 180gctgcaattg
catagtccaa aagctgactt tcccctccat gctctggggg gaattgctct
240ggcaactgat taatccactg agcaacagcc caagacacgc aaacaaaaac
caacgtcttg 300gcgatcgcca tcggcaccat gaaaccatcg taaaagctgg
ggaaagaata aaaaacagtg 360gttcaggaat tgcattgcca tggccacttc
acaaacctag ccaattttag cttgaccgca 420actttgacag attgtctttt
gactttgcct ggaccgcctc ccataatacc ttcgcgtctt 480gaagacttta
tccttgaaag gagaacatat gagaccccta agtcatcgca ccaaaattgt
540ggccaccatt ggccccgcca gtagttcggt agaggtgata cgtcagatgg
tggatgcggg 600catgaatgtg gcccggctga atttttccca cggtagttat
gaggaccatg ccaccatggt 660tcgcctacta cggtcagtgg agcaggaaat
ggacaccccc attacccttt tgcaagattt 720acaggggccc aaaattcgga
ttggtcagtt gccgggggga gaaaagcaac tgcgggaagg 780ggaaaaagtt
tctttggtgc cggtggaaat tggcgatcgc catcctggag cggtgggcat
840tgactatccc catttggcga cggaggcaaa agtaggggaa agaattttat
tggacgatgg 900tttactggaa atgaaagttg tgtccattca agatccagag
gtaatttgtg aagtggtgac 960cgggggcatc ctcaaaagtc gcaaaggggt
caatctaccg ggtttagtcc taaccctacc 1020ttccatgacg accaaagaca
agcaagactt agaatttggt ttgagccaag gcattgactg 1080ggtttccctt
agttttgtcc gcaaagggga agatatccac acccttaaac aatttctcgc
1140tgaacggggc catcctgatc tgccggtcat tgccaaaatt gaaaaacccc
aggcgatcga 1200taatctagaa gaaatcgtgg cagtttccaa cggcattatg
gtggccaggg gggatctggg 1260ggtggaagta aacccagaaa aggttccccg
tttgcaaaag gaaattattc ggcgctgtaa 1320cgtgcgggcc attccggtca
tcaccgctac ccaaatgcta gatagcatga ttcaaaattc 1380ccgacccacc
agggcggaag ccagtgacgt ggccaacgct attttggacg gcaccgatgc
1440ggtgatgtta tcgggggaat cggcagtggg acaatatccc gttaaatcag
tgcaaatgtt 1500gcgaaaaatt gccgaagaga cggaagtggg tctacatctt
gttaataatc ccccaataga 1560aaatacggaa acccatgccc taagtgaagc
gttagtggta attgacggaa ttctagattt 1620aaaatacatt gtcacgttca
ccacctcagg ttttacttct ctcctcgctt ccaaccaaag 1680accgtcggtg
ccggtgattg ctttcactcc ctcagaaaag gtttaccata gcctcaactt
1740agtttggggt attattccct ttttaattaa cgaagaattt gacacctttg
aggatttaat 1800ccaacaggcg gaggtactac tgcgggatag aaaaatggtg
gaaaagggcg atcagttgct 1860aatcatggcg ggaattccca ctaaaatacc
caggggcact aatttcctca agattcaccg 1920tatttcttaa gagctcgtgt
ttggagcatt acacaccgat gttaagtaaa gtccccgcca 1980ccattgaaga
aatcgccgcc cgggaaattt tagactccag gggtcgcccc accatcgaag
2040cggaagtccg gctggaaagt ggggcccacg gcattgccca ggtgcccagt
ggtgcttcca 2100caggcagttt tgaagcccat gaattgcggg acggagaccc
caaacgctat gacgggaaag 2160gggtagaaaa agcagtacgg aacgtgacag
aaaaaattgc cccggtggtg gaaggcctgg 2220atgccttcga tcaaatggcg
gtggatcagg ccatgattga ccgggatgga acggacaata 2280aaaaagaatt
gggggccaat gccattttgg gggtttcttt agccaccgct aaggccgccg
2340ccgctgagct agccattccc ctctaccgct acctgggagg ccccctggct
aacgtactgc 2400cggtaccgat gatgaacgtg attaacggtg gggcccatgc
cgacaataac gtcgattttc 2460aggaattcat gatcatgccg gtgggggcag
aaacctttaa agaagctctg cgctgggggg 2520cggaagtttt cgctgtgttg
ggcaaagtgt tgaaagaacg aaaactgctc tccggtgggg 2580tgggggacga
agggggttat gctcccaatt tgacctcgaa tcaacaggcc ctagatattc
2640tcatcgaggc gattgaacaa gctggttaca aacccggtag tcaaattgcc
ttggccatgg 2700acattgccgc cagtgaattt ttcaaaaatg gtcagtatga
atacgacggt ggttcccatt 2760ctccccagga attcatcgac tatcaggcca
agctagtgag tcaatatccc attgtctcca 2820ttgaagacgg tttgcacgaa
gacgattggg aaagttggaa gggtttaacc acttccctgg 2880gcaccaaaac
ccagttggtg ggggatgact tgatggtgac caacccggtg cgtctgcaaa
2940aatccattga tttgggagtt gccaacgcca ttttgatcaa actcaatcaa
atcggcactt 3000tgagcgaaac tttagagacc atttccctag ctactcgcca
tagttaccgt tctgttattt 3060cccatcgctc cggtgaaacg gaggacacca
cgatcgccga cttggccgtg gccaccaggg 3120tagggcaaat taaaaccggt
tccctttgtc gttctgagcg ggttgctaaa tataaccgtt 3180tactccgcat
tgaagatgaa cttggcgatc gggccgttta tgcccctaaa attggcctgg
3240gtcccaaaca ttcttaaaaa gatctgcccc tctgggaaaa aatgaccaaa
gacgatgtgg 3300cccaccaata tcccgaacaa tatcgtctct ggcacgaagc
accggatcaa ttggccatga 3360ccgtagatgg agcggaatat taccccgttg
cggctctcta tgcccaggcc caaagatttt 3420ggcaggatgt gttaaccgat
gcggcgggac aaaccctgct gattgtggcc cacaatggca 3480tcaatcgttg
cctgttaatg agcgccattg gtatgcccgc ttcccattac caacgcctgc
3540aacagtccaa ctgcaatatt aatgtgttga attttagtgg tggctggggc
gatccggtgc 3600aactggaatc cttgaatcaa accgcccata tgggggtacc
tctgccacct ccccgcaagg 3660ataataatcg tctgcggtta ctgcttatcc
gccatgggga aacccaatgg aatcgggaag 3720gacggttcca aggtattcgg
gatattcccc tcaatgacaa tggccgccat caagcccaaa 3780aagcggcgga
attcctcaaa gatgtgccca ttaacctagg cattagcagt cccatggctc
3840ggcccaagga aacggcggag attattctgc aatatcaccc aagcatagag
ttggatttac 3900agccggaatt ggcggaaatt tgccatggcc tgtgggaagg
caagctagaa acggaaattg 3960aagcggaata tcccggatta ttgcaacagt
ggaaagatgc ccccgccaca gtgcagatgc 4020cggaagggga aaatttacaa
caggtctggg accgggcgat cgcctgttgg caggaccggg 4080tcaaattcta
tagccagggg gatggttcca cagtgggcat tgtggtggcc catgatgcca
4140tcaacaaggt gattttggct tatttgttgg gtcttactcc cgctcacttt
tggcaagtta 4200aacagggtaa tggcggggtg agcgtcattg actatcccca
gggtctagat aagcccccag 4260ttattcaagc cattaatttg atgggccatt
tgggcacagt gttggataaa accgccgccg 4320gagccctata gtcctgtcca
tagccaatta tccccccatt tgttccctaa ctcttgtttg 4380ctatgactca
ctttggtttg ctctgtccag caacgacggg tcatctcaat accatgttgc
4440ccttgggtaa ggaactgcaa cagcggggtc atactcgag
4479524834DNAartificialinsert of construct
pVZ322-PpetJ-pyk2-eno-pgm 52ctgcaggtcg actctagagg atccccgggt
acccctcatc gggggctgtg ttggccgaga 60cggcactgag gattttactc tccatggcat
tccaaggaat atctacccaa ctcacctgct 120ccggcggatt gttccgctca
aaagtactaa tcaagtcgtc aaaatactta ttaaattttg 180gctgcaattg
catagtccaa aagctgactt tcccctccat gctctggggg gaattgctct
240ggcaactgat taatccactg agcaacagcc caagacacgc aaacaaaaac
caacgtcttg 300gcgatcgcca tcggcaccat gaaaccatcg taaaagctgg
ggaaagaata aaaaacagtg 360gttcaggaat tgcattgcca tggccacttc
acaaacctag ccaattttag cttgaccgca 420actttgacag attgtctttt
gactttgcct ggaccgcctc ccataatacc ttcgcgtctt 480gaagacttta
tccttgaaag gagaacatat gcaaacgtct ccccttcccc gtcgtaccaa
540aatcgtcgct accattggcc ccgcgaccca aagcaaggaa gtgctgagac
aactgatcca 600agccggtgct accactttcc gcctcaattt ttcccacgga
gaccacgctt accaccaaca 660aagtatccgt ttgattcgcc agattgcctt
tgaactgaac caaccagtgg gcattctcca 720ggatttacag gggccaaaga
ttcgggtggg caaatttctc aatgatgccg gctctgtgca 780actcaaaaac
ggtgatccct ataccctcac cagtcgcccg gtggaatgta cggaaaccat
840tagttccatt agctacgaat atttagccga cgaagtacct tctggggcaa
gaattttgct 900cgacgacggc aaactggaaa tgttggtgga ggaagtggac
actgttgccc gggatctcca 960ctgtcgggtg attgtggggg gaaccctttc
cagcaataaa ggggttaatt ttcccggggt 1020ctgcctttcc gttaaggcca
tgaccgataa agataaggaa gatttgatgt tcgggctgga 1080ccaaggggtg
gactgggtgg ccctgagttt tgttcgtaat ccccaggata ttgatgagat
1140taaggggtta attgcggcgg cagggaaatc cgtgccggta atcgccaaaa
ttgagaagca 1200cgaagcgatt aaggatatgc aggcggtgct ggaaaaatgt
gacggtgtca tggtggcccg 1260gggggacttg ggggtagaac tacccgcaga
agatgtacct attttgcaaa agaaactcat 1320tgccactgct aaccggttgg
gcattcctgt cattactgct acccaaatgt tggacagtat 1380ggtcaacagc
ccccgaccta ctagggctga agtgtccgac gtggccaatg ccatcctcga
1440tggtaccgat gcggtgatgc tctccaacga aacggcgatc ggtaaatttc
ccgtggaagc 1500agtggctatt atggccaaaa ttgcggagcg cattgaacag
gaagatatca atccttccca 1560agcggaagcc agtcgcactt ctattcccaa
tgctatttcc agcgccgtta gccagattgc 1620ggaaaccctc aacgcggcgg
ctattatgtc tttgactaag accggatcca ccgcccgcca 1680tgtgtcaaag
ttccgcccca aaacccccat tctggccgtt acgccccatg tggatgtgtc
1740ccgtcagttg cagttggtgt ggggagttaa gcccctgttg gtattggatt
taccttccac 1800cagccaaacg ttccaagccg ccattaacgt ggcccaggaa
aaccattttc tccgggatgg 1860agatttggtg gtgatgaccg ccgggacatt
gcagggagtt gccggttcga cggatttaat 1920caaagtggaa gtggtcaagg
ccattcttgg tcggggtgta ggtattggcc aaggagctgt 1980aagtggccgg
gccagggttg ccagtcgtcc ccaggcgatc gcccaattta cccagggaga
2040aattttagta gttccctcta ccaacgctga ttgtgtggac atgatgcgac
gggcggcggg 2100cattatcacc gaagaagaaa gcctgactag ccatgcggcc
attattggtt tgcggctggg 2160ggtgccggtc attgtgggtt ttaaaggcgc
tacccaaaag attcgagatg gagccattgt 2220caccatcgat gcccaaaaag
gactgattta ttccggtgca ttacccccgg tgtccaaagg 2280atagggagct
cgtgtttgga gcattacaca ccgatgttaa gtaaagtccc cgccaccatt
2340gaagaaatcg ccgcccggga aattttagac tccaggggtc gccccaccat
cgaagcggaa 2400gtccggctgg aaagtggggc ccacggcatt gcccaggtgc
ccagtggtgc ttccacaggc 2460agttttgaag cccatgaatt gcgggacgga
gaccccaaac gctatgacgg gaaaggggta 2520gaaaaagcag tacggaacgt
gacagaaaaa attgccccgg tggtggaagg cctggatgcc 2580ttcgatcaaa
tggcggtgga tcaggccatg attgaccggg atggaacgga caataaaaaa
2640gaattggggg ccaatgccat tttgggggtt tctttagcca ccgctaaggc
cgccgccgct 2700gagctagcca ttcccctcta ccgctacctg ggaggccccc
tggctaacgt actgccggta 2760ccgatgatga acgtgattaa cggtggggcc
catgccgaca ataacgtcga ttttcaggaa 2820ttcatgatca tgccggtggg
ggcagaaacc tttaaagaag ctctgcgctg gggggcggaa 2880gttttcgctg
tgttgggcaa agtgttgaaa gaacgaaaac tgctctccgg tggggtgggg
2940gacgaagggg gttatgctcc caatttgacc tcgaatcaac aggccctaga
tattctcatc 3000gaggcgattg aacaagctgg ttacaaaccc ggtagtcaaa
ttgccttggc catggacatt 3060gccgccagtg aatttttcaa aaatggtcag
tatgaatacg acggtggttc ccattctccc 3120caggaattca tcgactatca
ggccaagcta gtgagtcaat atcccattgt ctccattgaa 3180gacggtttgc
acgaagacga ttgggaaagt tggaagggtt taaccacttc cctgggcacc
3240aaaacccagt tggtggggga tgacttgatg gtgaccaacc cggtgcgtct
gcaaaaatcc 3300attgatttgg gagttgccaa cgccattttg atcaaactca
atcaaatcgg cactttgagc 3360gaaactttag agaccatttc cctagctact
cgccatagtt accgttctgt tatttcccat 3420cgctccggtg aaacggagga
caccacgatc gccgacttgg ccgtggccac cagggtaggg 3480caaattaaaa
ccggttccct ttgtcgttct gagcgggttg ctaaatataa ccgtttactc
3540cgcattgaag atgaacttgg cgatcgggcc gtttatgccc ctaaaattgg
cctgggtccc 3600aaacattctt aaaaagatct gcccctctgg gaaaaaatga
ccaaagacga tgtggcccac 3660caatatcccg aacaatatcg tctctggcac
gaagcaccgg atcaattggc catgaccgta 3720gatggagcgg aatattaccc
cgttgcggct ctctatgccc aggcccaaag attttggcag 3780gatgtgttaa
ccgatgcggc gggacaaacc ctgctgattg tggcccacaa tggcatcaat
3840cgttgcctgt taatgagcgc cattggtatg cccgcttccc attaccaacg
cctgcaacag 3900tccaactgca atattaatgt gttgaatttt agtggtggct
ggggcgatcc ggtgcaactg 3960gaatccttga atcaaaccgc ccatatgggg
gtacctctgc cacctccccg caaggataat 4020aatcgtctgc ggttactgct
tatccgccat ggggaaaccc aatggaatcg ggaaggacgg 4080ttccaaggta
ttcgggatat tcccctcaat gacaatggcc gccatcaagc ccaaaaagcg
4140gcggaattcc tcaaagatgt gcccattaac ctaggcatta gcagtcccat
ggctcggccc 4200aaggaaacgg cggagattat tctgcaatat cacccaagca
tagagttgga tttacagccg 4260gaattggcgg aaatttgcca tggcctgtgg
gaaggcaagc tagaaacgga aattgaagcg 4320gaatatcccg gattattgca
acagtggaaa gatgcccccg ccacagtgca gatgccggaa 4380ggggaaaatt
tacaacaggt ctgggaccgg gcgatcgcct gttggcagga ccgggtcaaa
4440ttctatagcc agggggatgg ttccacagtg ggcattgtgg tggcccatga
tgccatcaac 4500aaggtgattt tggcttattt gttgggtctt actcccgctc
acttttggca agttaaacag 4560ggtaatggcg gggtgagcgt cattgactat
ccccagggtc tagataagcc cccagttatt 4620caagccatta atttgatggg
ccatttgggc acagtgttgg ataaaaccgc cgccggagcc 4680ctatagtcct
gtccatagcc aattatcccc ccatttgttc cctaactctt gtttgctatg
4740actcactttg gtttgctctg tccagcaacg acgggtcatc tcaataccat
gttgcccttg 4800ggtaaggaac tgcaacagcg gggtcatact cgag
483453821PRTSynechocystis sp. strain PCC6803 53Met Gly Ser Thr Leu
Val Gly Lys Cys Thr Ser Leu Gly Val Phe Ser 1 5 10 15 Met Val Thr
Ser Pro Phe Ser Leu Ser Pro Phe Gly Gln Ala Arg Ser 20 25 30 Thr
Val Thr Gly Asn Pro Leu Asp Pro Thr Glu Leu Asn Gln Met His 35 40
45 Gly Phe Trp Arg Ala Ala Asn Tyr Leu Ala Val Gly Met Ile Tyr Leu
50 55 60 Arg Asp Asn Pro Leu Leu Arg Glu Pro Leu Gln Pro Glu Gln
Ile Lys 65 70 75 80 His Arg Leu Leu Gly His Trp Gly Ser Ser Pro Gly
Ile Ser Phe Leu 85 90 95 Tyr Thr His Leu Asn Arg Ile Ile Arg Lys
Phe Asp Gln Asp Met Leu 100 105 110 Tyr Met Val Gly Pro Gly His Gly
Ala Pro Gly Phe Leu Gly Pro Cys 115 120 125 Tyr Leu Glu Gly Ser Tyr
Ser Arg Phe Phe Ala Glu Cys Ser Glu Asp 130 135 140 Glu Asp Gly Met
Lys Arg Phe Phe Lys Gln Phe Ser Phe Pro Gly Gly 145 150 155 160 Ile
Gly Ser His Cys Thr Pro Glu Thr Pro Gly Ser Ile His Glu Gly 165 170
175 Gly Glu Leu Gly Tyr Cys Leu Ser His Ala Tyr Gly Ala Ala Phe Asp
180 185 190 Asn Pro Asn Leu Ile Val Val Gly Leu Ala Gly Asp Gly Glu
Ser Glu 195 200 205 Thr Gly Pro Leu Ala Thr Ser Trp His Ser Asn Lys
Phe Ile Asn Pro 210 215 220 Ile Arg Asp Gly Ala Val Leu Pro Val Leu
His Leu Asn Gly Tyr Lys 225 230 235 240 Ile Asn Asn Pro Ser Val Leu
Ser Arg Ile Ser His Glu Glu Leu Lys 245 250 255 Ala Leu Phe Glu Gly
Tyr Gly Tyr Thr Pro Tyr Phe Val Glu Gly Ser 260 265 270 Asp Pro Glu
Ser Met His Gln Ala Met Ala Ala Thr Leu Asp His Cys 275 280 285 Val
Ser Glu Ile His Gln Ile Gln Gln Glu Ala Arg Ser Thr Gly Ile 290 295
300 Ala Val Arg Pro Arg Trp Pro Met Val Val Met Arg Thr Pro Lys Gly
305 310 315 320 Trp Thr Gly Pro Asp Tyr Val Asp Gly His Lys Val Glu
Gly Phe Trp 325 330 335 Arg Ser His Gln Val Pro Met Gly Gly Met His
Glu Asn Pro Ala His 340 345 350 Leu Gln Gln Leu Glu Ala Trp Met Arg
Ser Tyr Lys Pro Glu Glu Leu 355 360 365 Phe Asp Glu Gln Gly Thr Leu
Lys Pro Gly Phe Lys Ala Ile Ala Pro 370 375 380 Glu Gly Asp Lys Arg
Leu Gly Ser Thr Pro Tyr Ala Asn Gly Gly Leu 385 390 395 400 Leu Arg
Arg Gly Leu Lys Met Pro Asp Phe Arg Gln Tyr Gly Ile Asp 405 410 415
Val Asp Gln Pro Gly Thr Ile Glu Ala Pro Asn Thr Ala Pro Leu Gly 420
425 430 Val Phe Leu Arg Asp Val Met Ala Asn Asn Met Thr Asn Phe Arg
Leu 435 440 445 Phe Gly Pro Asp Glu Asn Ser Ser Asn Lys Leu His Ala
Val Tyr Glu 450 455 460 Val Ser Lys Lys Phe Trp Ile Ala Glu Tyr Leu
Glu Glu Asp Gln Asp 465 470 475 480 Gly Gly Glu Leu Ser Pro Asp Gly
Arg Val Met Glu Met Leu Ser Glu 485 490 495 His Thr Leu Glu Gly Trp
Leu Glu Ala Tyr Leu Leu Thr Gly Arg His 500 505 510 Gly Phe Phe Ala
Thr Tyr Glu Ser Phe Ala His Val Ile Thr Ser Met 515 520 525 Val Asn
Gln His Ala Lys Trp Leu Asp Ile Cys Arg His Leu Asn Trp 530 535 540
Arg Ala Asp Ile Ser Ser Leu Asn Ile Leu Met Thr Ser Thr Val Trp 545
550 555 560 Arg Gln Asp His Asn Gly Phe Thr His Gln Asp Pro Gly Phe
Leu Asp 565 570 575 Val Ile Leu Asn Lys Ser Pro Asp Val Val Arg Ile
Tyr Leu Pro Pro 580 585 590 Asp Val Asn Ser Leu Leu Ser Val Ala Asp
His Cys Leu Gln Ser Lys 595 600 605 Asn Tyr Ile Asn Ile Ile Val Cys
Asp Lys Gln Ala His Leu Gln Tyr 610 615 620 Gln Asp Met Thr Ser Ala
Ile Arg Asn Cys Thr Lys Gly Val Asp Ile 625 630 635 640 Trp Glu Trp
Ala Ser Asn Asp Ala Gly Thr Glu Pro Asp Val Val Met 645 650 655 Ala
Ala Ala Gly Asp Ile Pro Thr Lys Glu Ala Leu Ala Ala Thr Ala 660 665
670 Met Leu Arg Gln Phe Phe Pro Asn Leu Arg Ile Arg Phe Val Ser Val
675 680 685 Ile Asp Leu Leu Lys Leu Gln Pro Glu Ser Glu His Pro His
Gly Leu 690 695 700 Ser Asp Arg Asp Phe Asp Ser Leu Phe Thr Thr Asp
Lys Pro Ile Ile 705 710 715 720 Phe Asn Phe His Ala Tyr Pro Trp Leu
Ile His Arg Leu Thr Tyr Arg 725 730 735 Arg Thr Asn His Gly Asn Leu
His Val Arg Gly Tyr Lys Glu Lys Gly 740 745 750 Asn Ile Asn Thr Pro
Met Asp Leu Ala Ile Gln Asn Gln Ile Asp Arg 755 760 765 Phe Ser Leu
Ala Ile Asp Val Ile Asp Arg Leu Pro Gln Leu Arg Val 770 775 780 Ala
Gly Ala His Ile Lys Glu Met Leu Lys Asp Met Gln Ile Asp Cys 785 790
795 800 Thr Asn Tyr Ala Tyr Glu His Gly Ile Asp Met Pro Glu Ile Val
Asn 805 810 815 Trp Arg Trp Pro Leu 820 543234DNAartificialinsert
of the construct pVZ322 PpetJ-phk 54ctgcaggtcg actctagagg
atccccgggt acccctcatc gggggctgtg ttggccgaga 60cggcactgag gattttactc
tccatggcat tccaaggaat atctacccaa ctcacctgct 120ccggcggatt
gttccgctca aaagtactaa tcaagtcgtc aaaatactta ttaaattttg
180gctgcaattg catagtccaa aagctgactt tcccctccat gctctggggg
gaattgctct 240ggcaactgat taatccactg agcaacagcc caagacacgc
aaacaaaaac caacgtcttg 300gcgatcgcca tcggcaccat gaaaccatcg
taaaagctgg ggaaagaata aaaaacagtg 360gttcaggaat tgcattgcca
tggccacttc acaaacctag ccaattttag cttgaccgca 420actttgacag
attgtctttt gactttgcct ggaccgcctc ccataatacc ttcgcgtctt
480gaagacttta tccttgaaag gagaacatat ggttacatcc cccttttccc
ttagtccctt 540tggtcaagct agatccaccg tcactggcaa tccccttgac
ccgacagaac ttaaccaaat 600gcacggtttt tggcgggcag ccaactactt
ggcagtgggc atgatttatc tgcgggataa 660tccccttttg cgggaaccgc
ttcaaccgga acagatcaag catcgcctgt tgggtcactg 720gggttctagt
cccggcatta gttttctcta cacccatctc aaccgcatta tcaggaaatt
780tgaccaggat atgctgtaca tggtggggcc tggccacggc gcaccaggct
ttttggggcc 840ctgctaccta gaagggagct attctcgctt ttttgccgag
tgtagtgaag atgaggacgg 900catgaagcgc tttttcaaac aattttcctt
tcccggtggc attggcagtc attgcactcc 960cgaaacccct ggttccatcc
acgagggggg agaattgggc tactgcctat cccatgccta 1020tggcgctgcc
tttgataatc ccaatttaat tgtggtcggt ttagcggggg atggggagtc
1080ggaaacaggc cccttggcta cctcctggca ttccaataag tttattaacc
cgattcggga 1140tggggcagtt ttaccggttc tgcatctcaa tgggtacaag
attaacaatc caagtgtttt 1200atctcgcatt agccatgaag aattaaaggc
tttatttgaa ggttacggtt atacccccta 1260ctttgttgaa ggctctgacc
cggaatctat gcaccaagcc atggcagcca cgttggatca 1320ttgtgtgagc
gaaattcatc aaatccaaca agaagctcgt agtacgggca ttgccgtgcg
1380cccccgttgg cccatggttg tgatgcggac tcccaaggga tggacggggc
ctgactatgt 1440tgatggccat aaggtagaag gtttttggcg atcgcaccaa
gttcccatgg ggggcatgca 1500cgagaatcca gcccatttgc aacagttgga
agcttggatg cggagttata agccggaaga 1560attgttcgac gagcaaggta
ctttaaaacc gggatttaag gcgatcgccc cggagggaga 1620taagcgttta
ggctctactc cctacgccaa tggtggtttg ttacggcggg gtttgaaaat
1680gccggacttt cgtcaatatg gtattgatgt ggaccaacca ggcaccatcg
aagcccctaa 1740tactgcaccc ctgggagtat ttctgcggga tgtgatggcc
aacaacatga ccaatttccg 1800cctgtttggc cccgatgaaa atagttccaa
taaactccat gccgtctacg aggttagcaa 1860aaaattctgg attgctgaat
atctagaaga agaccaggat gggggggaat taagtcccga 1920tggtcgggtg
atggaaatgt taagcgagca caccttagaa ggttggttag aggcctatct
1980tttaaccggg cgtcacggct ttttcgccac ctatgaatcc tttgcccatg
tgatcacttc 2040catggttaac caacacgcta aatggttgga tatttgtcga
cacctcaact ggcgggcaga 2100tatttcctcg ttaaatatct tgatgacgtc
caccgtgtgg cgacaggatc acaacgggtt 2160tacccaccaa gatcccggtt
ttctcgatgt cattctcaat aaaagccccg atgtggtgcg 2220aatttattta
ccccccgatg ttaattctct gctttccgta gcggaccatt gtttacagag
2280caaaaactac atcaacatca tcgtttgcga taagcaagcc cacctgcaat
accaggacat 2340gacttccgct atccgtaact gcactaaagg ggtggacatt
tgggaatggg ccagtaatga 2400tgccggtacg gaaccggatg tggtgatggc
agcggcgggg gatattccca ccaaagaggc 2460cttggcggcc acagccatgc
taaggcaatt ttttcctaat ctgagaattc gctttgtcag 2520cgtgattgat
ttgctcaaac tgcaaccgga atcggagcat ccccatggcc tgagcgatcg
2580ggattttgac tccctcttta ccaccgataa accgattatt tttaacttcc
acgcctatcc 2640ctggttaatt catcggttga cctatcgacg gactaaccat
ggcaatctcc atgtgcgggg 2700ctacaaggaa aagggcaaca tcaacacccc
catggattta gcgattcaaa accagattga 2760ccgtttcagc ctcgccattg
atgtgatcga tcgcctgccc caattgcggg tggccggagc 2820ccacatcaag
gaaatgctca aggatatgca gattgactgc accaactacg cctacgaaca
2880cggcattgat atgccagaaa tcgttaattg gcgctggccc ctctagacct
taactaaaat 2940ccctgacatc gttctagttt ctgttccaat aggttagcta
ggccatgggg gacaacgctg 3000gtccagcaaa attttggcag aagctagagc
aaagttgggg agcttttccc gttaaagatc 3060ggcaaaggtt tcccctgcca
gtaagccagc attaattttg ttggtgacca gttcccggta 3120catggctagt
tcctgggata gtaattcata ccggggcttg gagtggagtg ccaaggcctt
3180aatgttggat tcttcttcct tggtgaccac accatcggcc acggcctgct cgag
323455697PRTSynechocystis sp. strain PCC6803 55Met Thr Ser Ser Leu
Tyr Leu Ser Thr Thr Glu Ala Arg Ser Gly Lys 1 5 10 15 Ser Leu Val
Val Leu Gly Ile Leu Asp Leu Ile Leu Lys Lys Thr Thr 20 25 30 Arg
Ile Ala Tyr Phe Arg Pro Ile Ile Gln Asp Pro Val Asn Gly Lys 35 40
45 His Asp Asn Asn Ile Ile Leu Val Leu Glu Asn Phe Arg Leu Gln Gln
50 55 60 Thr Tyr Thr Asp Ser Phe Gly Leu Tyr Phe His Glu Ala Val
Ser Leu 65 70 75 80 Ala Ser Asp Gly Ala Ile Asp Gln Val Leu Asp Arg
Ile Leu Ala Lys 85 90 95 Tyr Arg His Leu Ala Asp Gln Val Asp Phe
Ile Leu Cys Glu Gly Ser 100 105 110 Asp Tyr Leu Gly Glu Glu Ser Ala
Phe Glu Phe Asp Leu Asn Thr Thr 115 120 125 Ile Ala Lys Met Leu Asn
Cys Pro Ile Leu Leu Leu Gly Asn Ala Met 130 135 140 Gly Asn Thr Ile
Ala Asp Ser Leu Gln Pro Ile Asp Met Ala Leu Asn 145 150 155 160 Ser
Tyr Asp Gln Glu Ser Cys Gln Val Val Gly Val Ile Ile Asn Arg 165 170
175 Val Gln Pro Glu Leu Ala Thr Glu Ile Gln Ala Gln Leu Glu Gln Arg
180 185 190 Tyr Gly Asp Arg Pro Met Val Leu Gly Thr Ile Pro Gln Asp
Ile Met 195 200 205 Leu Lys Ser Leu Arg Leu Arg Glu Ile Val Ser Gly
Leu Asn Ala Gln 210 215 220 Val Leu Ser Gly Ala Asp Leu Leu Asp Asn
Leu Val Tyr His His Leu 225 230 235 240 Val Val Ala Met His Ile Ala
His Ala Leu His Trp Leu His Glu Lys 245 250 255 Asn Thr Leu Ile Ile
Thr Pro Gly Asp Arg Gly Asp Ile Ile Leu Gly 260 265 270 Val Met Gln
Ala His Arg Ser Leu Asn Tyr Pro Ser Ile Ala Gly Ile 275 280 285 Leu
Leu Thr Ala Asp Tyr His Pro Glu Pro Ala Ile Met Lys Leu Ile 290 295
300 Glu Gly Leu Pro Asp Ala Pro Pro Leu Leu Leu Thr Ser Thr His Thr
305 310 315 320 His Glu Thr Ser Ala Arg Leu Glu Thr Leu His Pro Ala
Leu Ser Pro 325 330 335 Thr Asp Asn Tyr Lys Ile Arg His Ser Ile Ala
Leu Phe Gln Gln Gln 340 345 350 Ile Asp Gly Glu Lys Leu Leu Asn Tyr
Leu Lys Thr Ile Arg Ser Lys 355 360 365 Gly Ile Thr Pro Lys Leu Phe
Leu Tyr Asn Leu Val Gln Ala Ala Thr 370 375 380 Ala Ala Gln Arg His
Ile Val Leu Pro Glu Gly Glu Glu Ile Arg Ile 385 390 395 400 Leu Lys
Ala Ala Ala Ser Leu Ile Asn His Gly Ile Val Arg Leu Thr 405 410 415
Leu Leu Gly Asn Ile Glu Ala Ile Glu Gln Thr Val Lys Ile Asn His 420
425 430 Ile Asp Leu Asp Leu Ser Lys Val Arg Leu Ile Asn Pro Lys Thr
Ser 435 440 445 Pro Asp Arg Glu Arg Tyr Ala Glu Thr Tyr Tyr Gln Leu
Arg Lys His 450 455 460 Lys Gly Val Thr Leu Ala Met Ala Arg Asp Ile
Leu Thr Asp Ile Ser 465 470 475 480 Tyr Phe Gly Thr Met Met Val His
Leu Gly Glu Ala Asp Gly Met Val 485 490 495 Ser Gly Ser Val Asn Thr
Thr Gln His Thr Val Arg Pro Ala Leu Gln 500 505 510 Ile Ile Lys Thr
Gln Pro Gly Phe Ser Leu Val Ser Ser Val Phe Phe 515 520 525 Met Cys
Leu Glu Asp Arg Val Leu Val Tyr Gly Asp Cys Ala Val Asn 530 535 540
Pro Asp Pro Asn Ala Glu Gln Leu Ala Glu Ile Ala Leu Thr Ser Ala 545
550 555 560 Ala Thr Ala Lys Asn Phe Gly Ile Glu Pro Arg Val Ala Leu
Leu Ser 565 570 575 Tyr Ser Ser Gly Ser Ser Gly Gln Gly Ala Asp Val
Glu Lys Val Arg 580 585 590 Gln Ala Thr Ala Ile Ala Lys Glu Arg Glu
Pro Asp Leu Ala Leu Glu 595 600 605 Gly Pro Ile Gln Tyr Asp Ala Ala
Val Asp Ser Thr Val Ala Ala Gln 610 615 620 Lys Met Pro Gly Ser Ala
Val Ala Gly Lys Ala Thr Val Phe Ile Phe 625 630 635 640 Pro Asp Leu
Asn Thr Gly Asn Asn Thr Tyr Lys Ala Val Gln Arg Glu 645 650 655 Thr
Lys Ala Ile Ala Ile Gly Pro Ile Leu Gln Gly Leu Asn Lys Pro 660 665
670 Val Asn Asp Leu Ser Arg Gly Cys Leu Val Glu Asp Ile Ile Asn Thr
675 680 685 Val Val Ile Thr Ala Leu Gln Val Lys 690 695
562857DNAartificialinsert of construct pVZ322 PpetJ pta
56gtcgactcta gaggatcccc gggtacccct catcgggggc tgtgttggcc gagacggcac
60tgaggatttt actctccatg gcattccaag gaatatctac ccaactcacc tgctccggcg
120gattgttccg ctcaaaagta ctaatcaagt cgtcaaaata cttattaaat
tttggctgca 180attgcatagt ccaaaagctg actttcccct ccatgctctg
gggggaattg ctctggcaac 240tgattaatcc actgagcaac agcccaagac
acgcaaacaa aaaccaacgt cttggcgatc 300gccatcggca ccatgaaacc
atcgtaaaag ctggggaaag aataaaaaac agtggttcag 360gaattgcatt
gccatggcca cttcacaaac ctagccaatt ttagcttgac cgcaactttg
420acagattgtc ttttgacttt gcctggaccg cctcccataa taccttcgcg
tcttgaagac 480tttatccttg aaaggagaac atatgacgag ttccctttat
ttaagcacca ccgaagcccg 540cagcggtaaa tctctagtag tattgggcat
tttagactta attctcaaaa aaaccacccg 600tattgcctat tttcgtccca
ttattcaaga cccagttaat ggcaaacatg ataacaacat 660tattctggtg
ctggaaaatt ttcgtctcca acaaacctat accgattcct ttggtttgta
720tttccatgaa gcggtgagtt tagcctccga tggagctatt gatcaggtat
tagaccgaat 780tttggctaaa tatcgccatt tggcagatca agtagatttt
attctctgtg aaggctcaga 840ctatttgggg gaggaatcgg cttttgaatt
tgatctcaac accacgatcg ccaagatgtt 900gaactgcccc attttgctgt
tgggcaatgc catgggcaac accattgccg atagtttgca 960acccatcgat
atggccctga atagctatga ccaagagtct tgtcaggtgg tgggggtaat
1020cattaaccga gtgcagcccg aattagccac agaaattcaa gcccaactgg
aacagcgtta 1080tggcgatcgc ccgatggtgt tgggcactat tccccaggac
attatgctca aaagtctgcg 1140cctgagggaa attgtcagcg ggctcaatgc
ccaagtactc agcggtgcgg atttgctcga 1200taacttggtc tatcaccatt
tagtggtggc gatgcacatt gcccacgccc tccattggtt 1260gcacgaaaaa
aataccctaa ttattacccc tggcgatcgg ggcgacatca ttctgggggt
1320gatgcaggcc caccgctccc tcaactatcc cagcattgcc ggtattttgc
tcactgcaga 1380ttaccatccc gaaccggcca ttatgaaact aattgaaggg
ctacccgacg cccctcccct 1440gttgctgact agcacccaca cccatgaaac
ttccgcccgt ttggaaactc tccaccctgc 1500cctgagccct acggataatt
ataaaattcg ccacagtatt gcgctgtttc aacaacaaat 1560tgatggggag
aaattactca attaccttaa aaccatccgc agtaaaggta ttacccccaa
1620actgtttctc tacaatttag ttcaagccgc caccgccgcc caacgacata
ttgtcctacc 1680ggaaggggaa gaaattcgta ttctcaaggc ggccgctagc
ttaattaacc acggcattgt 1740ccgtttgact ttactcggta acattgaggc
gatcgagcaa acggtaaaaa ttaatcacat 1800tgacttagat ttgagcaaag
ttcgcctcat taatcctaaa actagcccag accgagagcg 1860ctacgccgaa
acctattacc agctacgtaa acataagggg gtaaccctgg ccatggctcg
1920ggatatcctc accgatattt cctattttgg aacgatgatg gtgcatttgg
gagaggccga 1980tggcatggtt tctggctccg tcaataccac ccaacatacc
gtgcgtcctg ctttacaaat 2040tattaaaacc cagccaggtt tttccttggt
ttcttcagtc ttttttatgt gtttagaaga 2100ccgagttttg gtctatggag
attgtgctgt taatcccgat cccaatgcag aacagttagc 2160agaaattgcc
cttacttctg cggctacggc caagaatttt ggcattgagc ccagggtagc
2220tctattgtcc tattcttccg gttcttctgg gcaaggggcc gatgtggaaa
aagtgcggca 2280agccacggcg atcgccaagg aaagagagcc agatttagca
ttggaagggc cgatccagta 2340tgatgcggcg gtggattcca cagtggcggc
ccaaaaaatg cctgggtcag cggtggcggg 2400taaagcaacg gtgtttattt
ttcccgattt aaataccggt aacaatactt acaaggcagt 2460gcaaagagaa
acaaaggcga tcgccattgg ccccatttta caaggattaa ataaaccagt
2520taatgatcta agtcggggtt gtttagtgga ggatattatt aatacggtgg
taattacagc 2580tttgcaagtt aaataatttt actcttaatt agttaaaatg
atcccttgaa ttaccttgat 2640tttgccctcc aaactaccaa tagctgggcc
gaaaattggc atcatttaaa atcaccaacg 2700tgtccccgga cggagctagc
acaaacagac ccttaccata ggcatagctg accacttctt 2760ggcttaacac
catggctgcc actgcaccta aagctttaac atcccggtag cggggcataa
2820actgtttgaa tttacccaac cgttccagat gctcgag
2857575385DNAartificialinsert of construct pVZ322 PpetJ phk pta
57cccgggtacc cctcatcggg ggctgtgttg gccgagacgg cactgaggat tttactctcc
60atggcattcc aaggaatatc tacccaactc acctgctccg gcggattgtt ccgctcaaaa
120gtactaatca agtcgtcaaa atacttatta aattttggct gcaattgcat
agtccaaaag 180ctgactttcc cctccatgct ctggggggaa ttgctctggc
aactgattaa tccactgagc 240aacagcccaa gacacgcaaa caaaaaccaa
cgtcttggcg atcgccatcg gcaccatgaa 300accatcgtaa aagctgggga
aagaataaaa aacagtggtt caggaattgc attgccatgg 360ccacttcaca
aacctagcca attttagctt gaccgcaact ttgacagatt gtcttttgac
420tttgcctgga ccgcctccca taataccttc gcgtcttgaa gactttatcc
ttgaaaggag 480aacatatggt tacatccccc ttttccctta gtccctttgg
tcaagctaga tccaccgtca 540ctggcaatcc ccttgacccg acagaactta
accaaatgca cggtttttgg cgggcagcca 600actacttggc agtgggcatg
atttatctgc gggataatcc ccttttgcgg gaaccgcttc 660aaccggaaca
gatcaagcat cgcctgttgg gtcactgggg ttctagtccc ggcattagtt
720ttctctacac ccatctcaac cgcattatca ggaaatttga ccaggatatg
ctgtacatgg 780tggggcctgg ccacggcgca ccaggctttt tggggccctg
ctacctagaa gggagctatt 840ctcgcttttt tgccgagtgt agtgaagatg
aggacggcat gaagcgcttt ttcaaacaat 900tttcctttcc cggtggcatt
ggcagtcatt gcactcccga aacccctggt tccatccacg 960aggggggaga
attgggctac tgcctatccc atgcctatgg cgctgccttt gataatccca
1020atttaattgt ggtcggttta gcgggggatg gggagtcgga aacaggcccc
ttggctacct 1080cctggcattc caataagttt attaacccga ttcgggatgg
ggcagtttta ccggttctgc 1140atctcaatgg gtacaagatt aacaatccaa
gtgttttatc tcgcattagc catgaagaat 1200taaaggcttt atttgaaggt
tacggttata ccccctactt tgttgaaggc tctgacccgg 1260aatctatgca
ccaagccatg gcagccacgt tggatcattg tgtgagcgaa attcatcaaa
1320tccaacaaga agctcgtagt acgggcattg ccgtgcgccc ccgttggccc
atggttgtga 1380tgcggactcc caagggatgg acggggcctg actatgttga
tggccataag gtagaaggtt 1440tttggcgatc gcaccaagtt cccatggggg
gcatgcacga gaatccagcc catttgcaac 1500agttggaagc ttggatgcgg
agttataagc cggaagaatt gttcgacgag caaggtactt 1560taaaaccggg
atttaaggcg atcgccccgg agggagataa gcgtttaggc tctactccct
1620acgccaatgg tggtttgtta cggcggggtt tgaaaatgcc ggactttcgt
caatatggta 1680ttgatgtgga ccaaccaggc accatcgaag cccctaatac
tgcacccctg ggagtatttc 1740tgcgggatgt gatggccaac aacatgacca
atttccgcct gtttggcccc gatgaaaata 1800gttccaataa actccatgcc
gtctacgagg ttagcaaaaa attctggatt gctgaatatc 1860tagaagaaga
ccaggatggg ggggaattaa gtcccgatgg tcgggtgatg gaaatgttaa
1920gcgagcacac cttagaaggt tggttagagg cctatctttt aaccgggcgt
cacggctttt 1980tcgccaccta tgaatccttt gcccatgtga tcacttccat
ggttaaccaa cacgctaaat 2040ggttggatat ttgtcgacac ctcaactggc
gggcagatat ttcctcgtta aatatcttga 2100tgacgtccac cgtgtggcga
caggatcaca acgggtttac ccaccaagat cccggttttc 2160tcgatgtcat
tctcaataaa agccccgatg tggtgcgaat ttatttaccc cccgatgtta
2220attctctgct ttccgtagcg gaccattgtt tacagagcaa aaactacatc
aacatcatcg 2280tttgcgataa gcaagcccac ctgcaatacc aggacatgac
ttccgctatc cgtaactgca 2340ctaaaggggt ggacatttgg gaatgggcca
gtaatgatgc cggtacggaa ccggatgtgg 2400tgatggcagc ggcgggggat
attcccacca aagaggcctt ggcggccaca gccatgctaa 2460ggcaattttt
tcctaatctg agaattcgct ttgtcagcgt gattgatttg ctcaaactgc
2520aaccggaatc ggagcatccc catggcctga gcgatcggga ttttgactcc
ctctttacca 2580ccgataaacc gattattttt aacttccacg cctatccctg
gttaattcat cggttgacct 2640atcgacggac taaccatggc aatctccatg
tgcggggcta caaggaaaag ggcaacatca 2700acacccccat ggatttagcg
attcaaaacc agattgaccg tttcagcctc gccattgatg 2760tgatcgatcg
cctgccccaa ttgcgggtgg ccggagccca catcaaggaa atgctcaagg
2820atatgcagat tgactgcacc aactacgcct acgaacacgg cattgatatg
ccagaaatcg 2880ttaattggcg ctggcccctc tagaccttaa ctaaaatccc
tgacatcgtt ctagtttctg 2940ttccaatagg ttagctaggc catgggggac
aacagatctg gatacgttga ggttatttaa 3000attatgacga gttcccttta
tttaagcacc accgaagccc gcagcggtaa atctctagta 3060gtattgggca
ttttagactt aattctcaaa aaaaccaccc gtattgccta ttttcgtccc
3120attattcaag acccagttaa tggcaaacat gataacaaca ttattctggt
gctggaaaat 3180tttcgtctcc aacaaaccta taccgattcc tttggtttgt
atttccatga agcggtgagt 3240ttagcctccg atggagctat tgatcaggta
ttagaccgaa ttttggctaa atatcgccat 3300ttggcagatc aagtagattt
tattctctgt gaaggctcag actatttggg ggaggaatcg 3360gcttttgaat
ttgatctcaa caccacgatc gccaagatgt tgaactgccc cattttgctg
3420ttgggcaatg ccatgggcaa caccattgcc gatagtttgc aacccatcga
tatggccctg 3480aatagctatg accaagagtc ttgtcaggtg gtgggggtaa
tcattaaccg agtgcagccc 3540gaattagcca cagaaattca agcccaactg
gaacagcgtt atggcgatcg cccgatggtg 3600ttgggcacta ttccccagga
cattatgctc aaaagtctgc gcctgaggga aattgtcagc 3660gggctcaatg
cccaagtact cagcggtgcg gatttgctcg ataacttggt ctatcaccat
3720ttagtggtgg cgatgcacat tgcccacgcc ctccattggt tgcacgaaaa
aaatacccta 3780attattaccc ctggcgatcg gggcgacatc attctggggg
tgatgcaggc ccaccgctcc 3840ctcaactatc ccagcattgc cggtattttg
ctcactgcag attaccatcc cgaaccggcc 3900attatgaaac taattgaagg
gctacccgac gcccctcccc tgttgctgac tagcacccac 3960acccatgaaa
cttccgcccg tttggaaact ctccaccctg ccctgagccc tacggataat
4020tataaaattc gccacagtat tgcgctgttt caacaacaaa ttgatgggga
gaaattactc 4080aattacctta aaaccatccg cagtaaaggt attaccccca
aactgtttct ctacaattta 4140gttcaagccg ccaccgccgc ccaacgacat
attgtcctac cggaagggga agaaattcgt 4200attctcaagg cggccgctag
cttaattaac cacggcattg tccgtttgac tttactcggt 4260aacattgagg
cgatcgagca aacggtaaaa attaatcaca ttgacttaga tttgagcaaa
4320gttcgcctca ttaatcctaa aactagccca gaccgagagc gctacgccga
aacctattac 4380cagctacgta aacataaggg ggtaaccctg gccatggctc
gggatatcct caccgatatt 4440tcctattttg gaacgatgat ggtgcatttg
ggagaggccg atggcatggt ttctggctcc 4500gtcaatacca cccaacatac
cgtgcgtcct gctttacaaa ttattaaaac ccagccaggt 4560ttttccttgg
tttcttcagt cttttttatg tgtttagaag accgagtttt ggtctatgga
4620gattgtgctg ttaatcccga tcccaatgca gaacagttag cagaaattgc
ccttacttct 4680gcggctacgg ccaagaattt tggcattgag cccagggtag
ctctattgtc ctattcttcc 4740ggttcttctg ggcaaggggc cgatgtggaa
aaagtgcggc aagccacggc gatcgccaag 4800gaaagagagc cagatttagc
attggaaggg ccgatccagt atgatgcggc ggtggattcc 4860acagtggcgg
cccaaaaaat gcctgggtca gcggtggcgg gtaaagcaac ggtgtttatt
4920tttcccgatt taaataccgg taacaatact tacaaggcag tgcaaagaga
aacaaaggcg 4980atcgccattg gccccatttt acaaggatta aataaaccag
ttaatgatct aagtcggggt 5040tgtttagtgg aggatattat taatacggtg
gtaattacag ctttgcaagt taaataattt 5100tactcttaat tagttaaaat
gatcccttga attaccttga ttttgccctc caaactacca 5160atagctgggc
cgaaaattgg catcatttaa aatcaccaac gtgtccccgg acggagctag
5220cacaaacaga cccttaccat aggcatagct gaccacttct tggcttaaca
ccatggctgc 5280cactgcacct aaagctttaa catcccggta gcggggcata
aactgtttga atttacccaa 5340ccgttccaga tgctcgagca accgatcttt
ctagaagatc tcgag 538558456PRTSynechocystis sp. strain PCC6803 58Met
Asn Thr Ala Lys Thr Val Val Ala Glu Gln Arg Asp Phe Phe Arg 1 5 10
15 Gln Gly Lys Thr Lys Ser Val Gln Asp Arg Leu Thr Ala Leu Ala Lys
20 25 30 Leu Lys Thr Gln Ile Gln Ala Gln Glu Glu Glu Ile Ile Lys
Ala Leu 35 40 45 Lys Gln Asp Phe Gly Lys Pro Thr Phe Glu Ser Tyr
Val Asn Glu Ile 50 55 60 Leu Gly Val Ile Arg Glu Ile Asn Tyr Tyr
Gln Lys His Leu Gln Gln 65 70 75 80 Trp Ser Lys Pro Gln Arg Val Gly
Thr Asn Leu Met Val Phe Pro Ala 85 90 95 Ser Ala Gln Leu Arg Pro
Glu Pro Leu Gly Val Val Leu Ile Ile Ser 100 105 110 Pro Trp Asn Tyr
Pro Phe Tyr Leu Cys Leu Met Pro Leu Ile Gly Ala 115 120 125 Ile Ala
Ala Gly Asn Cys Val Val Val Lys Pro Ser Glu Tyr Thr Pro 130 135 140
Ala Ile Ser Gly Val Ile Thr Arg Leu Ile Gln Asn Val Phe Ser Pro 145
150 155 160 Ala Trp Ala Thr Val Val Glu Gly Asp Glu Thr Ile Ser Gln
Gln Leu 165 170 175 Leu Gln Glu Lys Phe Asp His Ile Phe Phe Thr Gly
Ser Pro Arg Val 180 185 190 Gly Arg Leu Ile Met Ala Ala Ala Ala Glu
Gln Leu Thr Pro Val Thr 195 200
205 Leu Glu Leu Gly Gly Lys Ser Pro Cys Val Val Asp Arg Glu Ile Asn
210 215 220 Leu Gln Glu Thr Ala Lys Arg Ile Met Trp Gly Lys Leu Val
Asn Ala 225 230 235 240 Gly Gln Thr Cys Val Ala Pro Asp Tyr Leu Leu
Val Glu Gln Ser Cys 245 250 255 Leu Glu Gln Leu Leu Pro Ala Leu Gln
Gln Ala Ile Gln Met Leu Phe 260 265 270 Gly Glu Asn Pro Ala His Ser
Pro Asp Tyr Thr Arg Ile Val Asn Gln 275 280 285 Gln Gln Trp Ser Arg
Leu Val Ser Leu Leu Ser His Gly Lys Val Ile 290 295 300 Thr Arg Gly
Asp His Asn Glu Gly Asp Arg Tyr Ile Ala Pro Thr Leu 305 310 315 320
Ile Ile Asp Pro Asp Leu Asn Ser Pro Leu Met Gln Glu Glu Ile Phe 325
330 335 Gly Pro Ile Leu Pro Ile Leu Thr Tyr Gln Ser Leu Ser Glu Ala
Ile 340 345 350 Asp Phe Ile Asn Ile Lys Pro Lys Pro Leu Ala Leu Tyr
Phe Phe Ser 355 360 365 Asn Asn Arg Gln Lys Gln Glu Glu Ile Leu Gln
Ser Thr Ser Ser Gly 370 375 380 Ser Val Cys Leu Asn Asp Ile Leu Leu
His Leu Thr Val Thr Asp Leu 385 390 395 400 Pro Phe Gly Gly Val Gly
Glu Ser Gly Met Gly Arg Tyr His Gly Lys 405 410 415 Ala Thr Phe Asp
Thr Leu Ser Asn Tyr Lys Ser Ile Leu Arg Arg Pro 420 425 430 Phe Trp
Gly Glu Thr Asn Leu Arg Tyr Ser Pro Tyr Gly Lys Lys Met 435 440 445
Asn Leu Ile Lys Lys Leu Phe Ser 450 455 592089DNAartificialinsert
of construct pVZ 322 PpetJ aldh 59ctgcaggtcg actctagagg atccccgggt
acccctcatc gggggctgtg ttggccgaga 60cggcactgag gattttactc tccatggcat
tccaaggaat atctacccaa ctcacctgct 120ccggcggatt gttccgctca
aaagtactaa tcaagtcgtc aaaatactta ttaaattttg 180gctgcaattg
catagtccaa aagctgactt tcccctccat gctctggggg gaattgctct
240ggcaactgat taatccactg agcaacagcc caagacacgc aaacaaaaac
caacgtcttg 300gcgatcgcca tcggcaccat gaaaccatcg taaaagctgg
ggaaagaata aaaaacagtg 360gttcaggaat tgcattgcca tggccacttc
acaaacctag ccaattttag cttgaccgca 420actttgacag attgtctttt
gactttgcct ggaccgcctc ccataatacc ttcgcgtctt 480gaagacttta
tccttgaaag gagaacatat gaatactgct aaaactgttg ttgctgagca
540aagggacttt tttcgtcagg gcaaaactaa atcagtccaa gatagattaa
cagctctagc 600aaaattaaaa acgcaaattc aagcccagga agaggaaatt
attaaggccc ttaagcaaga 660ttttggtaag cccacctttg aaagctatgt
aaacgaaatt ttgggggtaa ttagggaaat 720taattattat caaaaacatc
ttcagcaatg gtctaagccc caacgggtag gtacgaatct 780gatggttttt
cctgccagtg cccagttaag accagaaccc cttggtgtag tgctaattat
840tagcccctgg aattatcctt tttatctttg tttaatgccc ttgatcgggg
cgatcgccgc 900tggaaattgt gtggtggtaa agccgtcgga atatactcca
gctattagtg gggtaattac 960cagattaatc caaaatgtat tttccccggc
ttgggcaaca gtggtggagg gagatgaaac 1020cattagccaa caattgttac
aggaaaaatt tgaccatatt ttctttaccg gcagccctag 1080ggtgggtcgg
ttaattatgg cagctgcggc agagcaatta accccagtta cgttggaatt
1140ggggggtaaa tctccctgtg tggtggatag ggaaatcaac ctccaggaaa
cagccaaacg 1200cattatgtgg ggcaagctag tcaatgctgg ccaaacctgt
gtggcaccgg attatttatt 1260ggtggagcaa tcctgcttag aacaactttt
accagcttta caacaggcaa ttcagatgct 1320tttcggggaa aatccagccc
atagccctga ctacactcgc attgttaacc aacaacaatg 1380gtcacggtta
gttagtttat taagccatgg caaagtaatt acaaggggag atcataacga
1440aggcgatcgc tacattgccc caactttaat catcgatcca gatttaaatt
ctcccttaat 1500gcaagaggaa atatttggcc caattttgcc aattttaact
tatcagagtt tgtcagaagc 1560aatagatttt attaacatca aacctaaacc
attggcactt tattttttta gcaataatcg 1620gcaaaaacag gaggaaattt
tgcaatctac cagttccggt agtgtttgtt tgaacgatat 1680tttgcttcat
ttaactgtga cagacttacc ctttggtggg gtgggagaaa gtggtatggg
1740acgctaccat ggcaaggcta cttttgacac attgagcaat tataaaagca
ttttacgacg 1800acccttttgg ggggaaacta atttacgcta ttctccctat
ggcaaaaaaa tgaatttaat 1860caaaaagttg ttctcctagg attattcatg
gccgaccgtc cccagttgag tattattatt 1920cccgtgttta atgaagcaaa
aattttacaa aagtctccga ctgaaaatac cagacaatat 1980ttgggacaat
ttaccgagga tcaacggata gaaattttaa ttattgatgg gggcagtcag
2040gatagcacag tggagttatg ccagacctat gctgattctt tacctcgag
2089601034PRTSynechocystis sp. strain PCC6803 60Met Asn Leu Ala Val
Pro Ala Phe Gly Leu Ser Thr Asn Trp Ser Gly 1 5 10 15 Asn Gly Asn
Gly Ser Asn Ser Glu Glu Glu Ser Val Leu Tyr Gln Arg 20 25 30 Leu
Lys Met Val Glu Glu Leu Trp Glu Arg Val Leu Gln Ser Glu Cys 35 40
45 Gly Gln Glu Leu Val Asp Leu Leu Thr Glu Leu Arg Leu Gln Gly Thr
50 55 60 His Glu Ala Ile Thr Ser Glu Ile Ser Glu Glu Val Ile Met
Gly Ile 65 70 75 80 Thr Gln Arg Ile Glu His Leu Glu Leu Asn Asp Ala
Ile Arg Ala Ala 85 90 95 Arg Ala Phe Ala Leu Tyr Phe Gln Leu Ile
Asn Ile Val Glu Gln His 100 105 110 Tyr Glu Gln Asn Glu Gln Gln Arg
Asn Arg Trp Glu Ala Ser Gln Glu 115 120 125 Thr Asn Phe Tyr Glu Gln
Ala Gly Asn Glu Glu Glu Met Val Pro Pro 130 135 140 Ser Arg Leu Gly
Ala Ser Thr Glu Pro Leu Pro Val Gly Ile Asp Gln 145 150 155 160 Asn
Glu Leu Gln Ala Ser Val Gly Thr Phe His Trp Leu Met Arg Glu 165 170
175 Leu Lys Arg Leu Asn Val Pro Pro Gln His Ile Gln Asn Leu Leu Asp
180 185 190 His Leu Asp Ile Arg Leu Val Ile Thr Ala His Pro Thr Glu
Ile Val 195 200 205 Arg His Thr Ile Arg Arg Lys Gln Arg Arg Val Asp
Arg Ile Leu Arg 210 215 220 Lys Leu Asp Gln Leu Gln Gly Ser Val Thr
Gly Arg Asp Trp Leu Asn 225 230 235 240 Thr Trp Asp Ala Lys Thr Ala
Ile Ala Gln Leu Thr Glu Glu Ile Arg 245 250 255 Phe Trp Trp Arg Thr
Asp Glu Leu His Gln Phe Lys Pro Thr Val Leu 260 265 270 Asp Glu Val
Asp Tyr Ser Leu His Tyr Phe Asp Glu Val Leu Phe Asp 275 280 285 Ala
Val Pro Glu Leu Ser Lys Arg Leu Gly Gln Ala Ile Lys Glu Thr 290 295
300 Phe Pro His Leu Arg Ala Pro Arg Ala Asn Phe Cys Tyr Phe Gly Ser
305 310 315 320 Trp Val Gly Gly Asp Arg Asp Gly Asn Pro Ser Val Thr
Pro Glu Val 325 330 335 Thr Trp Gln Thr Ala Cys Tyr Gln Arg Gly Leu
Val Leu Gly Lys Tyr 340 345 350 Leu Phe Ser Leu Gly Glu Leu Val Ala
Ile Leu Ser Pro Ser Leu His 355 360 365 Trp Cys Lys Val Ser Gln Glu
Leu Leu Asp Ser Leu Glu Arg Asp Arg 370 375 380 Ile Gln Leu Pro Glu
Ile Tyr Glu Glu Leu Ser Leu Arg Tyr Arg Gln 385 390 395 400 Glu Pro
Tyr Arg Met Lys Leu Ala Tyr Val Thr Lys Arg Leu Glu Asn 405 410 415
Thr Leu Arg Arg Asn Asn Arg Leu Ala Asn Pro Glu Glu Arg Gln Thr 420
425 430 Met Ile Thr Met Pro Ala Glu Asn His Tyr Arg Thr Gly Glu Glu
Leu 435 440 445 Leu Glu Glu Leu Arg Leu Ile Gln Arg Asn Leu Thr Glu
Thr Gly Leu 450 455 460 Thr Cys Leu Glu Leu Glu Asn Leu Ile Thr Gln
Leu Glu Val Tyr Gly 465 470 475 480 Phe Asn Leu Ala Gln Leu Asp Phe
Arg Gln Glu Ser Ser Arg His Ala 485 490 495 Glu Ala Ile Ala Glu Ile
Ala Glu Tyr Met Gly Val Leu Thr Thr Pro 500 505 510 Tyr Glu Glu Met
Ala Glu Glu Asp Lys Leu Ala Trp Leu Gly Val Glu 515 520 525 Leu Gln
Thr Arg Arg Pro Leu Ile Pro Gln Glu Met Pro Phe Ser Glu 530 535 540
Arg Thr Arg Glu Thr Ile Glu Thr Leu Arg Thr Leu Arg His Leu Gln 545
550 555 560 Met Glu Phe Gly Val Asp Ile Cys Gln Thr Tyr Ile Ile Ser
Met Thr 565 570 575 Asn Asp Ala Ser Asp Val Leu Glu Val Leu Leu Leu
Ala Lys Glu Ala 580 585 590 Gly Leu Tyr Asp Pro Ala Thr Ala Ser Asn
Ser Leu Arg Ile Val Pro 595 600 605 Leu Phe Glu Thr Val Glu Asp Leu
Lys Asn Ala Pro Gly Ile Met Asp 610 615 620 Ser Leu Phe Ser Leu Pro
Phe Tyr Arg Ala Thr Leu Ala Gly Ser Tyr 625 630 635 640 His Ser Leu
Lys Glu Leu Gln Asn Gln Pro Pro Asp Tyr Tyr Gln Ile 645 650 655 Pro
Thr Thr Thr Ala Leu Leu Asn Pro Gly Asn Leu Gln Glu Ile Met 660 665
670 Val Gly Tyr Ser Asp Ser Asn Lys Asp Ser Gly Phe Leu Ser Ser Asn
675 680 685 Trp Glu Ile His Lys Ala Gln Lys Ser Leu Gln Ala Val Ala
Gln Ser 690 695 700 His Arg Val Ile Leu Arg Leu Phe His Gly Arg Gly
Gly Ser Val Gly 705 710 715 720 Arg Gly Gly Gly Pro Ala Tyr Lys Ala
Ile Leu Ala Gln Pro Ala Gly 725 730 735 Thr Val Asp Gly Arg Ile Lys
Ile Thr Glu Gln Gly Glu Val Leu Ala 740 745 750 Ser Lys Tyr Ser Leu
Pro Glu Leu Ala Leu Tyr Asn Leu Glu Thr Leu 755 760 765 Thr Thr Ala
Val Ile Gln Ala Ser Leu Leu Lys Ser Ser Phe Asp Phe 770 775 780 Ile
Glu Pro Trp Asn Arg Ile Met Glu Glu Leu Ala Cys Thr Ala Arg 785 790
795 800 Arg Ala Tyr Arg Ser Leu Ile Tyr Glu Glu Pro Asp Phe Leu Asp
Phe 805 810 815 Phe Leu Thr Val Thr Pro Ile Pro Glu Ile Ser Glu Leu
Gln Ile Ser 820 825 830 Ser Arg Pro Ala Arg Arg Lys Gly Gly Lys Ala
Asp Leu Ser Ser Leu 835 840 845 Arg Ala Ile Pro Trp Val Phe Ser Trp
Thr Gln Thr Arg Phe Leu Leu 850 855 860 Pro Ala Trp Tyr Gly Val Gly
Thr Ala Leu Lys Ser Phe Val Asp Gln 865 870 875 880 Asp Pro Val Lys
Asn Met Lys Leu Leu Arg Tyr Phe Tyr Phe Lys Trp 885 890 895 Pro Phe
Phe Asn Met Val Ile Ser Lys Val Glu Met Thr Leu Ser Lys 900 905 910
Val Asp Leu Thr Ile Ala Ser His Tyr Val Gln Glu Leu Ser Lys Pro 915
920 925 Glu Asp Arg Glu Arg Phe Asp Arg Leu Phe Gln Gln Ile Lys Gln
Glu 930 935 940 Tyr Gln Leu Thr Arg Asp Phe Ala Met Glu Ile Thr Ala
His Pro His 945 950 955 960 Leu Leu Asp Gly Asp Arg Ser Leu Gln Arg
Ser Val Leu Leu Arg Asn 965 970 975 Arg Thr Ile Val Pro Leu Gly Leu
Leu Gln Ile Ser Leu Leu Lys Arg 980 985 990 Leu Arg Gln Val Thr Gln
Glu Ala Glu Thr Ser Gly Val Arg Tyr Arg 995 1000 1005 Arg Tyr Ser
Lys Glu Glu Leu Leu Arg Gly Ala Leu Leu Thr Ile 1010 1015 1020 Asn
Gly Ile Ala Ala Gly Met Arg Asn Thr Gly 1025 1030
613650DNAartificialinsert of construct pVZ321-PpetJ-ppc
61tcgactctag aggatccccg ggtacccctc atcgggggct gtgttggccg agacggcact
60gaggatttta ctctccatgg cattccaagg aatatctacc caactcacct gctccggcgg
120attgttccgc tcaaaagtac taatcaagtc gtcaaaatac ttattaaatt
ttggctgcaa 180ttgcatagtc caaaagctga ctttcccctc catgctctgg
ggggaattgc tctggcaact 240gattaatcca ctgagcaaca gcccaagaca
cgcaaacaaa aaccaacgtc ttggcgatcg 300ccatcggcac catgaaacca
tcgtaaaagc tggggaaaga ataaaaaaca gtggttcagg 360aattgcattg
ccatggccac ttcacaaacc tagccaattt tagcttgacc gcaactttga
420cagattgtct tttgactttg cctggaccgc ctcccataat accttcgcgt
cttgaagact 480ttatccttga aaggagaaca tatgaacttg gcagttcctg
cattcggtct ttccactaac 540tggtctggta atggcaatgg ttccaactct
gaagaagagt cggtgcttta ccagcggtta 600aagatggtgg aggaattgtg
ggaaagggtg ctccaaagcg aatgtggcca ggaattggtg 660gatttgctga
cggaattaag gcttcagggt acccatgagg cgatcaccag cgaaatttcc
720gaagaagtca tcatgggtat tacccagcgc attgagcatt tagaactcaa
tgatgccatc 780cgggcggctc gggcctttgc cctatatttc cagttgatca
acatcgttga acagcactac 840gaacaaaacg agcaacaacg gaatcgttgg
gaagcttccc aggaaaccaa cttctatgag 900caggcgggca atgaggaaga
aatggtcccc ccatcccgat taggcgcgtc aacggaacca 960ttgccagtgg
gcattgacca gaatgaattg caagcttctg taggtacgtt ccattggtta
1020atgagggagc taaaacgcct caatgtgccc ccccaacata tccaaaattt
attggatcat 1080ctggacattc gcctggtgat caccgctcac cccacggaaa
ttgtccgtca caccatccgg 1140cgcaaacaaa gaagggtgga ccgcattctt
cgtaaactag atcaactcca gggttctgtg 1200accggtcggg actggctcaa
cacctgggat gcaaaaacgg cgatcgccca attaacggag 1260gaaattcgct
tttggtggcg taccgacgaa cttcatcagt tcaaacccac tgtgttggac
1320gaagtggact attccctcca ttattttgat gaagtactgt tcgacgctgt
accggaattg 1380tccaaacggt taggacaagc tattaaagaa acctttcccc
atctgcgggc cccccgggct 1440aatttttgtt attttggctc ctgggtcggt
ggcgatcggg acggcaaccc ttcggtaacc 1500ccagaagtga cctggcagac
ggcctgttac cagcggggtt tagtgctggg gaaatatttg 1560tttagtttgg
gggaactggt ggccattctt agcccttccc tccattggtg caaagtctcc
1620caggaattgt tggactcttt ggaacgggac cgcattcaat taccggaaat
ttacgaagaa 1680ctttctctcc gctatcgcca ggaaccctat cggatgaagc
tggcctacgt taccaaacgg 1740ctggaaaaca ccctgcggcg taataatcgt
ctagccaacc cagaagaacg gcaaacgatg 1800atcaccatgc cggccgaaaa
tcactatcgc actggggaag aattattaga ggaattaaga 1860ctcattcagc
gtaatctgac cgaaactggt ttaacctgcc tggagttgga aaatttgatt
1920acccagttgg aagtctatgg ctttaaccta gcccagttgg attttcgcca
ggaatcttcc 1980cgccacgccg aggcgatcgc cgaaattgct gagtatatgg
gggtactcac cactccctac 2040gaagaaatgg ccgaagaaga taaattagcc
tggttagggg tagaactgca aacccgccgt 2100cctttaattc cccaggaaat
gcccttttcg gagcggacta gggaaaccat tgaaaccctc 2160cgcaccctgc
gccatctaca aatggaattt ggggtggata tttgccaaac ctacatcatc
2220agcatgacca acgatgccag tgatgtgttg gaagtattgc tgttagccaa
ggaagccgga 2280ttgtatgacc cggccaccgc ctccaattcc ctccgcattg
tgcccctgtt tgaaacagta 2340gaagatctca aaaacgctcc ggggattatg
gattctcttt tcagcttgcc tttttaccgg 2400gctacattgg cgggcagtta
ccattcctta aaagagttgc aaaatcagcc accggattat 2460taccaaattc
ccaccaccac agccctacta aatcccggca atctccagga aattatggtg
2520ggctattccg acagcaataa agactccggc tttttgagca gtaactggga
aattcataag 2580gcccaaaaat cactgcaggc agtggcccaa agccatcggg
taattctccg gctgttccac 2640ggtcgaggag gatctgttgg ccgggggggc
ggcccggcct ataaagccat tttggcccag 2700cccgcaggca ccgtggacgg
tcggatcaaa attaccgaac aaggggaagt gttagcttct 2760aaatattccc
tgccagagtt ggccctctac aacctggaaa ctttaaccac ggcggtcatc
2820caagctagtt tacttaaaag tagttttgat ttcattgagc cctggaaccg
gattatggag 2880gagttggcct gcactgcccg tcgagcctac cggagtttga
tttacgaaga accggacttt 2940ttagatttct tcctgacggt tacccccatt
cctgaaatta gcgagttaca gattagttcc 3000cgccctgccc gacgtaaggg
gggtaaagcg gatctcagca gtttgcgggc cattccctgg 3060gtgttcagtt
ggacccaaac ccgtttcctg ctgccggctt ggtatggggt gggcacggcg
3120ttgaaatcct ttgtggacca agacccggtc aaaaatatga agttgttgcg
ttacttctat 3180ttcaaatggc ctttcttcaa catggtgatc tcgaaggtgg
aaatgaccct ttccaaggtg 3240gacctcacca tcgcttccca ctatgtgcaa
gagctatcta agccagaaga ccgggaacga 3300ttcgatcgcc tttttcagca
gatcaagcaa gagtatcaat taaccaggga ctttgccatg 3360gaaattacgg
cccatcccca cctcctggac ggcgatcgct ctttgcaacg gtcggtactc
3420ctacgaaatc gtactattgt tcccctgggg ctactgcaga tttccctgct
gaaacgttta 3480cgccaagtaa cccaggaagc ggagaccagc ggcgtgcgtt
accgtcgtta ttccaaagaa 3540gaactactgc ggggagctct gttaaccatt
aacggtattg cggccggaat gcgtaatact 3600ggttgatcca gtgatatggt
gcctaatatt gggtaaggac ctgcccttgc 36506226DNAartificialprimer
SynRbc-BglII-fw 62agatctcaac ggctcacaag cccaac
266325DNAartificialprimer SynRbc-PstI-rev 63ctgcagaatt ttctccattc
aaccc 256426DNAartificialprimer SynRbc-SacI-fw 64gagctctgga
ggactgacct agatgg 26652984DNASynechocystis PCC 6803 65agatctcaac
ggctcacaag cccaactaat caccatttgg acaaaacatc aggaattcta 60attagaaagt
ccaaaaattg taatttaaaa aacagtcaat ggagagcatt gccataagta
120aaggcatccc ctgcgtgata agattacctt cagaaaacag atagttgctg
ggttatcgca 180gatttttctc gcaaccaaat aactgtaaat aataactgtc
tctggggcga cggtaggctt 240tatattgcca aatttcgccc gtgggagaaa
gctaggctat tcaagagctc tggaggactg 300acctagatgg tacaagccaa
agcagggttt aaggcgggcg tacaagatta tcgcctgacc 360tactataccc
ccgactacac ccccaaggat
accgacctgc tcgcctgctt ccgtatgacc 420ccccaaccgg gtgtacctgc
tgaagaagcc gctgctgcgg tggccgctga gtcttccacc 480ggtacctgga
ccaccgtttg gactgacaac ctaactgact tggaccgcta caaaggtcgt
540tgctatgacc tggaagctgt tcccaacgaa gataaccaat attttgcttt
tattgcctat 600cctctagatt tatttgaaga aggttccgtc accaacgttt
taacctcttt ggtcggtaac 660gtatttggtt ttaaggctct gcgggccctc
cgtttagaag atattcgttt tcccgttgct 720ttaattaaaa ccttccaagg
ccctccccac ggtattaccg ttgagcggga caaattaaac 780aaatacggtc
gtcctctgct tggttgtacc atcaaaccca aacttggtct gtccgccaag
840aactacggtc gggctgttta cgaatgtctc cggggtggtt tggacttcac
caaagacgac 900gaaaacatca actcccagcc cttcatgcgt tggcgcgatc
gtttcctctt cgttcaagag 960gcgatcgaaa aagcccaggc tgagaccaac
gaaatgaaag gtcactacct gaacgtcacc 1020gctggcacct gcgaagaaat
gatgaaacgg gccgagtttg ccaaggaaat tggcaccccc 1080atcatcatgc
atgacttctt caccggcggt ttcactgcca acaccaccct cgctcgttgg
1140tgtcgggaca acggcatttt gctccatatt caccgggcaa tgcacgccgt
agttgaccgt 1200cagaaaaacc acgggatcca cttccgggtt ttggccaagt
gtctgcgtct gtccggcggt 1260gaccacctcc actccggtac cgtggttggt
aaattggaag gggaacgggg tatcaccatg 1320ggcttcgttg acctcatgcg
cgaagattac gttgaggaag atcgctcccg gggtattttc 1380ttcacccaag
actatgcctc catgcctggc accatgcccg tagcttccgg tggtatccac
1440gtatggcaca tgcccgcgtt ggtggaaatc ttcggtgatg attcctgctt
acagtttggt 1500ggtggtactt tgggtcaccc ctggggtaat gctcccggtg
caaccgctaa ccgtgttgct 1560ttggaagctt gtgttcaagc tcggaacgaa
ggtcgtaacc tggctcgcga aggtaatgac 1620gttatccggg aagcctgtcg
ttggtcccct gagttggccg ccgcctgcga actctggaaa 1680gagatcaagt
ttgagttcga ggccatggat accctctaaa ccggtgtttg gattgtcgga
1740gttgtactcg tccgttaagg atgaacagtt cttcggggtt gagtctgcta
actaattagc 1800cattaacagc ggcttaacta acagttagtc attggcaatt
gtcaaaaaat tgttaatcag 1860ccaaaaccca ctgcttactg atgttcaact
tcgacagcaa tttaccaatt accgggtaga 1920gtgttcatgc aaactaagca
catagctcag gcaacagtga aagtactgca aagttacctc 1980acctaccaag
ccgttctcag gatccagagt gaactcgggg aaaccaaccc tccccaggcc
2040atttggttaa accagtattt agccagtcac agtattcaaa atggagaaac
gtttttgacg 2100gaactcctgg atgaaaataa agaactggta ctcaggatcc
tggcggtaag ggaagacatt 2160gccgaatcag tgttagattt tttgcccggt
atgacccgga atagcttagc ggaatctaac 2220atcgcccacc gccgccattt
gcttgaacgt ctgacccgta ccgtagccga agtcgataat 2280ttcccttcgg
aaacctccaa cggagaatca aacaacaacg attctccccc gtcctaacgt
2340agtcatcagc aaggaaaact tttaaatcga tgaaaacttt acccaaagag
cgccgctacg 2400aaaccctttc ttacctgccc cctttaaccg atcaacagat
tgctaaacag gttgagtttc 2460tgttagacca gggctttatt cccggcgtgg
aatttgaaga agacccccaa cccgaaaccc 2520acttctggac catgtggaaa
ctgcccttct ttggtggtgc cactgccaac gaagttctag 2580ccgaagtacg
ggaatgtcgt tctgagaatc ccaactgcta cattcgggtg attggtttcg
2640acaatatcaa acagtgccag actgtaagct ttattgtcca caaacccaac
caaaaccaag 2700gccgttacta agttacagtt ttggcaatta ctaaaaaact
gacttcaatt caatgttagc 2760ccgctcccgc gggttttttg ttgctttttc
acagtgacta taggtaatca gcaacacaat 2820acggccctgt tctttggaca
gtttttgtat aatgttgacc gcatcctgac cggatttttt 2880atctaagtgg
ggaattgtca attgtcaatt aaagctaagt tctactaatg ttttagaagg
2940cattgtcgat tgaaaataag ggttgaatgg agaaaattct gcag
298466470PRTSynechocystis PCC 6803 66Met Val Gln Ala Lys Ala Gly
Phe Lys Ala Gly Val Gln Asp Tyr Arg 1 5 10 15 Leu Thr Tyr Tyr Thr
Pro Asp Tyr Thr Pro Lys Asp Thr Asp Leu Leu 20 25 30 Ala Cys Phe
Arg Met Thr Pro Gln Pro Gly Val Pro Ala Glu Glu Ala 35 40 45 Ala
Ala Ala Val Ala Ala Glu Ser Ser Thr Gly Thr Trp Thr Thr Val 50 55
60 Trp Thr Asp Asn Leu Thr Asp Leu Asp Arg Tyr Lys Gly Arg Cys Tyr
65 70 75 80 Asp Leu Glu Ala Val Pro Asn Glu Asp Asn Gln Tyr Phe Ala
Phe Ile 85 90 95 Ala Tyr Pro Leu Asp Leu Phe Glu Glu Gly Ser Val
Thr Asn Val Leu 100 105 110 Thr Ser Leu Val Gly Asn Val Phe Gly Phe
Lys Ala Leu Arg Ala Leu 115 120 125 Arg Leu Glu Asp Ile Arg Phe Pro
Val Ala Leu Ile Lys Thr Phe Gln 130 135 140 Gly Pro Pro His Gly Ile
Thr Val Glu Arg Asp Lys Leu Asn Lys Tyr 145 150 155 160 Gly Arg Pro
Leu Leu Gly Cys Thr Ile Lys Pro Lys Leu Gly Leu Ser 165 170 175 Ala
Lys Asn Tyr Gly Arg Ala Val Tyr Glu Cys Leu Arg Gly Gly Leu 180 185
190 Asp Phe Thr Lys Asp Asp Glu Asn Ile Asn Ser Gln Pro Phe Met Arg
195 200 205 Trp Arg Asp Arg Phe Leu Phe Val Gln Glu Ala Ile Glu Lys
Ala Gln 210 215 220 Ala Glu Thr Asn Glu Met Lys Gly His Tyr Leu Asn
Val Thr Ala Gly 225 230 235 240 Thr Cys Glu Glu Met Met Lys Arg Ala
Glu Phe Ala Lys Glu Ile Gly 245 250 255 Thr Pro Ile Ile Met His Asp
Phe Phe Thr Gly Gly Phe Thr Ala Asn 260 265 270 Thr Thr Leu Ala Arg
Trp Cys Arg Asp Asn Gly Ile Leu Leu His Ile 275 280 285 His Arg Ala
Met His Ala Val Val Asp Arg Gln Lys Asn His Gly Ile 290 295 300 His
Phe Arg Val Leu Ala Lys Cys Leu Arg Leu Ser Gly Gly Asp His 305 310
315 320 Leu His Ser Gly Thr Val Val Gly Lys Leu Glu Gly Glu Arg Gly
Ile 325 330 335 Thr Met Gly Phe Val Asp Leu Met Arg Glu Asp Tyr Val
Glu Glu Asp 340 345 350 Arg Ser Arg Gly Ile Phe Phe Thr Gln Asp Tyr
Ala Ser Met Pro Gly 355 360 365 Thr Met Pro Val Ala Ser Gly Gly Ile
His Val Trp His Met Pro Ala 370 375 380 Leu Val Glu Ile Phe Gly Asp
Asp Ser Cys Leu Gln Phe Gly Gly Gly 385 390 395 400 Thr Leu Gly His
Pro Trp Gly Asn Ala Pro Gly Ala Thr Ala Asn Arg 405 410 415 Val Ala
Leu Glu Ala Cys Val Gln Ala Arg Asn Glu Gly Arg Asn Leu 420 425 430
Ala Arg Glu Gly Asn Asp Val Ile Arg Glu Ala Cys Arg Trp Ser Pro 435
440 445 Glu Leu Ala Ala Ala Cys Glu Leu Trp Lys Glu Ile Lys Phe Glu
Phe 450 455 460 Glu Ala Met Asp Thr Leu 465 470
67136PRTSynechocystis PCC 6803 67Met Gln Thr Lys His Ile Ala Gln
Ala Thr Val Lys Val Leu Gln Ser 1 5 10 15 Tyr Leu Thr Tyr Gln Ala
Val Leu Arg Ile Gln Ser Glu Leu Gly Glu 20 25 30 Thr Asn Pro Pro
Gln Ala Ile Trp Leu Asn Gln Tyr Leu Ala Ser His 35 40 45 Ser Ile
Gln Asn Gly Glu Thr Phe Leu Thr Glu Leu Leu Asp Glu Asn 50 55 60
Lys Glu Leu Val Leu Arg Ile Leu Ala Val Arg Glu Asp Ile Ala Glu 65
70 75 80 Ser Val Leu Asp Phe Leu Pro Gly Met Thr Arg Asn Ser Leu
Ala Glu 85 90 95 Ser Asn Ile Ala His Arg Arg His Leu Leu Glu Arg
Leu Thr Arg Thr 100 105 110 Val Ala Glu Val Asp Asn Phe Pro Ser Glu
Thr Ser Asn Gly Glu Ser 115 120 125 Asn Asn Asn Asp Ser Pro Pro Ser
130 135 68113PRTSynechocystis PCC 6803 68Met Lys Thr Leu Pro Lys
Glu Arg Arg Tyr Glu Thr Leu Ser Tyr Leu 1 5 10 15 Pro Pro Leu Thr
Asp Gln Gln Ile Ala Lys Gln Val Glu Phe Leu Leu 20 25 30 Asp Gln
Gly Phe Ile Pro Gly Val Glu Phe Glu Glu Asp Pro Gln Pro 35 40 45
Glu Thr His Phe Trp Thr Met Trp Lys Leu Pro Phe Phe Gly Gly Ala 50
55 60 Thr Ala Asn Glu Val Leu Ala Glu Val Arg Glu Cys Arg Ser Glu
Asn 65 70 75 80 Pro Asn Cys Tyr Ile Arg Val Ile Gly Phe Asp Asn Ile
Lys Gln Cys 85 90 95 Gln Thr Val Ser Phe Ile Val His Lys Pro Asn
Gln Asn Gln Gly Arg 100 105 110 Tyr 696267DNAartificialvector pSK9
69cacctaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag
60ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac
120cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa
agaacgtgga 180ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat
ggcccactac gtgaaccatc 240accctaatca agttttttgg ggtcgaggtg
ccgtaaagca ctaaatcgga accctaaagg 300gagcccccga tttagagctt
gacggggaaa gccggcgaac gtggcgagaa aggaagggaa 360gaaagcgaaa
ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac
420caccacaccc gccgcgctta atgcgccgct acagggcgcg tcccattcgc
cattcaggct 480gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg
ctattacgcc agctggcgaa 540agggggatgt gctgcaaggc gattaagttg
ggtaacgcca gggttttccc agtcacgacg 600ttgtaaaacg acggccagtg
aattgtaata cgactcacta tagggcgaat tggaggccag 660tgctggagga
atatgatttt gtcatcctcg actgtgcccc tggttataat ctgttgaccc
720gcagtggcat tgcggccagc gacttttatc tgttgccggc tcgtcctgaa
cccctatcgg 780tggtggggat gcagttactg gaaagaagaa ttgagaaact
gaaggaaagc cataaggcct 840ccgatgatcc cctgaatatc aatctgatcg
gagtggtgtt tattctgtcc ggcggcggtt 900tgatgagtcg ctactataac
caggtaatgc ggcgggtaca aacggatttc accccgggac 960aactttttca
gcagtccatt cccatggatg tcaatgtggc taaggcagtg gatagcttta
1020tgccggtggt tacctccatg cccaatacgg cgggttcaaa agcttttatt
aaattaaccc 1080aggaattttt acagaaagta gaagcttttg gctaaagcaa
agcccccatt gattaacaac 1140gggaggggta ccgaggtgct gctgaagttg
cccgcaacag agagtggaac caaccggtga 1200taccacgata ctatgactga
gagtcaacgc catgagcggc ctcatttctt attctgagtt 1260acaacagtcc
gcaccgctgt ccggtagctc cttccggtgg gcgcggggca tgactatcgt
1320cgccgcactt atgactgtct tctttatcat gcaactcgta ggacaggtgc
cggcagcgcc 1380caacagtccc ccggccacgg ggcctgccac catacccacg
ccgaaacaag cgccctgcac 1440cattatgttc cggatctgca tcgcaggatg
ctgctggcta ccctgtggaa cacctacatc 1500tgtattaacg aagcgctaac
cgtttttatc aggctctggg aggcagaata aatgatcata 1560tcgtcaatta
ttacctccac ggggagagcc tgagcaaact ggcctcaggc atttgagaag
1620cacacggtca cactgcttcc ggtagtcaat aaaccggtaa accagcaata
gacataagcg 1680gctatttaac gaccctgccc tgaaccgacg accgggtcga
atttgctttc gaatttctgc 1740cattcatccg cttattatca cttattcagg
cgtagcacca ggcgtttaag ggcaccaata 1800actgccttaa aaaaattacg
ccccgccctg ccactcatcg cagtactgtt gtaattcatt 1860aagcattctg
ccgacatgga agccatcaca gacggcatga tgaacctgaa tcgccagcgg
1920catcagcacc ttgtcgcctt gcgtataata tttgcccatg gtgaaaacgg
gggcgaagaa 1980gttgtccata ttggccacgt ttaaatcaaa actggtgaaa
ctcacccagg gattggctga 2040gacgaaaaac atattctcaa taaacccttt
agggaaatag gccaggtttt caccgtaaca 2100cgccacatct tgcgaatata
tgtgtagaaa ctgccggaaa tcgtcgtggt attcactcca 2160gagcgatgaa
aacgtttcag tttgctcatg gaaaacggtg taacaagggt gaacactatc
2220ccatatcacc agctcaccgt ctttcattgc catacggaat tccggatgag
cattcatcag 2280gcgggcaaga atgtgaataa aggccggata aaacttgtgc
ttatttttct ttacggtctt 2340taaaaaggcc gtaatatcca gctgaacggt
ctggttatag gtacattgag caactgactg 2400aaatgcctca aaatgttctt
tacgatgcca ttgggatata tcaacggtgg tatatccagt 2460gatttttttc
tccattttag cttccttagc tcctgaaaat ctcgataact caaaaaatac
2520gcccggtagt gatcttattt cattatggtg aaagttggaa cctcttacct
cggtacccct 2580catcgggggc tgtgttggcc gagacggcac tgaggatttt
actctccatg gcattccaag 2640gaatatctac ccaactcacc tgctccggcg
gattgttccg ctcaaaagta ctaatcaagt 2700cgtcaaaata cttattaaat
tttggctgca attgcatagt ccaaaagctg actttcccct 2760ccatgctctg
gggggaattg ctctggcaac tgattaatcc actgagcaac agcccaagac
2820acgcaaacaa aaaccaacgt cttggcgatc gccatcggca ccatgaaacc
atcgtaaaag 2880ctggggaaag aataaaaaac agtggttcag gaattgcatt
gccatggcca cttcacaaac 2940ctagccaatt ttagcttgac cgcaactttg
acagattgtc ttttgacttt gcctggaccg 3000cctcccataa taccttcgcg
tcttgaagac tttatccttg aaaggagaac atatgtttct 3060cggcaaaaat
taattatcga ttggctggaa cctggtcaaa ccagggcttt tcatccattg
3120gaaaagcgat tttgatcatc tagggtcagg agcaaagatc tgatcaaata
ttgatcattt 3180attaggaaag ctgaactttc accactttat ttttggcttc
ctctactttg ggcaaagtca 3240aagttaggat accggcatcg taattagctt
taacttctgt gttttggatt gctccaggta 3300caggaataac ccggcggaaa
ctgccatagc ggaactctgt gcgccgcacc ccatcttttt 3360cggtgctatg
ggtatcctgg cgatcgccgc tgacggtcac cgcatccctg gcggcttgga
3420tgtccaaatt atcggggtcc atgccaggta attctagttt gagcacatag
gcttcttcag 3480tttcagttag ttctgcttta ggattaaacc cttggcgatc
gccgtggcgg tccgtaggga 3540caaaaacttc ttcaaacagt tggttcatct
gctgctggaa attatccatt tcccgcaggg 3600gattgtaaag aatgagagac
ataatgttaa ctcctgatgt gtggaaggaa ttgattaccc 3660ttgaatggtt
ctatcttaaa atttcccctt ccaggttaga ttcggttttc aggaaagaag
3720gtggggggat tgccgaaatt acatttctag ccgcaatttt tagtaaaaaa
aagatgagtt 3780tttacctcac cttaagtaaa tatttgagtg gcaaaacaaa
atggtaaaaa tagctaagct 3840tccaccgccc tatggatttt tggaaggaag
tcttaggttg tgaaaaacta taaaaaccaa 3900ccataggaat ggagaccttt
acccaacaag ttgaccccta ggtaacaaat ccaaaccacc 3960gtaaaaccgc
tggcggccaa aatagcgggc ttgcggcctt gccaaccttt ggtaatgcgg
4020gcatggagat aggcggcaaa tactagccag gtgattaggg cccggtaccc
agcttttgtt 4080ccctttagtg agggttaatt tcgagcttgg cgtaatcatg
gtcatagctg tttcctgtgt 4140gaaattgtta tccgctcaca attccacaca
acatacgagc cggaagcata aagtgtaaag 4200cctggggtgc ctaatgagtg
agctaactca cattaattgc gttgcgctca ctgcccgctt 4260tccagtcggg
aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
4320gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg
cgctcggtcg 4380ttcggctgcg gcgagcggta tcagctcact caaaggcggt
aatacggtta tccacagaat 4440caggggataa cgcaggaaag aacatgtgag
caaaaggcca gcaaaaggcc aggaaccgta 4500aaaaggccgc gttgctggcg
tttttccata ggctccgccc ccctgacgag catcacaaaa 4560atcgacgctc
aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
4620cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
ggatacctgt 4680ccgcctttct cccttcggga agcgtggcgc tttctcatag
ctcacgctgt aggtatctca 4740gttcggtgta ggtcgttcgc tccaagctgg
gctgtgtgca cgaacccccc gttcagcccg 4800accgctgcgc cttatccggt
aactatcgtc ttgagtccaa cccggtaaga cacgacttat 4860cgccactggc
agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
4920cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta
tttggtatct 4980gcgctctgct gaagccagtt accttcggaa aaagagttgg
tagctcttga tccggcaaac 5040aaaccaccgc tggtagcggt ggtttttttg
tttgcaagca gcagattacg cgcagaaaaa 5100aaggatctca agaagatcct
ttgatctttt ctacggggtc tgacgctcag tggaacgaaa 5160actcacgtta
agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
5220taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
tggtctgaca 5280gttaccaatg cttaatcagt gaggcaccta tctcagcgat
ctgtctattt cgttcatcca 5340tagttgcctg actccccgtc gtgtagataa
ctacgatacg ggagggctta ccatctggcc 5400ccagtgctgc aatgataccg
cgagacccac gctcaccggc tccagattta tcagcaataa 5460accagccagc
cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
5520agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
agtttgcgca 5580acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
gtcgtttggt atggcttcat 5640tcagctccgg ttcccaacga tcaaggcgag
ttacatgatc ccccatgttg tgcaaaaaag 5700cggttagctc cttcggtcct
ccgatcgttg tcagaagtaa gttggccgca gtgttatcac 5760tcatggttat
ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
5820ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
cgaccgagtt 5880gctcttgccc ggcgtcaata cgggataata ccgcgccaca
tagcagaact ttaaaagtgc 5940tcatcattgg aaaacgttct tcggggcgaa
aactctcaag gatcttaccg ctgttgagat 6000ccagttcgat gtaacccact
cgtgcaccca actgatcttc agcatctttt actttcacca 6060gcgtttctgg
gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
6120cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc
atttatcagg 6180gttattgtct catgagcgga tacatatttg aatgtattta
gaaaaataaa caaatagggg 6240ttccgcgcac atttccccga aaagtgc
6267709231DNAartificialpVZ321 vector 70gatctgtaat ccgggcagcg
caacggaaca ttcatcagtg taaaaatgga atcaataaag 60ccctgcgcag cgcgcagggt
cagcctgaat acgcgtttaa tgaccagcac agtcgtgatg 120gcaaggtcag
aatagcgctg aggtctgcct cgtgaagaag gtgttgctga ctcataccag
180gcctgaatcg ccccatcatc cagccagaaa gtgagggagc cacggttgat
gagagctttg 240ttgtaggtgg accagttggt gattttgaac ttttgctttg
ccacggaacg gtctgcgttg 300tcgggaagat gcgtgatctg atccttcaac
tcagcaaaag ttcgatttat tcaacaaagc 360cgccgtcccg tcaagtcagc
gtaatgctct gccagtgtta caaccaatta accaattctg 420attagaaaaa
ctcatcgagc atcaaatgaa actgcaattt attcatatca ggattatcaa
480taccatattt ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg
aggcagttcc 540ataggatggc aagatcctgg tatcggtctg cgattccgac
tcgtccaaca tcaatacaac 600ctattaattt cccctcgtca aaaataaggt
tatcaagtga gaaatcacca tgagtgacga 660ctgaatccgg tgagaatggc
aaaagcttat gcatttcttt ccagacttgt tcaacaggcc 720agccattacg
ctcgtcatca aaatcactcg catcaaccaa accgttattc attcgtgatt
780gcgcctgagc gagacgaaat acgcgatcgc tgttaaaagg acaattacaa
acaggaatcg 840aatgcaaccg gcgcaggaac actgccagcg catcaacaat
attttcacct gaatcaggat 900attcttctaa tacctggaat gctgttttcc
cggggatcgc agtggtgagt aaccatgcat 960catcaggagt acggataaaa
tgcttgatgg tcggaagagg cataaattcc gtcagccagt 1020ttagtctgac
catctcatct gtaacatcat tggcaacgct acctttgcca tgtttcagaa
1080acaactctgg cgcatcgggc ttcccataca atcgatagat tgtcgcacct
gattgcccga 1140cattatcgcg agcccattta tacccatata aatcagcatc
catgttggaa tttaatcgcg 1200gcctcgagca agacgtttcc cgttgaatat
ggctcataac accccttgta ttactgttta 1260tgtaagcaga cagttttatt
gttcatgatg
atatattttt atcttgtgca atgtaacatc 1320agagattttg agacacaacg
tggctttgtt gaataaatcg aacttttgct gagttgaagg 1380atcagatcac
gcatcttccc gacaacgcag accgttccgt ggcaaagcaa aagttcaaaa
1440tcaccaactg gtccacctac aacaaagctc tcatcaaccg tggctccctc
actttctggc 1500tggatgatgg ggcgattcag gcctggtatg agtcagcaac
accttcttca cgaggcagac 1560ctcagcgcta ttctgacctt gccatcacga
ctgtgctggt cattaaacgc gtattcaggc 1620tgaccctgcg cgctgcgcag
ggctttattg attccatttt tacactgatg aatgttccgt 1680tgcgctgccc
ggattacagc tgaaagcgac caggtgctcg gcgtggcaag actcgcagcg
1740aacccgtaga aagccatgct ccagccgccc gcattggaga aattcttcaa
attcccgttg 1800cacatagccc ggcaattcct ttccctgctc tgccataagc
gcagcgaatg ccgggtaata 1860ctcgtcaacg atctgataga gaagggtttg
ctcgggtcgg tggctctggt aacgaccagt 1920atcccgatcc cggctggccg
tcctggccgc cacatgaggc atgttccgcg tccttgcaat 1980actgtgttta
catacagtct atcgcttagc ggaaagttct tttaccctca gccgaaatgc
2040ctgccgttgc tagacattgc cagccagtgc ccgtcactcc cgtactaact
gtcacgaacc 2100cctgcaataa ctgtcacgcc cccctgcaat aactgtcacg
aacccctgca ataactgtca 2160cgcccccaaa cctgcaaacc cagcaggggc
gggggctggc ggggtgttgg aaaaatccat 2220ccatgattat ctaagaataa
tccactaggc gcggttatca gcgcccttgt ggggcgctgc 2280tgcccttgcc
caatatgccc ggccagaggc cggatagctg gtctattcgc tgcgctaggc
2340tacacaccgc cccaccgctg cgcggcaggg ggaaaggcgg gcaaagcccg
ctaaacccca 2400caccaaaccc cgcagaaata cgctggagcg cttttagccg
ctttagcggc ctttccccct 2460acccgaaggg tgggggcgcg tgtgcagccc
cgcagggcct gtctcggtcg atcattcagc 2520ccggctcatc cttctggcgt
ggcggcagac cgaacaaggc gcggtcgtgg tcgcgttcaa 2580ggtacgcatc
cattgccgcc atgagccgat cctccggcca ctcgctgctg ttcaccttgg
2640ccaaaatcat ggcccccacc agcaccttgc gccttgtttc gttcttgcgc
tcttgctgct 2700gttcccttgc ccgcacccgc tgaatttcgg cattgattcg
cgctcgttgt tcttcgagct 2760tggccagccg atccgccgcc ttgttgctcc
ccttaaccat cttgacaccc cattgttaat 2820gtgctgtctc gtaggctatc
atggaggcac agcggcggca atcccgaccc tactttgtag 2880gggagggcgc
acttaccggt ttctcttcga gaaactggcc taacggccac ccttcgggcg
2940gtgcgctctc cgagggccat tgcatggagc cgaaaagcaa aagcaacagc
gaggcagcat 3000ggcgatttat caccttacgg cgaaaaccgg cagcaggtcg
ggcggccaat cggccagggc 3060caaggccgac tacatccagc gcgaaggcaa
gtatgcccgc gacatggatg aagtcttgca 3120cgccgaatcc gggcacatgc
cggagttcgt cgagcggccc gccgactact gggatgctgc 3180cgacctgtat
gaacgcgcca atgggcggct gttcaaggag gtcgaatttg ccctgccggt
3240cgagctgacc ctcgaccagc agaaggcgct ggcgtccgag ttcgcccagc
acctgaccgg 3300tgccgagcgc ctgccgtata cgctggccat ccatgccggt
ggcggcgaga acccgcactg 3360ccacctgatg atctccgagc ggatcaatga
cggcatcgag cggcccgccg ctcagtggtt 3420caagcggtac aacggcaaga
ccccggagaa gggcggggca cagaagaccg aagcgctcaa 3480gcccaaggca
tggcttgagc agacccgcga ggcatgggcc gaccatgcca accgggcatt
3540agagcgggct ggccacgacg cccgcattga ccacagaaca cttgaggcgc
agggcatcga 3600gcgcctgccc ggtgttcacc tggggccgaa cgtggtggag
atggaaggcc ggggcatccg 3660caccgaccgg gcagacgtgg ccctgaacat
cgacaccgcc aacgcccaga tcatcgactt 3720acaggaatac cgggaggcaa
tagaccatga acgcaatcga cagagtgaag aaatccagag 3780gcatcaacga
gttagcggag cagatcgaac cgctggccca gagcatggcg acactggccg
3840acgaagcccg gcaggtcatg agccagaccc agcaggccag cgaggcgcag
gcggcggagt 3900ggctgaaagc ccagcgccag acaggggcgg catgggtgga
gctggccaaa gagttgcggg 3960aggtagccgc cgaggtgagc agcgccgcgc
agagcgcccg gagcgcgtcg cgggggtggc 4020actggaagct atggctaacc
gtgatgctgg cttccatgat gcctacggtg gtgctgctga 4080tcgcatcgtt
gctcttgctc gacctgacgc cactgacaac cgaggacggc tcgatctggc
4140tgcgcttggt ggcccgatga agaacgacag gactttgcag gccataggcc
gacagctcaa 4200ggccatgggc tgtgagcgct tcgatatcgg cgtcagggac
gccaccaccg gccagatgat 4260gaaccgggaa tggtcagccg ccgaagtgct
ccagaacacg ccatggctca agcggatgaa 4320tgcccagggc aatgacgtgt
atatcaggcc cgccgagcag gagcggcatg gtctggtgct 4380ggtggacgac
ctcagcgagt ttgacctgga tgacatgaaa gccgagggcc gggagcctgc
4440cctggtagtg gaaaccagcc cgaagaacta tcaggcatgg gtcaaggtgg
ccgacgccgc 4500aggcggtgaa cttcgggggc agattgcccg gacgctggcc
agcgagtacg acgccgaccc 4560ggccagcgcc gacagccgcc actatggccg
cttggcgggc ttcaccaacc gcaaggacaa 4620gcacaccacc cgcgccggtt
atcagccgtg ggtgctgctg cgtgaatcca agggcaagac 4680cgccaccgct
ggcccggcgc tggtgcagca ggctggccag cagatcgagc aggcccagcg
4740gcagcaggag aaggcccgca ggctggccag cctcgaactg cccgagcggc
agcttagccg 4800ccaccggcgc acggcgctgg acgagtaccg cagcgagatg
gccgggctgg tcaagcgctt 4860cggtgatgac ctcagcaagt gcgactttat
cgccgcgcag aagctggcca gccggggccg 4920cagtgccgag gaaatcggca
aggccatggc cgaggccagc ccagcgctgg cagagcgcaa 4980gcccggccac
gaagcggatt acatcgagcg caccgtcagc aaggtcatgg gtctgcccag
5040cgtccagctt gcgcgggccg agctggcacg ggcaccggca ccccgccagc
gaggcatgga 5100caggggcggg ccagatttca gcatgtagtg cttgcgttgg
tactcacgcc tgttatacta 5160tgagtactca cgcacagaag ggggttttat
ggaatacgaa aaaagcgctt cagggtcggt 5220ctacctgatc aaaagtgaca
agggctattg gttgcccggt ggctttggtt atacgtcaaa 5280caaggccgag
gctggccgct tttcagtcgc tgatatggcc agccttaacc ttgacggctg
5340caccttgtcc ttgttccgcg aagacaagcc tttcggcccc ggcaagtttc
tcggtgactg 5400atatgaaaga ccaaaaggac aagcagaccg gcgacctgct
ggccagccct gacgctgtac 5460gccaagcgcg atatgccgag cgcatgaagg
ccaaagggat gcgtcagcgc aagttctggc 5520tgaccgacga cgaatacgag
gcgctgcgcg agtgcctgga agaactcaga gcggcgcagg 5580gcgggggtag
tgaccccgcc agcgcctaac caccaactgc ctgcaaagga ggcaatcaat
5640ggctacccat aagcctatca atattctgga ggcgttcgca gcagcgccgc
caccgctgga 5700ctacgttttg cccaacatgg tggccggtac ggtcggggcg
ctggtgtcgc ccggtggtgc 5760cggtaaatcc atgctggccc tgcaactggc
cgcacagatt gcaggcgggc cggatctgct 5820ggaggtgggc gaactgccca
ccggcccggt gatctacctg cccgccgaag acccgcccac 5880cgccattcat
caccgcctgc acgcccttgg ggcgcacctc agcgccgagg aacggcaagc
5940cgtggctgac ggcctgctga tccagccgct gatcggcagc ctgcccaaca
tcatggcccc 6000ggagtggttc gacggcctca agcgcgccgc cgagggccgc
cgcctgatgg tgctggacac 6060gctgcgccgg ttccacatcg aggaagaaaa
cgccagcggc cccatggccc aggtcatcgg 6120tcgcatggag gccatcgccg
ccgataccgg gtgctctatc gtgttcctgc accatgccag 6180caagggcgcg
gccatgatgg gcgcaggcga ccagcagcag gccagccggg gcagctcggt
6240actggtcgat aacatccgct ggcagtccta cctgtcgagc atgaccagcg
ccgaggccga 6300ggaatggggt gtggacgacg accagcgccg gttcttcgtc
cgcttcggtg tgagcaaggc 6360caactatggc gcaccgttcg ctgatcggtg
gttcaggcgg catgacggcg gggtgctcaa 6420gcccgccgtg ctggagaggc
agcgcaagag caagggggtg ccccgtggtg aagcctaaga 6480acaagcacag
cctcagccac gtccggcacg acccggcgca ctgtctggcc cccggcctgt
6540tccgtgccct caagcggggc gagcgcaagc gcagcaagct ggacgtgacg
tatgactacg 6600gcgacggcaa gcggatcgag ttcagcggcc cggagccgct
gggcgctgat gatctgcgca 6660tcctgcaagg gctggtggcc atggctgggc
ctaatggcct agtgcttggc ccggaaccca 6720agaccgaagg cggacggcag
ctccggctgt tcctggaacc caagtgggag gccgtcaccg 6780ctgaatgcca
tgtggtcaaa ggtagctatc gggcgctggc aaaggaaatc ggggcagagg
6840tcgatagtgg tggggcgctc aagcacatac aggactgcat cgagcgcctt
tggaaggtat 6900ccatcatcgc ccagaatggc cgcaagcggc aggggtttcg
gctgctgtcg gagtacgcca 6960gcgacgaggc ggacgggcgc ctgtacgtgg
ccctgaaccc cttgatcgcg caggccgtca 7020tgggtggcgg ccagcatgtg
cgcatcagca tggacgaggt gcgggcgctg gacagcgaaa 7080ccgcccgcct
gctgcaccag cggctgtgtg gctggatcga ccccggcaaa accggcaagg
7140cttccataga taccttgtgc ggctatgtct ggccgtcaga ggccagtggt
tcgaccatgc 7200gcaagcgccg ccagcgggtg cgcgaggcgt tgccggagct
ggtcgcgctg ggctggacgg 7260taaccgagtt cgcggcgggc aagtacgaca
tcacccggcc caaggcggca ggctgacccc 7320ccccactcta ttgtaaacaa
gacattttta tcttttatat tcaatggctt attttcctgc 7380taattggtaa
taccatgaaa aataccatgc tcagaaaagg cttaacaata ttttgaaaaa
7440ttgcctactg agcgctgccg cacagctcca taggccgctt tcctggcttt
gcttccagat 7500gtatgctctt ctgctcctgc aggcatcgtg gtgtcacgct
cgtcgtttgg tatggcttca 7560ttcagctccg gttcccaacg atcaaggcga
gttacatgat cccccatgtt gtgcaaaaaa 7620gcggttagct ccttcggtcc
tccgatcgtt gtcagaagta agttggccgc agtgttatca 7680ctcatggtta
tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
7740tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
gcgaccgagt 7800tgctcttgcc cggcgtcaac acgggataat accgcgccac
atagcagaac tttaaaagtg 7860ctcatcattg gaaaacgttc ttcggggcga
aaactctcaa ggatcttacc gctgttgaga 7920tccagttcga tgtaacccac
tcgtgcaccc aactgatctt cagcatcttt tactttcacc 7980agcgtttctg
ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
8040acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
catttatcag 8100ggttattgtc tcatgagcgg atacatattt gaatgtattt
agaaaaataa acaaataggg 8160gttccgcgca catttccccg aaaagtgcca
cctgacgtct aagaaaccat tattatcatg 8220acattaacct ataaaaatag
gcgtatcacg aggccctttc gtcttcgaat aaatacctgt 8280gacggaagat
cacttcgcag aataaataaa tcctggtgtc cctgttgata ccgggaagcc
8340ctgggccaac ttttggcgaa aatgagacgt tgatcggcac gtaagaggtt
ccaactttca 8400ccataatgaa ataagatcac taccgggcgt attttttgag
ttatcgagat tttcaggagc 8460taaggaagct aaaatggaga aaaaaatcac
tggatatacc accgttgata tatcccaatg 8520gcatcgtaaa gaacattttg
aggcatttca gtcagttgct caatgtacct ataaccagac 8580cgttcagctg
gatattacgg cctttttaaa gaccgtaaag aaaaataagc acaagtttta
8640tccggccttt attcacattc ttgcccgcct gatgaatgct catccggaat
tccgtatggc 8700aatgaaagac ggtgagctgg tgatatggga tagtgttcac
ccttgttaca ccgttttcca 8760tgagcaaact gaaacgtttt catcgctctg
gagtgaatac cacgacgatt tccggcagtt 8820tctacacata tattcgcaag
atgtggcgtg ttacggtgaa aacctggcct atttccctaa 8880agggtttatt
gagaatatgt ttttcgtctc agccaatccc tgggtgagtt tcaccagttt
8940tgatttaaac gtggccaata tggacaactt cttcgccccc gttttcacca
tgggcaaata 9000ttatacgcaa ggcgacaagg tgctgatgcc gctggcgatt
caggttcatc atgccgtctg 9060tgatggcttc catgtcggca gaatgcttaa
tgaattacaa cagtactgcg atgagtggca 9120gggcggggcg taattttttt
aaggcagtta ttggtgccct taaacgcctg gtgctacgcc 9180tgaataagtg
ataataagcg gatgaatggc agaaattcgt cgactctaga g
9231718390DNAartificialpVZ322 vector 71gatctgtaat ccgggcagcg
caacggaaca ttcatcagtg taaaaatgga atcaataaag 60ccctgcgcag cgcgcagggt
cagcctgaat acgcgtttaa tgaccagcac agtcgtgatg 120gcaaggtcag
aatagcgctg aggtctgcct cgtgaagaag gtgttgctga ctcataccag
180gcctgaatcg ccccatcatc cagccagaaa gtgagggagc cacggttgat
gagagctttg 240ttgtaggtgg accagttggt gattttgaac ttttgctttg
ccacggaacg gtctgcgttg 300tcgggaagat gcgtgatctg atccttcaac
tcagcaaaag ttcgatttat tcaacaaagc 360cgccgtcccg tcaagtcagc
gtaatgctct gccagtgtta caaccaatta accaattctg 420attagaaaaa
ctcatcgagc atcaaatgaa actgcaattt attcatatca ggattatcaa
480taccatattt ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg
aggcagttcc 540ataggatggc aagatcctgg tatcggtctg cgattccgac
tcgtccaaca tcaatacaac 600ctattaattt cccctcgtca aaaataaggt
tatcaagtga gaaatcacca tgagtgacga 660ctgaatccgg tgagaatggc
aaaagcttat gcatttcttt ccagacttgt tcaacaggcc 720agccattacg
ctcgtcatca aaatcactcg catcaaccaa accgttattc attcgtgatt
780gcgcctgagc gagacgaaat acgcgatcgc tgttaaaagg acaattacaa
acaggaatcg 840aatgcaaccg gcgcaggaac actgccagcg catcaacaat
attttcacct gaatcaggat 900attcttctaa tacctggaat gctgttttcc
cggggatcgc agtggtgagt aaccatgcat 960catcaggagt acggataaaa
tgcttgatgg tcggaagagg cataaattcc gtcagccagt 1020ttagtctgac
catctcatct gtaacatcat tggcaacgct acctttgcca tgtttcagaa
1080acaactctgg cgcatcgggc ttcccataca atcgatagat tgtcgcacct
gattgcccga 1140cattatcgcg agcccattta tacccatata aatcagcatc
catgttggaa tttaatcgcg 1200gcctcgagca agacgtttcc cgttgaatat
ggctcataac accccttgta ttactgttta 1260tgtaagcaga cagttttatt
gttcatgatg atatattttt atcttgtgca atgtaacatc 1320agagattttg
agacacaacg tggctttgtt gaataaatcg aacttttgct gagttgaagg
1380atcagatcac gcatcttccc gacaacgcag accgttccgt ggcaaagcaa
aagttcaaaa 1440tcaccaactg gtccacctac aacaaagctc tcatcaaccg
tggctccctc actttctggc 1500tggatgatgg ggcgattcag gcctggtatg
agtcagcaac accttcttca cgaggcagac 1560ctcagcgcta ttctgacctt
gccatcacga ctgtgctggt cattaaacgc gtattcaggc 1620tgaccctgcg
cgctgcgcag ggctttattg attccatttt tacactgatg aatgttccgt
1680tgcgctgccc ggattacagg ggtaccgagc tcgaattgac ataagcctgt
tcggttcgta 1740aactgtaatg caagtagcgt atgcgctcac gcaactggtc
cagaaccttg accgaacgca 1800gcggtggtaa cggcgcagtg gcggttttca
tggcttgtta tgactgtttt tttgtacagt 1860ctatgcctcg ggcatccaag
cagcaagcgc gttacgccgt gggtcgatgt ttgatgttat 1920ggagcagcaa
cgatgttacg cagcagcaac gatgttacgc agcagggcag tcgccctaaa
1980acaaagttag gtggctcaag tatgggcatc attcgcacat gtaggctcgg
ccctgaccaa 2040gtcaaatcca tgcgggctgc tcttgatctt ttcggtcgtg
agttcggaga cgtagccacc 2100tactcccaac atcagccgga ctccgattac
ctcgggaact tgctccgtag taagacattc 2160atcgcgcttg ctgccttcga
ccaagaagcg gttgttggcg ctctcgcggc ttacgttctg 2220cccaggtttg
agcagccgcg tagtgagatc tatatctatg atctcgcagt ctccggcgag
2280caccggaggc agggcattgc caccgcgctc atcaatctcc tcaagcatga
ggccaacgcg 2340cttggtgctt atgtgatcta cgtgcaagca gattacggtg
acgatcccgc agtggctctc 2400tatacaaagt tgggcatacg ggaagaagtg
atgcactttg atatcgaccc aagtaccgcc 2460acctaacaat tcgttcaagc
cgagatcggc ttcccggccg cggagttgtt cggtaaattg 2520tcacaacgcc
gcggccaatt cgagctcggt acccctgaaa gcgaccaggt gctcggcgtg
2580gcaagactcg cagcgaaccc gtagaaagcc atgctccagc cgcccgcatt
ggagaaattc 2640ttcaaattcc cgttgcacat agcccggcaa ttcctttccc
tgctctgcca taagcgcagc 2700gaatgccggg taatactcgt caacgatctg
atagagaagg gtttgctcgg gtcggtggct 2760ctggtaacga ccagtatccc
gatcccggct ggccgtcctg gccgccacat gaggcatgtt 2820ccgcgtcctt
gcaatactgt gtttacatac agtctatcgc ttagcggaaa gttcttttac
2880cctcagccga aatgcctgcc gttgctagac attgccagcc agtgcccgtc
actcccgtac 2940taactgtcac gaacccctgc aataactgtc acgcccccct
gcaataactg tcacgaaccc 3000ctgcaataac tgtcacgccc ccaaacctgc
aaacccagca ggggcggggg ctggcggggt 3060gttggaaaaa tccatccatg
attatctaag aataatccac taggcgcggt tatcagcgcc 3120cttgtggggc
gctgctgccc ttgcccaata tgcccggcca gaggccggat agctggtcta
3180ttcgctgcgc taggctacac accgccccac cgctgcgcgg cagggggaaa
ggcgggcaaa 3240gcccgctaaa ccccacacca aaccccgcag aaatacgctg
gagcgctttt agccgcttta 3300gcggcctttc cccctacccg aagggtgggg
gcgcgtgtgc agccccgcag ggcctgtctc 3360ggtcgatcat tcagcccggc
tcatccttct ggcgtggcgg cagaccgaac aaggcgcggt 3420cgtggtcgcg
ttcaaggtac gcatccattg ccgccatgag ccgatcctcc ggccactcgc
3480tgctgttcac cttggccaaa atcatggccc ccaccagcac cttgcgcctt
gtttcgttct 3540tgcgctcttg ctgctgttcc cttgcccgca cccgctgaat
ttcggcattg attcgcgctc 3600gttgttcttc gagcttggcc agccgatccg
ccgccttgtt gctcccctta accatcttga 3660caccccattg ttaatgtgct
gtctcgtagg ctatcatgga ggcacagcgg cggcaatccc 3720gaccctactt
tgtaggggag ggcgcactta ccggtttctc ttcgagaaac tggcctaacg
3780gccacccttc gggcggtgcg ctctccgagg gccattgcat ggagccgaaa
agcaaaagca 3840acagcgaggc agcatggcga tttatcacct tacggcgaaa
accggcagca ggtcgggcgg 3900ccaatcggcc agggccaagg ccgactacat
ccagcgcgaa ggcaagtatg cccgcgacat 3960ggatgaagtc ttgcacgccg
aatccgggca catgccggag ttcgtcgagc ggcccgccga 4020ctactgggat
gctgccgacc tgtatgaacg cgccaatggg cggctgttca aggaggtcga
4080atttgccctg ccggtcgagc tgaccctcga ccagcagaag gcgctggcgt
ccgagttcgc 4140ccagcacctg accggtgccg agcgcctgcc gtatacgctg
gccatccatg ccggtggcgg 4200cgagaacccg cactgccacc tgatgatctc
cgagcggatc aatgacggca tcgagcggcc 4260cgccgctcag tggttcaagc
ggtacaacgg caagaccccg gagaagggcg gggcacagaa 4320gaccgaagcg
ctcaagccca aggcatggct tgagcagacc cgcgaggcat gggccgacca
4380tgccaaccgg gcattagagc gggctggcca cgacgcccgc attgaccaca
gaacacttga 4440ggcgcagggc atcgagcgcc tgcccggtgt tcacctgggg
ccgaacgtgg tggagatgga 4500aggccggggc atccgcaccg accgggcaga
cgtggccctg aacatcgaca ccgccaacgc 4560ccagatcatc gacttacagg
aataccggga ggcaatagac catgaacgca atcgacagag 4620tgaagaaatc
cagaggcatc aacgagttag cggagcagat cgaaccgctg gcccagagca
4680tggcgacact ggccgacgaa gcccggcagg tcatgagcca gacccagcag
gccagcgagg 4740cgcaggcggc ggagtggctg aaagcccagc gccagacagg
ggcggcatgg gtggagctgg 4800ccaaagagtt gcgggaggta gccgccgagg
tgagcagcgc cgcgcagagc gcccggagcg 4860cgtcgcgggg gtggcactgg
aagctatggc taaccgtgat gctggcttcc atgatgccta 4920cggtggtgct
gctgatcgca tcgttgctct tgctcgacct gacgccactg acaaccgagg
4980acggctcgat ctggctgcgc ttggtggccc gatgaagaac gacaggactt
tgcaggccat 5040aggccgacag ctcaaggcca tgggctgtga gcgcttcgat
atcggcgtca gggacgccac 5100caccggccag atgatgaacc gggaatggtc
agccgccgaa gtgctccaga acacgccatg 5160gctcaagcgg atgaatgccc
agggcaatga cgtgtatatc aggcccgccg agcaggagcg 5220gcatggtctg
gtgctggtgg acgacctcag cgagtttgac ctggatgaca tgaaagccga
5280gggccgggag cctgccctgg tagtggaaac cagcccgaag aactatcagg
catgggtcaa 5340ggtggccgac gccgcaggcg gtgaacttcg ggggcagatt
gcccggacgc tggccagcga 5400gtacgacgcc gacccggcca gcgccgacag
ccgccactat ggccgcttgg cgggcttcac 5460caaccgcaag gacaagcaca
ccacccgcgc cggttatcag ccgtgggtgc tgctgcgtga 5520atccaagggc
aagaccgcca ccgctggccc ggcgctggtg cagcaggctg gccagcagat
5580cgagcaggcc cagcggcagc aggagaaggc ccgcaggctg gccagcctcg
aactgcccga 5640gcggcagctt agccgccacc ggcgcacggc gctggacgag
taccgcagcg agatggccgg 5700gctggtcaag cgcttcggtg atgacctcag
caagtgcgac tttatcgccg cgcagaagct 5760ggccagccgg ggccgcagtg
ccgaggaaat cggcaaggcc atggccgagg ccagcccagc 5820gctggcagag
cgcaagcccg gccacgaagc ggattacatc gagcgcaccg tcagcaaggt
5880catgggtctg cccagcgtcc agcttgcgcg ggccgagctg gcacgggcac
cggcaccccg 5940ccagcgaggc atggacaggg gcgggccaga tttcagcatg
tagtgcttgc gttggtactc 6000acgcctgtta tactatgagt actcacgcac
agaagggggt tttatggaat acgaaaaaag 6060cgcttcaggg tcggtctacc
tgatcaaaag tgacaagggc tattggttgc ccggtggctt 6120tggttatacg
tcaaacaagg ccgaggctgg ccgcttttca gtcgctgata tggccagcct
6180taaccttgac ggctgcacct tgtccttgtt ccgcgaagac aagcctttcg
gccccggcaa 6240gtttctcggt gactgatatg aaagaccaaa aggacaagca
gaccggcgac ctgctggcca 6300gccctgacgc tgtacgccaa gcgcgatatg
ccgagcgcat gaaggccaaa gggatgcgtc 6360agcgcaagtt ctggctgacc
gacgacgaat acgaggcgct gcgcgagtgc ctggaagaac 6420tcagagcggc
gcagggcggg ggtagtgacc ccgccagcgc ctaaccacca actgcctgca
6480aaggaggcaa tcaatggcta cccataagcc tatcaatatt ctggaggcgt
tcgcagcagc 6540gccgccaccg ctggactacg ttttgcccaa catggtggcc
ggtacggtcg gggcgctggt 6600gtcgcccggt ggtgccggta aatccatgct
ggccctgcaa ctggccgcac agattgcagg 6660cgggccggat ctgctggagg
tgggcgaact gcccaccggc ccggtgatct acctgcccgc 6720cgaagacccg
cccaccgcca ttcatcaccg cctgcacgcc cttggggcgc acctcagcgc
6780cgaggaacgg caagccgtgg ctgacggcct gctgatccag ccgctgatcg
gcagcctgcc 6840caacatcatg gccccggagt ggttcgacgg cctcaagcgc
gccgccgagg gccgccgcct 6900gatggtgctg gacacgctgc gccggttcca
catcgaggaa gaaaacgcca gcggccccat 6960ggcccaggtc atcggtcgca
tggaggccat cgccgccgat accgggtgct ctatcgtgtt 7020cctgcaccat
gccagcaagg gcgcggccat gatgggcgca ggcgaccagc agcaggccag
7080ccggggcagc tcggtactgg tcgataacat ccgctggcag tcctacctgt
cgagcatgac 7140cagcgccgag gccgaggaat ggggtgtgga cgacgaccag
cgccggttct tcgtccgctt 7200cggtgtgagc aaggccaact atggcgcacc
gttcgctgat cggtggttca ggcggcatga 7260cggcggggtg ctcaagcccg
ccgtgctgga gaggcagcgc aagagcaagg gggtgccccg 7320tggtgaagcc
taagaacaag cacagcctca gccacgtccg gcacgacccg gcgcactgtc
7380tggcccccgg cctgttccgt gccctcaagc ggggcgagcg caagcgcagc
aagctggacg 7440tgacgtatga ctacggcgac ggcaagcgga tcgagttcag
cggcccggag ccgctgggcg 7500ctgatgatct gcgcatcctg caagggctgg
tggccatggc tgggcctaat ggcctagtgc 7560ttggcccgga acccaagacc
gaaggcggac ggcagctccg gctgttcctg gaacccaagt 7620gggaggccgt
caccgctgaa tgccatgtgg tcaaaggtag ctatcgggcg ctggcaaagg
7680aaatcggggc agaggtcgat agtggtgggg cgctcaagca catacaggac
tgcatcgagc 7740gcctttggaa ggtatccatc atcgcccaga atggccgcaa
gcggcagggg tttcggctgc 7800tgtcggagta cgccagcgac gaggcggacg
ggcgcctgta cgtggccctg aaccccttga 7860tcgcgcaggc cgtcatgggt
ggcggccagc atgtgcgcat cagcatggac gaggtgcggg 7920cgctggacag
cgaaaccgcc cgcctgctgc accagcggct gtgtggctgg atcgaccccg
7980gcaaaaccgg caaggcttcc atagatacct tgtgcggcta tgtctggccg
tcagaggcca 8040gtggttcgac catgcgcaag cgccgccagc gggtgcgcga
ggcgttgccg gagctggtcg 8100cgctgggctg gacggtaacc gagttcgcgg
cgggcaagta cgacatcacc cggcccaagg 8160cggcaggctg acccccccca
ctctattgta aacaagacat ttttatcttt tatattcaat 8220ggcttatttt
cctgctaatt ggtaatacca tgaaaaatac catgctcaga aaaggcttaa
8280caatattttg aaaaattgcc tactgagcgc tgccgcacag ctccataggc
cgctttcctg 8340gctttgcttc cagatgtatg ctcttctgct cctgcaggtc
gactctagag 8390723206DNAartificialconstruct pIC PpetJ 72gcgcccaata
cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca 60cgacaggttt
cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct
120cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt
tgtgtggaat 180tgtgagcgga taacaatttc acacaggaaa cagctatgac
catgattacg ccaagcttgc 240atgcctgcag gtcgactcta gaggatcccc
gggtacccct catcgggggc tgtgttggcc 300gagacggcac tgaggatttt
actctccatg gcattccaag gaatatctac ccaactcacc 360tgctccggcg
gattgttccg ctcaaaagta ctaatcaagt cgtcaaaata cttattaaat
420tttggctgca attgcatagt ccaaaagctg actttcccct ccatgctctg
gggggaattg 480ctctggcaac tgattaatcc actgagcaac agcccaagac
acgcaaacaa aaaccaacgt 540cttggcgatc gccatcggca ccatgaaacc
atcgtaaaag ctggggaaag aataaaaaac 600agtggttcag gaattgcatt
gccatggcca cttcacaaac ctagccaatt ttagcttgac 660cgcaactttg
acagattgtc ttttgacttt gcctggaccg cctcccataa taccttcgcg
720tcttgaagac tttatccttg aaaggagaac atatgtttct cggcaaaaat
taattatcga 780tatctagatc tcgagctcgc gaaagcttgg cactggccgt
cgttttacaa cgtcgtgact 840gggaaaaccc tggcgttacc caacttaatc
gccttgcagc acatccccct ttcgccagct 900ggcgtaatag cgaagaggcc
cgcaccgatc gcccttccca acagttgcgc agcctgaatg 960gcgaatggcg
cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca
1020tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc
ccgacacccg 1080ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc
cggcatccgc ttacagacaa 1140gctgtgaccg tctccgggag ctgcatgtgt
cagaggtttt caccgtcatc accgaaacgc 1200gcgagacgaa agggcctcgt
gatacgccta tttttatagg ttaatgtcat gataataatg 1260gtttcttaga
cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
1320tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
ataaatgctt 1380caataatatt gaaaaaggaa gagtatgagt attcaacatt
tccgtgtcgc ccttattccc 1440ttttttgcgg cattttgcct tcctgttttt
gctcacccag aaacgctggt gaaagtaaaa 1500gatgctgaag atcagttggg
tgcacgagtg ggttacatcg aactggatct caacagcggt 1560aagatccttg
agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt
1620ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact
cggtcgccgc 1680atacactatt ctcagaatga cttggttgag tactcaccag
tcacagaaaa gcatcttacg 1740gatggcatga cagtaagaga attatgcagt
gctgccataa ccatgagtga taacactgcg 1800gccaacttac ttctgacaac
gatcggagga ccgaaggagc taaccgcttt tttgcacaac 1860atgggggatc
atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca
1920aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg
caaactatta 1980actggcgaac tacttactct agcttcccgg caacaattaa
tagactggat ggaggcggat 2040aaagttgcag gaccacttct gcgctcggcc
cttccggctg gctggtttat tgctgataaa 2100tctggagccg gtgagcgtgg
gtctcgcggt atcattgcag cactggggcc agatggtaag 2160ccctcccgta
tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat
2220agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc
agaccaagtt 2280tactcatata tactttagat tgatttaaaa cttcattttt
aatttaaaag gatctaggtg 2340aagatccttt ttgataatct catgaccaaa
atcccttaac gtgagttttc gttccactga 2400gcgtcagacc ccgtagaaaa
gatcaaagga tcttcttgag atcctttttt tctgcgcgta 2460atctgctgct
tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
2520gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
accaaatact 2580gtccttctag tgtagccgta gttaggccac cacttcaaga
actctgtagc accgcctaca 2640tacctcgctc tgctaatcct gttaccagtg
gctgctgcca gtggcgataa gtcgtgtctt 2700accgggttgg actcaagacg
atagttaccg gataaggcgc agcggtcggg ctgaacgggg 2760ggttcgtgca
cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
2820cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
gtatccggta 2880agcggcaggg tcggaacagg agagcgcacg agggagcttc
cagggggaaa cgcctggtat 2940ctttatagtc ctgtcgggtt tcgccacctc
tgacttgagc gtcgattttt gtgatgctcg 3000tcaggggggc ggagcctatg
gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc 3060ttttgctggc
cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac
3120cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
cgagcgcagc 3180gagtcagtga gcgaggaagc ggaaga
3206733093DNAartificialinsert of the vector pCB4-LR(TF)pa that
encodes Z. mobilis adhII and pdc genes 73atgaattctt atactgtcgg
tacctattta gcggagcggc ttgtccagat tggtctcaag 60catcacttcg cagtcgcggg
cgactacaac ctcgtccttc ttgacaacct gcttttgaac 120aaaaacatgg
agcaggttta ttgctgtaac gaactgaact gcggtttcag tgcagaaggt
180tatgctcgtg ccaaaggcgc agcagcagcc gtcgttacct acagcgtcgg
tgcgctttcc 240gcatttgatg ctatcggtgg cgcctatgca gaaaaccttc
cggttatcct gatctccggt 300gctccgaaca acaatgatca cgctgctggt
cacgtgttgc atcacgctct tggcaaaacc 360gactatcact atcagttgga
aatggccaag aacatcacgg ccgcagctga agcgatttac 420accccagaag
aagctccggc taaaatcgat cacgtgatta aaactgctct tcgtgagaag
480aagccggttt atctcgaaat cgcttgcaac attgcttcca tgccctgcgc
cgctcctgga 540ccggcaagcg cattgttcaa tgacgaagcc agcgacgaag
cttctttgaa tgcagcggtt 600gaagaaaccc tgaaattcat cgccaaccgc
gacaaagttg ccgtcctcgt cggcagcaag 660ctgcgcgcag ctggtgctga
agaagctgct gtcaaatttg ctgatgctct cggtggcgca 720gttgctacca
tggctgctgc aaaaagcttc ttcccagaag aaaacccgca ttacatcggt
780acctcatggg gtgaagtcag ctatccgggc gttgaaaaga cgatgaaaga
agccgatgcg 840gttatcgctc tggctcctgt cttcaacgac tactccacca
ctggttggac ggatattcct 900gatcctaaga aactggttct cgctgaaccg
cgttctgtcg tcgttaacgg cgttcgcttc 960cccagcgttc atctgaaaga
ctatctgacc cgtttggctc agaaagtttc caagaaaacc 1020ggtgctttgg
acttcttcaa atccctcaat gcaggtgaac tgaagaaagc cgctccggct
1080gatccgagtg ctccgttggt caacgcagaa atcgcccgtc aggtcgaagc
tcttctgacc 1140ccgaacacga cggttattgc tgaaaccggt gactcttggt
tcaatgctca gcgcatgaag 1200ctcccgaacg gtgctcgcgt tgaatatgaa
atgcagtggg gtcacatcgg ttggtccgtt 1260cctgccgcct tcggttatgc
cgtcggtgct ccggaacgtc gcaacatcct catggttggt 1320gatggttcct
tccagctgac ggctcaggaa gtcgctcaga tggttcgcct gaaactgccg
1380gttatcatct tcttgatcaa taactatggt tacaccatcg aagttatgat
ccatgatggt 1440ccgtacaaca acatcaagaa ctgggattat gccggtctga
tggaagtgtt caacggtaac 1500ggtggttatg acagcggtgc tggtaaaggc
ctgaaggcta aaaccggtgg cgaactggca 1560gaagctatca aggttgctct
ggcaaacacc gacggcccaa ccctgatcga atgcttcatc 1620ggtcgtgaag
actgcactga agaattggtc aaatggggta agcgcgttgc tgccgccaac
1680agccgtaagc ctgttaacaa gctcctctag tttttgggga tcaattcgag
ctcggtaccc 1740aaactagtat gtagggtgag gttatagcta tggcttcttc
aactttttat attcctttcg 1800tcaacgaaat gggcgaaggt tcgcttgaaa
aagcaatcaa ggatcttaac ggcagcggct 1860ttaaaaatgc gctgatcgtt
tctgatgctt tcatgaacaa atccggtgtt gtgaagcagg 1920ttgctgacct
gttgaaagca cagggtatta attctgctgt ttatgatggc gttatgccga
1980acccgactgt taccgcagtt ctggaaggcc ttaagatcct gaaggataac
aattcagact 2040tcgtcatctc cctcggtggt ggttctcccc atgactgcgc
caaagccatc gctctggtcg 2100caaccaatgg tggtgaagtc aaagactacg
aaggtatcga caaatctaag aaacctgccc 2160tgcctttgat gtcaatcaac
acgacggctg gtacggcttc tgaaatgacg cgtttctgca 2220tcatcactga
tgaagtccgt cacgttaaga tggccattgt tgaccgtcac gttaccccga
2280tggtttccgt caacgatcct ctgttgatgg ttggtatgcc aaaaggcctg
accgccgcca 2340ccggtatgga tgctctgacc cacgcatttg aagcttattc
ttcaacggca gctactccga 2400tcaccgatgc ttgcgccttg aaggctgcgt
ccatgatcgc taagaatctg aagaccgctt 2460gcgacaacgg taaggatatg
ccagctcgtg aagctatggc ttatgcccaa ttcctcgctg 2520gtatggcctt
caacaacgct tcgcttggtt atgtccatgc tatggctcac cagttgggcg
2580gctactacaa cctgccgcat ggtgtctgca acgctgttct gcttccgcat
gttctggctt 2640ataacgcctc tgtcgttgct ggtcgtctga aagacgttgg
tgttgctatg ggtctcgata 2700tcgccaatct cggtgataaa gaaggcgcag
aagccaccat tcaggctgtt cgcgatctgg 2760ctgcttccat tggtattcca
gcaaatctga ccgagctggg tgctaagaaa gaagatgtgc 2820cgcttcttgc
tgaccacgct ctgaaagatg cttgtgctct gaccaacccg cgtcagggtg
2880atcagaaaga agttgaagaa ctcttcctga gcgctttcta atttcaaaac
aggaaaacgg 2940ttttccgtcc tgtcttgatt ttcaagcaaa caatgcctcc
gatttctaat cggaggcatt 3000tgtttttgtt tattgcaaaa acaaaaaata
ttgttacaaa tttttacagg ctattaagcc 3060taccgtcata aataatttgc
catttgggga tcc 309374569PRTZymomonas mobilis 74Met Asn Ser Tyr Thr
Val Gly Thr Tyr Leu Ala Glu Arg Leu Val Gln 1 5 10 15 Ile Gly Leu
Lys His His Phe Ala Val Ala Gly Asp Tyr Asn Leu Val 20 25 30 Leu
Leu Asp Asn Leu Leu Leu Asn Lys Asn Met Glu Gln Val Tyr Cys 35 40
45 Cys Asn Glu Leu Asn Cys Gly Phe Ser Ala Glu Gly Tyr Ala Arg Ala
50 55 60 Lys Gly Ala Ala Ala Ala Val Val Thr Tyr Ser Val Gly Ala
Leu Ser 65 70 75 80 Ala Phe Asp Ala Ile Gly Gly Ala Tyr Ala Glu Asn
Leu Pro Val Ile 85 90 95 Leu Ile Ser Gly Ala Pro Asn Asn Asn Asp
His Ala Ala Gly His Val 100 105 110 Leu His His Ala Leu Gly Lys Thr
Asp Tyr His Tyr Gln Leu Glu Met 115 120 125 Ala Lys Asn Ile Thr Ala
Ala Ala Glu Ala Ile Tyr Thr Pro Glu Glu 130 135 140 Ala Pro Ala Lys
Ile Asp His Val Ile Lys Thr Ala Leu Arg Glu Lys 145 150 155 160 Lys
Pro Val Tyr Leu Glu Ile Ala Cys Asn Ile Ala Ser Met Pro Cys 165 170
175 Ala Ala Pro Gly Pro Ala Ser Ala Leu Phe Asn Asp Glu Ala Ser Asp
180 185 190 Glu Ala Ser Leu Asn Ala Ala Val Glu Glu Thr Leu Lys Phe
Ile Ala 195 200 205 Asn Arg Asp Lys Val Ala Val Leu Val Gly Ser Lys
Leu Arg Ala Ala 210 215 220 Gly Ala Glu Glu Ala Ala Val Lys Phe Ala
Asp Ala Leu Gly Gly Ala 225 230 235 240 Val Ala Thr Met Ala Ala Ala
Lys Ser Phe Phe Pro Glu Glu Asn Pro 245 250 255 His Tyr Ile Gly Thr
Ser Trp Gly Glu Val Ser Tyr Pro Gly Val Glu 260 265 270 Lys Thr Met
Lys Glu Ala Asp Ala Val Ile Ala Leu Ala Pro Val Phe 275 280 285 Asn
Asp Tyr Ser Thr Thr Gly Trp Thr Asp Ile Pro Asp Pro Lys Lys 290 295
300 Leu Val Leu Ala Glu Pro Arg Ser Val Val Val Asn Gly Val Arg Phe
305 310 315 320 Pro Ser Val His Leu Lys Asp Tyr Leu Thr Arg Leu Ala
Gln Lys Val 325 330 335 Ser Lys Lys Thr Gly Ala Leu Asp Phe Phe Lys
Ser Leu Asn Ala Gly 340 345 350 Glu Leu Lys Lys Ala Ala Pro Ala Asp
Pro Ser Ala Pro Leu Val Asn 355 360 365 Ala Glu Ile Ala Arg Gln Val
Glu Ala Leu Leu Thr Pro Asn Thr Thr 370 375 380 Val Ile Ala Glu Thr
Gly Asp Ser Trp Phe Asn Ala Gln Arg Met Lys 385 390 395 400 Leu Pro
Asn Gly Ala Arg Val Glu Tyr Glu Met Gln Trp Gly His Ile 405 410 415
Gly Trp Ser Val Pro Ala Ala Phe Gly Tyr Ala Val Gly Ala Pro Glu 420
425 430 Arg Arg Asn Ile Leu Met Val Gly Asp Gly Ser Phe Gln Leu Thr
Ala 435 440 445 Gln Glu Val Ala Gln Met Val Arg Leu Lys Leu Pro Val
Ile Ile Phe 450 455 460 Leu Ile Asn Asn Tyr Gly Tyr Thr Ile Glu Val
Met Ile His Asp Gly 465 470 475 480 Pro Tyr Asn Asn Ile Lys Asn Trp
Asp Tyr Ala Gly Leu Met Glu Val 485 490 495 Phe Asn Gly Asn Gly Gly
Tyr Asp Ser Gly Ala Gly Lys Gly Leu Lys 500 505 510 Ala Lys Thr Gly
Gly Glu Leu Ala Glu Ala Ile Lys Val Ala Leu Ala 515 520 525 Asn Thr
Asp Gly Pro Thr Leu Ile Glu Cys Phe Ile Gly Arg Glu Asp 530 535 540
Cys Thr Glu Glu Leu Val Lys Trp Gly Lys Arg Val Ala Ala Ala Asn 545
550 555 560 Ser Arg Lys Pro Val Asn Lys Leu Leu 565
75383PRTZymomonas mobilis 75Met Ala Ser Ser Thr Phe Tyr Ile Pro Phe
Val Asn Glu Met Gly Glu 1 5 10 15 Gly Ser Leu Glu Lys Ala Ile Lys
Asp Leu Asn Gly Ser Gly Phe Lys 20 25 30 Asn Ala Leu Ile Val Ser
Asp Ala Phe Met Asn Lys Ser Gly Val Val 35 40 45 Lys Gln Val Ala
Asp Leu Leu Lys Ala Gln Gly Ile Asn Ser Ala Val 50 55 60 Tyr Asp
Gly Val Met Pro Asn Pro Thr Val Thr Ala Val Leu Glu Gly 65 70 75 80
Leu Lys Ile Leu Lys Asp Asn Asn Ser Asp Phe Val Ile Ser Leu Gly 85
90 95 Gly Gly Ser Pro His Asp Cys Ala Lys Ala Ile Ala Leu Val Ala
Thr 100 105 110 Asn Gly Gly Glu Val Lys Asp Tyr Glu Gly Ile Asp Lys
Ser Lys Lys 115 120 125 Pro Ala Leu Pro Leu Met Ser Ile Asn Thr Thr
Ala Gly Thr Ala Ser 130 135 140 Glu Met Thr Arg Phe Cys Ile Ile Thr
Asp Glu Val Arg His Val Lys 145 150 155 160 Met Ala Ile Val Asp Arg
His Val Thr Pro Met Val Ser Val Asn Asp 165 170 175 Pro Leu Leu Met
Val Gly Met Pro Lys Gly Leu Thr Ala Ala Thr Gly 180 185 190 Met Asp
Ala Leu Thr His Ala Phe Glu Ala Tyr Ser Ser Thr Ala Ala 195 200 205
Thr Pro Ile Thr Asp Ala Cys Ala Leu Lys Ala Ala Ser Met Ile Ala 210
215 220 Lys Asn Leu Lys Thr Ala Cys Asp Asn Gly Lys Asp Met Pro Ala
Arg 225 230 235 240 Glu Ala Met Ala Tyr Ala Gln Phe Leu Ala Gly Met
Ala Phe Asn Asn 245 250 255 Ala Ser Leu Gly Tyr Val His Ala Met Ala
His Gln Leu Gly Gly Tyr 260 265 270 Tyr Asn Leu Pro His Gly Val Cys
Asn Ala Val Leu Leu Pro His Val 275 280 285 Leu Ala Tyr Asn Ala Ser
Val Val Ala Gly Arg Leu Lys Asp Val Gly 290 295 300 Val Ala Met Gly
Leu Asp Ile Ala Asn Leu Gly Asp Lys Glu Gly Ala 305 310 315 320 Glu
Ala Thr Ile Gln Ala Val Arg Asp Leu Ala Ala Ser Ile Gly Ile 325 330
335 Pro Ala Asn Leu Thr Glu Leu Gly Ala Lys Lys Glu Asp Val Pro Leu
340 345 350 Leu Ala Asp His Ala Leu Lys Asp Ala Cys Ala Leu Thr Asn
Pro Arg 355 360 365 Gln Gly Asp Gln Lys Glu Val Glu Glu Leu Phe Leu
Ser Ala Phe 370 375 380 76614DNASynechocystis sp. PCC6803
76gtcgaccttc cagcaccacg tcaactttgt ttaactgctc ccggagttgt ctttccgctt
60tggcaatgtg cccgggatac cattggatta aagccatgag ttgttcactt ttttactgac
120gagggcttcc ggaggccacg ctcccaccca taacagcttg ccacatcccc
gtcggaagtt 180acgttaccct tgggcgatcg ccaaaaatca gcatatatac
accaattcta aataagatct 240tttacaccgc tactgcaatc aacctcatca
acaaaattcc cctctagcat ccctggaggc 300aaatcctcac ctggccatgg
gttcaaccct gcttaacatt tcttaataat tttagttgct 360ataaattctc
atttatgccc ctataataat tcgggagtaa gtgctaaaga ttctcaactg
420ctccatcagt ggtttgagct tagtcctagg gaaagattgg cgatcgccgt
tgtggttaag 480ccagaatagg tctcgggtgg acagagaacg ctttattctt
tgcctccatg gcggcatccc 540acctaggttt ctcggcactt attgccataa
tttattattt gtcgtctcaa ttaaggaggc 600aattctgtga attc
61477614DNASynechocystis sp. PCC6803 77gtcgacttat ggttgattcg
cattgttttg ctcctgaaat tttcggcaaa tacaaatact 60tcgctcttct agccctatta
accattttaa cgacaaattg atggggcaac gattaacaaa 120taatgaataa
attttatgtt tttcaagatg aaaatttgaa aatttgattt ccttatattt
180ctactataga agactaatac aattagatct aaaatttgca agtataaaaa
tcagcaaata
240gttatattgt taataattca atgacccaat aactcgtact gttatctacg
tggtgaaagc 300caaaaagacg aacagtttag cctcctcctc ctcggcgatc
gccaagcgaa atgtcatggg 360agatgttcag attgagcatt tttttctaaa
agcccttgct aaaacaaacc acatgtgcag 420ggtgtccccg atgttgacta
aattcagcgg acttaaaacc tattttttcc ctgggttgct 480aggtttgccc
cccgttttgg gcaagcttgt ataagcagat actgttaatt gggtcaactt
540tttgttacat ttatttacaa ttgattgttt acaattgaaa ggtagtcgcc
ttggagggca 600acagctatga attc 61478614DNASynechocystis sp. PCC6803
78gtcgacaacg acggaggttt aagggaaaag tgaccatgga agctatatcg agattaatta
60aagggggggg atcaaccaag ttgctggact ggagcagtgg gcaagaaacc tgagctaatt
120ctaggcaatt ttgttgccat tgcccccaac gtaaagttat ttttttctcc
attacctttt 180gttacctaaa atttaatgga gccaacctag gctggacctt
ttgtcaccgt tgctctgtcc 240acgggatgga ggataatcta aacgaaatca
ttagggaccc tgaccatgca aaaagccgac 300gaatttgcca tccatctgtt
tttggccaat ggccatcgag aggaagtccg tttcatgacc 360attcaagatt
tccaaaaatg gtatagcaac gaagttgtgc ccaagcacga ttcccaggaa
420tttatcagtg tgcctatcag aaatattcag ggcgagtaca tggtggtgcg
accctcggcg 480atcgttgcca ttcgggtgga accaattttc tttggcagtg
tggagcgcat gtaatccgtt 540gtgtataatc ttgatacaga atggggtttt
gcacttccct tgatgccccc attaccgtga 600acgtgcatga attc
6147910502DNAartificialcloning vector pVZ321b 79gtcgacgaat
ttctgccatt catccgctta ttatcactta ttcaggcgta gcaccaggcg 60tttaagggca
ccaataactg ccttaaaaaa attacgcccc gccctgccac tcatcgcagt
120actgttgtaa ttcattaagc attctgccga catggaagcc atcacagacg
gcatgatgaa 180cctgaatcgc cagcggcatc agcaccttgt cgccttgcgt
ataatatttg cccatggtga 240aaacgggggc gaagaagttg tccatattgg
ccacgtttaa atcaaaactg gtgaaactca 300cccagggatt ggctgagacg
aaaaacatat tctcaataaa ccctttaggg aaataggcca 360ggttttcacc
gtaacacgcc acatcttgcg aatatatgtg tagaaactgc cggaaatcgt
420cgtggtattc actccagagc gatgaaaacg tttcagtttg ctcatggaaa
acggtgtaac 480aagggtgaac actatcccat atcaccagct caccgtcttt
cattgccata cggaattccg 540gatgagcatt catcaggcgg gcaagaatgt
gaataaaggc cggataaaac ttgtgcttat 600ttttctttac ggtctttaaa
aaggccgtaa tatccagctg aacggtctgg ttataggtac 660attgagcaac
tgactgaaat gcctcaaaat gttctttacg atgccattgg gatatatcaa
720cggtggtata tccagtgatt tttttctcca ttttagcttc cttagctcct
gaaaatctcg 780ataactcaaa aaatacgccc ggtagtgatc ttatttcatt
atggtgaaag ttggaacctc 840ttacgtgccg atcaacgtct cattttcgcc
aaaagttggc ccagggcttc ccggtatcaa 900cagggacacc aggatttatt
tattctgcga agtgatcttc cgtcacaggt atttattcga 960agacgaaagg
gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt
1020tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat
ttgtttattt 1080ttctaaatac attcaaatat gtatccgctc atgagacaat
aaccctgata aatgcttcaa 1140taatattgaa aaaggaagag tatgagtatt
caacatttcc gtgtcgccct tattcccttt 1200tttgcggcat tttgccttcc
tgtttttgct cacccagaaa cgctggtgaa agtaaaagat 1260gctgaagatc
agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag
1320atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt
taaagttctg 1380ctatgtggcg cggtattatc ccgtgttgac gccgggcaag
agcaactcgg tcgccgcata 1440cactattctc agaatgactt ggttgagtac
tcaccagtca cagaaaagca tcttacggat 1500ggcatgacag taagagaatt
atgcagtgct gccataacca tgagtgataa cactgcggcc 1560aacttacttc
tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg
1620ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc
cataccaaac 1680gacgagcgtg acaccacgat gcctgcagga gcagaagagc
atacatctgg aagcaaagcc 1740aggaaagcgg cctatggagc tgtgcggcag
cgctcagtag gcaatttttc aaaatattgt 1800taagcctttt ctgagcatgg
tatttttcat ggtattacca attagcagga aaataagcca 1860ttgaatataa
aagataaaaa tgtcttgttt acaatagagt ggggggggtc agcctgccgc
1920cttgggccgg gtgatgtcgt acttgcccgc cgcgaactcg gttaccgtcc
agcccagcgc 1980gaccagctcc ggcaacgcct cgcgcacccg ctggcggcgc
ttgcgcatgg tcgaaccact 2040ggcctctgac ggccagacat agccgcacaa
ggtatctatg gaagccttgc cggttttgcc 2100ggggtcgatc cagccacaca
gccgctggtg cagcaggcgg gcggtttcgc tgtccagcgc 2160ccgcacctcg
tccatgctga tgcgcacatg ctggccgcca cccatgacgg cctgcgcgat
2220caaggggttc agggccacgt acaggcgccc gtccgcctcg tcgctggcgt
actccgacag 2280cagccgaaac ccctgccgct tgcggccatt ctgggcgatg
atggatacct tccaaaggcg 2340ctcgatgcag tcctgtatgt gcttgagcgc
cccaccacta tcgacctctg ccccgatttc 2400ctttgccagc gcccgatagc
tacctttgac cacatggcat tcagcggtga cggcctccca 2460cttgggttcc
aggaacagcc ggagctgccg tccgccttcg gtcttgggtt ccgggccaag
2520cactaggcca ttaggcccag ccatggccac cagcccttgc aggatgcgca
gatcatcagc 2580gcccagcggc tccgggccgc tgaactcgat ccgcttgccg
tcgccgtagt catacgtcac 2640gtccagcttg ctgcgcttgc gctcgccccg
cttgagggca cggaacaggc cgggggccag 2700acagtgcgcc gggtcgtgcc
ggacgtggct gaggctgtgc ttgttcttag gcttcaccac 2760ggggcacccc
cttgctcttg cgctgcctct ccagcacggc gggcttgagc accccgccgt
2820catgccgcct gaaccaccga tcagcgaacg gtgcgccata gttggccttg
ctcacaccga 2880agcggacgaa gaaccggcgc tggtcgtcgt ccacacccca
ttcctcggcc tcggcgctgg 2940tcatgctcga caggtaggac tgccagcgga
tgttatcgac cagtaccgag ctgccccggc 3000tggcctgctg ctggtcgcct
gcgcccatca tggccgcgcc cttgctggca tggtgcagga 3060acacgataga
gcacccggta tcggcggcga tggcctccat gcgaccgatg acctgggcca
3120tggggccgct ggcgttttct tcctcgatgt ggaaccggcg cagcgtgtcc
agcaccatca 3180ggcggcggcc ctcggcggcg cgcttgaggc cgtcgaacca
ctccggggcc atgatgttgg 3240gcaggctgcc gatcagcggc tggatcagca
ggccgtcagc cacggcttgc cgttcctcgg 3300cgctgaggtg cgccccaagg
gcgtgcaggc ggtgatgaat ggcggtgggc gggtcttcgg 3360cgggcaggta
gatcaccggg ccggtgggca gttcgcccac ctccagcaga tccggcccgc
3420ctgcaatctg tgcggccagt tgcagggcca gcatggattt accggcacca
ccgggcgaca 3480ccagcgcccc gaccgtaccg gccaccatgt tgggcaaaac
gtagtccagc ggtggcggcg 3540ctgctgcgaa cgcctccaga atattgatag
gcttatgggt agccattgat tgcctccttt 3600gcaggcagtt ggtggttagg
cgctggcggg gtcactaccc ccgccctgcg ccgctctgag 3660ttcttccagg
cactcgcgca gcgcctcgta ttcgtcgtcg gtcagccaga acttgcgctg
3720acgcatccct ttggccttca tgcgctcggc atatcgcgct tggcgtacag
cgtcagggct 3780ggccagcagg tcgccggtct gcttgtcctt ttggtctttc
atatcagtca ccgagaaact 3840tgccggggcc gaaaggcttg tcttcgcgga
acaaggacaa ggtgcagccg tcaaggttaa 3900ggctggccat atcagcgact
gaaaagcggc cagcctcggc cttgtttgac gtataaccaa 3960agccaccggg
caaccaatag cccttgtcac ttttgatcag gtagaccgac cctgaagcgc
4020ttttttcgta ttccataaaa cccccttctg tgcgtgagta ctcatagtat
aacaggcgtg 4080agtaccaacg caagcactac atgctgaaat ctggcccgcc
cctgtccatg cctcgctggc 4140ggggtgccgg tgcccgtgcc agctcggccc
gcgcaagctg gacgctgggc agacccatga 4200ccttgctgac ggtgcgctcg
atgtaatccg cttcgtggcc gggcttgcgc tctgccagcg 4260ctgggctggc
ctcggccatg gccttgccga tttcctcggc actgcggccc cggctggcca
4320gcttctgcgc ggcgataaag tcgcacttgc tgaggtcatc accgaagcgc
ttgaccagcc 4380cggccatctc gctgcggtac tcgtccagcg ccgtgcgccg
gtggcggcta agctgccgct 4440cgggcagttc gaggctggcc agcctgcggg
ccttctcctg ctgccgctgg gcctgctcga 4500tctgctggcc agcctgctgc
accagcgccg ggccagcggt ggcggtcttg cccttggatt 4560cacgcagcag
cacccacggc tgataaccgg cgcgggtggt gtgcttgtcc ttgcggttgg
4620tgaagcccgc caagcggcca tagtggcggc tgtcggcgct ggccgggtcg
gcgtcgtact 4680cgctggccag cgtccgggca atctgccccc gaagttcacc
gcctgcggcg tcggccacct 4740tgacccatgc ctgatagttc ttcgggctgg
tttccactac cagggcaggc tcccggccct 4800cggctttcat gtcatccagg
tcaaactcgc tgaggtcgtc caccagcacc agaccatgcc 4860gctcctgctc
ggcgggcctg atatacacgt cattgccctg ggcattcatc cgcttgagcc
4920atggcgtgtt ctggagcact tcggcggctg accattcccg gttcatcatc
tggccggtgg 4980tggcgtccct gacgccgata tcgaagcgct cacagcccat
ggccttgagc tgtcggccta 5040tggcctgcaa agtcctgtcg ttcttcatcg
ggccaccaag cgcagccaga tcgagccgtc 5100ctcggttgtc agtggcgtca
ggtcgagcaa gagcaacgat gcgatcagca gcaccaccgt 5160aggcatcatg
gaagccagca tcacggttag ccatagcttc cagtgccacc cccgcgacgc
5220gctccgggcg ctctgcgcgg cgctgctcac ctcggcggct acctcccgca
actctttggc 5280cagctccacc catgccgccc ctgtctggcg ctgggctttc
agccactccg ccgcctgcgc 5340ctcgctggcc tgctgggtct ggctcatgac
ctgccgggct tcgtcggcca gtgtcgccat 5400gctctgggcc agcggttcga
tctgctccgc taactcgttg atgcctctgg atttcttcac 5460tctgtcgatt
gcgttcatgg tctattgcct cccggtattc ctgtaagtcg atgatctggg
5520cgttggcggt gtcgatgttc agggccacgt ctgcccggtc ggtgcggatg
ccccggcctt 5580ccatctccac cacgttcggc cccaggtgaa caccgggcag
gcgctcgatg ccctgcgcct 5640caagtgttct gtggtcaatg cgggcgtcgt
ggccagcccg ctctaatgcc cggttggcat 5700ggtcggccca tgcctcgcgg
gtctgctcaa gccatgcctt gggcttgagc gcttcggtct 5760tctgtgcccc
gcccttctcc ggggtcttgc cgttgtaccg cttgaaccac tgagcggcgg
5820gccgctcgat gccgtcattg atccgctcgg agatcatcag gtggcagtgc
gggttctcgc 5880cgccaccggc atggatggcc agcgtatacg gcaggcgctc
ggcaccggtc aggtgctggg 5940cgaactcgga cgccagcgcc ttctgctggt
cgagggtcag ctcgaccggc agggcaaatt 6000cgacctcctt gaacagccgc
ccattggcgc gttcatacag gtcggcagca tcccagtagt 6060cggcgggccg
ctcgacgaac tccggcatgt gcccggattc ggcgtgcaag acttcatcca
6120tgtcgcgggc atacttgcct tcgcgctgga tgtagtcggc cttggccctg
gccgattggc 6180cgcccgacct gctgccggtt ttcgccgtaa ggtgataaat
cgccatgctg cctcgctgtt 6240gcttttgctt ttcggctcca tgcaatggcc
ctcggagagc gcaccgcccg aagggtggcc 6300gttaggccag tttctcgaag
agaaaccggt aagtgcgccc tcccctacaa agtagggtcg 6360ggattgccgc
cgctgtgcct ccatgatagc ctacgagaca gcacattaac aatggggtgt
6420caagatggtt aaggggagca acaaggcggc ggatcggctg gccaagctcg
aagaacaacg 6480agcgcgaatc aatgccgaaa ttcagcgggt gcgggcaagg
gaacagcagc aagagcgcaa 6540gaacgaaaca aggcgcaagg tgctggtggg
ggccatgatt ttggccaagg tgaacagcag 6600cgagtggccg gaggatcggc
tcatggcggc aatggatgcg taccttgaac gcgaccacga 6660ccgcgccttg
ttcggtctgc cgccacgcca gaaggatgag ccgggctgaa tgatcgaccg
6720agacaggccc tgcggggctg cacacgcgcc cccacccttc gggtaggggg
aaaggccgct 6780aaagcggcta aaagcgctcc agcgtatttc tgcggggttt
ggtgtggggt ttagcgggct 6840ttgcccgcct ttccccctgc cgcgcagcgg
tggggcggtg tgtagcctag cgcagcgaat 6900agaccagcta tccggcctct
ggccgggcat attgggcaag ggcagcagcg ccccacaagg 6960gcgctgataa
ccgcgcctag tggattattc ttagataatc atggatggat ttttccaaca
7020ccccgccagc ccccgcccct gctgggtttg caggtttggg ggcgtgacag
ttattgcagg 7080ggttcgtgac agttattgca ggggggcgtg acagttattg
caggggttcg tgacagttag 7140tacgggagtg acgggcactg gctggcaatg
tctagcaacg gcaggcattt cggctgaggg 7200taaaagaact ttccgctaag
cgatagactg tatgtaaaca cagtattgca aggacgcgga 7260acatgcctca
tgtggcggcc aggacggcca gccgggatcg ggatactggt cgttaccaga
7320gccaccgacc cgagcaaacc cttctctatc agatcgttga cgagtattac
ccggcattcg 7380ctgcgcttat ggcagagcag ggaaaggaat tgccgggcta
tgtgcaacgg gaatttgaag 7440aatttctcca atgcgggcgg ctggagcatg
gctttctacg ggttcgctgc gagtcttgcc 7500acgccgagca cctggtcgct
ttcagctgta atccgggcag cgcaacggaa cattcatcag 7560tgtaaaaatg
gaatcaataa agccctgcgc agcgcgcagg gtcagcctga atacgcgttt
7620aatgaccagc acagtcgtga tggcaaggtc agaatagcgc tgaggtctgc
ctcgtgaaga 7680aggtgttgct gactcatacc aggcctgaat cgccccatca
tccagccaga aagtgaggga 7740gccacggttg atgagagctt tgttgtaggt
ggaccagttg gtgattttga acttttgctt 7800tgccacggaa cggtctgcgt
tgtcgggaag atgcgtgatc tgatccttca actcagcaaa 7860agttcgattt
attcaacaaa gccacgttgt gtctcaaaat ctctgatgtt acattgcaca
7920agataaaaat atatcatcat gaacaataaa actgtctgct tacataaaca
gtaatacaag 7980gggtgttatg agccatattc aacgggaaac gtcttgctcg
aggccgcgat taaattccaa 8040catggatgct gatttatatg ggtataaatg
ggctcgcgat aatgtcgggc aatcaggtgc 8100gacaatctat cgattgtatg
ggaagcccga tgcgccagag ttgtttctga aacatggcaa 8160aggtagcgtt
gccaatgatg ttacagatga gatggtcaga ctaaactggc tgacggaatt
8220tatgcctctt ccgaccatca agcattttat ccgtactcct gatgatgcat
ggttactcac 8280cactgcgatc cccgggaaaa cagcattcca ggtattagaa
gaatatcctg attcaggtga 8340aaatattgtt gatgcgctgg cagtgttcct
gcgccggttg cattcgattc ctgtttgtaa 8400ttgtcctttt aacagcgatc
gcgtatttcg tctcgctcag gcgcaatcac gaatgaataa 8460cggtttggtt
gatgcgagtg attttgatga cgagcgtaat ggctggcctg ttgaacaagt
8520ctggaaagaa atgcataagc ttttgccatt ctcaccggat tcagtcgtca
ctcatggtga 8580tttctcactt gataacctta tttttgacga ggggaaatta
ataggttgta ttgatgttgg 8640acgagtcgga atcgcagacc gataccagga
tcttgccatc ctatggaact gcctcggtga 8700gttttctcct tcattacaga
aacggctttt tcaaaaatat ggtattgata atcctgatat 8760gaataaattg
cagtttcatt tgatgctcga tgagtttttc taatcagaat tggttaattg
8820gttgtaacac tggcagagca ttacgctgac ttgacgggac ggcggctttg
ttgaataaat 8880cgaacttttg ctgagttgaa ggatcagatc acgcatcttc
ccgacaacgc agaccgttcc 8940gtggcaaagc aaaagttcaa aatcaccaac
tggtccacct acaacaaagc tctcatcaac 9000cgtggctccc tcactttctg
gctggatgat ggggcgattc aggcctggta tgagtcagca 9060acaccttctt
cacgaggcag acctcagcgc tattctgacc ttgccatcac gactgtgctg
9120gtcattaaac gcgtattcag gctgaccctg cgcgctgcgc agggctttat
tgattccatt 9180tttacactga tgaatgttcc gttgcgctgc ccggattaca
gatcctctag atctagaaga 9240acagcaaggc cgccaatgcc tgacgatgcg
tggagaccga aaccttgcgc tcgttcgcca 9300gccaggacag aaatgcctcg
acttcgctgc tgcccaaggt tgccgggtga cgcacaccgt 9360ggaaacggat
gaaggcacga acccagtgga cataagcctg ttcggttcgt aagctgtaat
9420gcaagtagcg tatgcgctca cgcaactggt ccagaacctt gaccgaacgc
agcggtggta 9480acggcgcagt ggcggttttc atggcttgtt atgactgttt
ttttggggta cagtctatgc 9540ctcgggcatc caagcagcaa gcgcgttacg
ccgtgggtcg atgtttgatg ttatggagca 9600gcaacgatgt tacgcagcag
ggcagtcgcc ctaaaacaaa gttaaacatc atgagggaag 9660cggtgatcgc
cgaagtatcg actcaactat cagaggtagt tggcgtcatc gagcgccatc
9720tcgaaccgac gttgctggcc gtacatttgt acggctccgc agtggatggc
ggcctgaagc 9780cacacagtga tattgatttg ctggttacgg tgaccgtaag
gcttgatgaa acaacgcggc 9840gagctttgat caacgacctt ttggaaactt
cggcttcccc tggagagagc gagattctcc 9900gcgctgtaga agtcaccatt
gttgtgcacg acgacatcat tccgtggcgt tatccagcta 9960agcgcgaact
gcaatttgga gaatggcagc gcaatgacat tcttgcaggt atcttcgagc
10020cagccacgat cgacattgat ctggctatct tgctgacaaa agcaagagaa
catagcgttg 10080ccttggtagg tccagcggcg gaggaactct ttgatccggt
tcctgaacag gatctatttg 10140aggcgctaaa tgaaacctta acgctatgga
actcgccgcc cgactgggct ggcgatgagc 10200gaaatgtagt gcttacgttg
tcccgcattt ggtacagcgc agtaaccggc aaaatcgcgc 10260cgaaggatgt
cgctgccgac tgggcaatgg agcgcctgcc ggcccagtat cagcccgtca
10320tacttgaagc tagacaggct tatcttggac aagaagaaga tcgcttggcc
tcgcgcgcag 10380atcagttgga agaatttgtc cactacgtga aaggcgagat
caccaaggta gtcggcaaat 10440aatgtctaac aattcgttca agccgacgcc
gcttcgcggc gcggcttaac tcaagctcta 10500ga 1050280294DNASynechocystis
sp. PCC 6803 80gtcgacggga attgctctgg caactgatta atccactgag
caacagccca agacacgcaa 60acaaaaacca acgtcttggc gatcgccatc ggcaccatga
aaccatcgta aaagctgggg 120aaagaataaa aaacagtggt tcaggaattg
cattgccatg gccacttcac aaacctagcc 180aattttagct tgaccgcaac
tttgacagat tgtcttttga ctttgcctgg accgcctccc 240ataatacctt
cgcgtcttga agactttatc cttgaaagga gaactaatga attc
29481608DNASynechocystis sp. PCC 6803 81gtcgactgtc cgaccaattg
gttcatcaaa gttgatttac ccacattggg acggccgaca 60atggccacaa agccggaacg
aaaacccgca ggagcctggg gaatagttgc aatggttgcg 120gtggtgttgg
gaatatccat tgaaaaaatc aagcctaaaa attccttagt ttatggaggg
180tcaagcggaa aaacgttaaa aactccactg agttaatcaa ccagaggaaa
aagtcaagga 240ggtaaactat ccgcctggaa aacggcttgc cagcttgaca
aaaaaatatg ttgggttaac 300cccactgtgc cattcggtaa tccttcatct
tggcccttgt ggaatccctt aatgattcgt 360catcatggtg atattgattt
tttgggtatc tttttagcta tgcggctgta ggagcgtggt 420attggtttcg
gcggtaacgc cccagcctag aaccacaaaa attattattt attcccgaac
480cttgtcacca tttgcggcgt ctaaaggccc actcgttagg acacggtgta
aaaaaaattg 540acgactgcac taccctattc tccaccatca atgacttagt
ctaagacatt tttgggaaag 600atgaattc 60882581DNASynechocystis sp. PCC
6803 82gtcgacgctg atgtgacggt taagggaggc ggaattaaac tgggtaagga
cgtaaatttt 60aacgatttct gagttgatgc aattactgac gggaatatcg atgaggcgat
actttccggc 120caagggaact gcgggtttgg ctctgagttt ggttaaagga
tagaggcggg tcccggcccc 180accgcccagg ataatcgcta agacacgttt
cacaagcaga cctctcgatt gccaacaaca 240cacttcgaag tcaagtttag
aaccgagggg gacatctgga aagggaatct ggacggaaat 300tccggctaac
cagcgggttt taatgcccca agcaagaatg gcgatcgccg ttgggattcg
360gagctgagtt gtcagatcac tgtgggggta cggataaccg aaatggcaaa
ggtcggaaac 420tgccgctgag taaactgtcc ctggcttcgt atgatgatgg
ggttaccccc attgctgggg 480cgctgggcaa atctggggag ctgactaggt
tcctggaagt tttgctaatc cactaaattt 540cctaacaatc ctaaacatta
aatctaaaga cctatgaatt c 58183590DNASynechocystis sp. PCC 6803
83gtcgacccaa caacattagt ccgtcctccc gttggcgatc gcgctgtttg gctctgaccc
60atcgccgctg atattgccaa gcttggcagt agggcacaag tccgaacgat aataaacgac
120aggaaggatt ggccatggcg ctacaaagga aacggaatca aactaaagtt
caaagttggc 180agaaattaag aaacgtaaag agatgcaaag gaaagtcaaa
atcacctgac cgattaggtc 240ttattcaata catagtgcta atctgaagat
agtcttagga gttaattatt taccaccaca 300attttctgga aaactttacc
tctaccctag ggatgattaa aagtaaacta gagaataaca 360aggttgggtt
tataattcat caccaagctc aaatttatgg tgttttttca atgatccatg
420cttttgatat ctttagcaga aaggcatttt aagtaatgat tccacctcac
tgtttctcgg 480aaaaattgcc caatctaact tagtttttat aacttaagtt
tagatctgcg gaaaaccaac 540cattgctcat tttttattaa ttttacgaag
ggagaattta gtatgaattc 59084572DNASynechocystis sp. PCC 6803
84gtcgacagaa tccttgccca gatgcaggcc ttctggcgat cgccatggtg agcaacgatt
60gcggctttag cgttccagtg gatatttgct gggggttaat gaaacattgt ggcggaaccc
120agggacaatg tgaccaaaaa attcagggat atcaataagt attaggtata
tggatcataa 180ttgtatgccc gactattgct taaactgact gaccactgac
cttaagagta atggcgtgca 240aggcccagtg atcaatttca ttatttttca
ttatttcatc tccattgtcc ctgaaaatca 300gttgtgtcgc ccctctacac
agcccagaac tatggtaaag gcgcacgaaa aaccgccagg 360taaactcttc
tcaaccccca aaacgccctc tgtttaccca tggaaaaaac gacaattaca
420agaaagtaaa acttatgtca tctataagct tcgtgtatat taacttcctg
ttacaaagct 480ttacaaaact ctcattaatc ctttagacta agtttagtca
gttccaatct gaacatcgac 540aaatacataa ggaattataa ccaaatgaat tc
57285273DNASynechocystis sp. PCC 6803 85gtcgacatca ggaattgtaa
ttagaaagtc caaaaattgt aatttaaaaa acagtcaatg 60gagagcattg ccataagtaa
aggcatcccc tgcgtgataa gattaccttc agaaaacaga 120tagttgctgg
gttatcgcag atttttctcg caaccaaata actgtaaata ataactgtct
180ctggggcgac ggtaggcttt atattgccaa atttcgcccg tgggagaaag
ctaggctatt 240caatgtttat ggaggactga cctagatgaa ttc
27386408DNASynechocystis sp. PCC6803 86gtcgacattt cttaaaatta
aagctgttat agcaacaaca ttgattaatt tctatctaat 60ttttgacggt
gcccattgct
atcagttgta agttgatgaa aatgctgtaa atttttgtaa 120caaagttcaa
ctttgtcttg acttttgtaa gtctttgcaa aatctaggag ctagaactgg
180tcagggctgg ggcaattttt aattattgtt acgcaggtct tgcctagggg
gggggaggcc 240gtattatctt ctagtgatgt ttgctgaaaa cgcctatctg
tgcaaggttt aacatcgtta 300ttatgaagcg aaaactaatt ccctttttta
cgcttcctct attacactat tctgcatagg 360aaacccttaa tagttcattg
tcgagcgagg agaaccctgc atgaattc 40887614DNASynechocystis sp. PCC6803
87gtcgacaaaa aaactgcaaa aattatcctg actgaatgga agtcaaaaag actggaaaat
60gggatcaaac aacaagaaaa aatcaattta ccctgcccat ggcaatagtt ttaaggttaa
120caaaaaaaat agaatttacc gcaatcgacg ggtaaatttc cagaggatac
ccccaactcc 180agaagcagaa atcttgccag aaaagctttt tctgttacta
tacttaacaa gtaactactt 240tttccatagt ccaggggcgg ctttccaaaa
accagagatt ggtggcttgc cgctgctgtt 300ctcctctgga gtaaggggaa
aaggtaatta gtgttacggc attttactga cgggttaagt 360aatctttaac
aaagatttat gagccgttac cgtaattgcc cccacagggg aacgcgatgt
420ctgtggactc gcccaggacg taatcaattt ttctgtaccg atattagcgg
tgaaaagttt 480tattcaacgt actaaaatgc cccggcggga attaacttgg
gttccgggaa gtcgggtgca 540ttagccgtac tagactaacc caatagttac
tttgtttgat tcttgatttt ggagaccgct 600gattttatga attc
61488367DNASynechocystis sp. PCC6803 88gtcgacggaa aacaagctca
gaatgctgcg gggagaaggg caactcccca ccagccccaa 60atttttgctg gcgataaata
tttttcggtt taattgttca caaagctttt tgaatttgag 120tttatagaaa
tttattggct ggtaatgctt ttttgccccc ctgcaggact tcattgatcc
180ttgcctatac catcaatatc attggtcaat aatgatgatg attgactaaa
acatgtttaa 240caaaatttaa cgcatatgct aaatgcgtaa actgcatatg
ccttggctga gtgtaattta 300cgttacaaat tttaacgaaa cgggaaccct
atattgatct ctatctggct tgaagcgttg 360tgaattc 36789359DNAAnabaena sp.
PCC7120 89gtcgactttt ttgctgaggt actgagtaca cagctaataa aattgggcaa
tctccgcgcc 60tctatgactt gaaggagagt gtaggggtat aggggaaaga tatcttttat
ctacatcaca 120taaataaaaa atttaatttg tcgctctggc tgcatatatt
gatgtatttt tagccataag 180ttttttagtg ccatgtaatt atagtgattt
ttagcgatcg cagagcattt ttccctggat 240ttatcgcgat ctcaaaaaaa
atttgcccga agtatgacag attgtcatat ttggtgtcga 300ttttatttaa
aatgaaataa gaaaaataaa actacaggtt aggagaacgc catgaattc
35990273DNASynechocystis sp. PCC6803 90gtcgacacaa cctaagactt
ccttccaaaa atccataggg cggtggaagc ttagctattt 60ttaccatttt gttttgccac
tcaaatattt acttaaggtg aggtaaaaac tcatcttttt 120tttactaaaa
attgcggcta gaaatgtaat ttcggcaatc cccccacctt ctttcctgaa
180aaccgaatct aacctggaag gggaaatttt aagatagaac cattcaaggg
taatcaattc 240cttccacaca tcaggagtta acattatgaa ttc
27391342DNASynechocystis sp. PCC6803 91gtcgacgcac ttctggtcag
tttatagcaa aaatgctggg gaaaggaaga caactaggga 60aaaagaacag gacatcaaat
ggtcattccc cagaccctgg cgtctttgcc agagtaatct 120ccctggcgcg
gatgttacac aaatgtaacg aaaaatattt tccctctcag aatttaggca
180aagtgcccaa acccatccta ggcaagcaat tcgtccacca acaaaaagct
cttttggtca 240acagacttga caaaaatctt aacaatacgt tacatttatt
tacataaggt tacaaaataa 300aaacctcaaa tacccaatca aggagatcaa
cactatgaat tc 34292273DNASynechocystis sp. PCC6803 92gtcgacaaga
ttagccctta gcttacaaga aaggggcttt ggggcctagt tgaatggcac 60aaattttcct
tccctgactg tttttgcgcc attgtctagc tcaaagtcag cctccggcat
120cctctagaaa gacttccatc ccctggttga gcaagggtaa accccaccac
tgcattggga 180aaaccctcct tcctagctcc ggattccacc ccctaaaatt
gatttggtag tccttacaca 240cccaatagcc aatatagaaa attttatgaa ttc
273931216DNAZymomonas mobilis ZM4 93gagctctata tcaacaaaag
gtagtcacca tgtcagccgc agatttgtcg actgacctct 60atctctccga gatatatcaa
caaaaggtag tcaccatgaa agcagccgtc ataactaaag 120atcatacgat
cgaagtgaaa gacaccaaat tacgccctct gaaatacggg gaagcgcttt
180tggaaatgga atattgcggg gtatgtcata ccgatctcca cgtgaaaaac
ggggattttg 240gcgatgaaac cggcagaatt accggccatg aaggcatcgg
tatcgtcaag caggtcgggg 300aaggggttac ttctctgaaa gtcggtgacc
gtgccagtgt tgcatggttc ttcaaaggct 360gcggccattg cgaatattgt
gtcagtggaa atgaaacgct ttgccgcaac gttgaaaatg 420ccggttatac
ggttgacggc gctatggcag aagaatgcat cgtcgttgcc gattactcgg
480tcaaagtgcc agatggtctt gatcctgcgg ttgccagcag catcacttgc
gcgggtgtaa 540ccacctataa agcagtcaaa gtttctcaga tacagccggg
acaatggctg gctatctatg 600gcttgggcgg tttaggcaat ctagcccttc
aatatgccaa gaatgttttc aacgccaaag 660tgatcgcgat cgatgtcaat
gatgaacagc tcgcttttgc caaagagctg ggcgcagata 720tggtcatcaa
tccgaaaaac gaagatgctg ccaaaatcat tcaggaaaaa gtcggcggcg
780cacatgcgac ggtggtgaca gctgttgcca aatccgcctt taactcggct
gttgaggcta 840tccgcgcggg tggccgtgtt gtcgccgttg gtctgcctcc
tgaaaaaatg gatttgagca 900ttcctcgctt ggtgcttgac ggtatcgaag
tcttaggttc tttggtcgga acgcgggaag 960atttgaaaga agccttccag
tttgcagccg aaggtaaggt caaaccgaaa gtcaccaagc 1020gtaaagtcga
agaaatcaac caaatctttg acgaaatgga acatggtaaa ttcacaggcc
1080gtatggttgt tgattttacc catcactagg tttccgtgaa ggcggaagca
taaacggaaa 1140aagcctttct cttaccagaa aggctttttc tttgtcgtct
gataaaaatt ttcatacaga 1200atttaataca ctgcag 121694337PRTZymomonas
mobilis 94Met Lys Ala Ala Val Ile Thr Lys Asp His Thr Ile Glu Val
Lys Asp 1 5 10 15 Thr Lys Leu Arg Pro Leu Lys Tyr Gly Glu Ala Leu
Leu Glu Met Glu 20 25 30 Tyr Cys Gly Val Cys His Thr Asp Leu His
Val Lys Asn Gly Asp Phe 35 40 45 Gly Asp Glu Thr Gly Arg Ile Thr
Gly His Glu Gly Ile Gly Ile Val 50 55 60 Lys Gln Val Gly Glu Gly
Val Thr Ser Leu Lys Val Gly Asp Arg Ala 65 70 75 80 Ser Val Ala Trp
Phe Phe Lys Gly Cys Gly His Cys Glu Tyr Cys Val 85 90 95 Ser Gly
Asn Glu Thr Leu Cys Arg Asn Val Glu Asn Ala Gly Tyr Thr 100 105 110
Val Asp Gly Ala Met Ala Glu Glu Cys Ile Val Val Ala Asp Tyr Ser 115
120 125 Val Lys Val Pro Asp Gly Leu Asp Pro Ala Val Ala Ser Ser Ile
Thr 130 135 140 Cys Ala Gly Val Thr Thr Tyr Lys Ala Val Lys Val Ser
Gln Ile Gln 145 150 155 160 Pro Gly Gln Trp Leu Ala Ile Tyr Gly Leu
Gly Gly Leu Gly Asn Leu 165 170 175 Ala Leu Gln Tyr Ala Lys Asn Val
Phe Asn Ala Lys Val Ile Ala Ile 180 185 190 Asp Val Asn Asp Glu Gln
Leu Ala Phe Ala Lys Glu Leu Gly Ala Asp 195 200 205 Met Val Ile Asn
Pro Lys Asn Glu Asp Ala Ala Lys Ile Ile Gln Glu 210 215 220 Lys Val
Gly Gly Ala His Ala Thr Val Val Thr Ala Val Ala Lys Ser 225 230 235
240 Ala Phe Asn Ser Ala Val Glu Ala Ile Arg Ala Gly Gly Arg Val Val
245 250 255 Ala Val Gly Leu Pro Pro Glu Lys Met Asp Leu Ser Ile Pro
Arg Leu 260 265 270 Val Leu Asp Gly Ile Glu Val Leu Gly Ser Leu Val
Gly Thr Arg Glu 275 280 285 Asp Leu Lys Glu Ala Phe Gln Phe Ala Ala
Glu Gly Lys Val Lys Pro 290 295 300 Lys Val Thr Lys Arg Lys Val Glu
Glu Ile Asn Gln Ile Phe Asp Glu 305 310 315 320 Met Glu His Gly Lys
Phe Thr Gly Arg Met Val Val Asp Phe Thr His 325 330 335 His
951144DNASynechocystis sp. PCC 6803 95gagctctctg gataaaacta
ataaactcta ttacccatga ttaaagccta cgctgccctg 60gaagccaacg gaaaactcca
accctttgaa tacgaccccg gtgccctggg tgctaatgag 120gtggagattg
aggtgcagta ttgtggggtg tgccacagtg atttgtccat gattaataac
180gaatggggca tttccaatta ccccctagtg ccgggtcatg aggtggtggg
tactgtggcc 240gccatgggcg aaggggtgaa ccatgttgag gtgggggatt
tagtggggct gggttggcat 300tcgggctact gcatgacctg ccatagttgt
ttatctggct accacaacct ttgtgccacg 360gcggaatcga ccattgtggg
ccactacggt ggctttggcg atcgggttcg ggccaaggga 420gtcagcgtgg
tgaaattacc taaaggcatt gacctagcca gtgccgggcc ccttttctgt
480ggaggaatta ccgttttcag tcctatggtg gaactgagtt taaagcccac
tgcaaaagtg 540gcagtgatcg gcattggggg cttgggccat ttagcggtgc
aatttctccg ggcctggggc 600tgtgaagtga ctgcctttac ctccagtgcc
aggaagcaaa cggaagtgtt ggaattgggc 660gctcaccaca tactagattc
caccaatcca gaggcgatcg ccagtgcgga aggcaaattt 720gactatatta
tctccactgt gaacctgaag cttgactgga acttatacat cagcaccctg
780gcgccccagg gacatttcca ctttgttggg gtggtgttgg agcctttgga
tctaaatctt 840tttccccttt tgatgggaca acgctccgtt tctgcctccc
cagtgggtag tcccgccacc 900attgccacca tgttggactt tgctgtgcgc
catgacatta aacccgtggt ggaacaattt 960agctttgatc agatcaacga
ggcgatcgcc catctagaaa gcggcaaagc ccattatcgg 1020gtagtgctca
gccatagtaa aaattagctc tgcaaaggtt gcttctgggt ccgtggaatg
1080gtcaaacgga gtcgatctca gttttgatac gctctatctg gaaagcttga
cattcgatct 1140gcag 114496336PRTSynechocystis sp. PCC 6803 96Met
Ile Lys Ala Tyr Ala Ala Leu Glu Ala Asn Gly Lys Leu Gln Pro 1 5 10
15 Phe Glu Tyr Asp Pro Gly Ala Leu Gly Ala Asn Glu Val Glu Ile Glu
20 25 30 Val Gln Tyr Cys Gly Val Cys His Ser Asp Leu Ser Met Ile
Asn Asn 35 40 45 Glu Trp Gly Ile Ser Asn Tyr Pro Leu Val Pro Gly
His Glu Val Val 50 55 60 Gly Thr Val Ala Ala Met Gly Glu Gly Val
Asn His Val Glu Val Gly 65 70 75 80 Asp Leu Val Gly Leu Gly Trp His
Ser Gly Tyr Cys Met Thr Cys His 85 90 95 Ser Cys Leu Ser Gly Tyr
His Asn Leu Cys Ala Thr Ala Glu Ser Thr 100 105 110 Ile Val Gly His
Tyr Gly Gly Phe Gly Asp Arg Val Arg Ala Lys Gly 115 120 125 Val Ser
Val Val Lys Leu Pro Lys Gly Ile Asp Leu Ala Ser Ala Gly 130 135 140
Pro Leu Phe Cys Gly Gly Ile Thr Val Phe Ser Pro Met Val Glu Leu 145
150 155 160 Ser Leu Lys Pro Thr Ala Lys Val Ala Val Ile Gly Ile Gly
Gly Leu 165 170 175 Gly His Leu Ala Val Gln Phe Leu Arg Ala Trp Gly
Cys Glu Val Thr 180 185 190 Ala Phe Thr Ser Ser Ala Arg Lys Gln Thr
Glu Val Leu Glu Leu Gly 195 200 205 Ala His His Ile Leu Asp Ser Thr
Asn Pro Glu Ala Ile Ala Ser Ala 210 215 220 Glu Gly Lys Phe Asp Tyr
Ile Ile Ser Thr Val Asn Leu Lys Leu Asp 225 230 235 240 Trp Asn Leu
Tyr Ile Ser Thr Leu Ala Pro Gln Gly His Phe His Phe 245 250 255 Val
Gly Val Val Leu Glu Pro Leu Asp Leu Asn Leu Phe Pro Leu Leu 260 265
270 Met Gly Gln Arg Ser Val Ser Ala Ser Pro Val Gly Ser Pro Ala Thr
275 280 285 Ile Ala Thr Met Leu Asp Phe Ala Val Arg His Asp Ile Lys
Pro Val 290 295 300 Val Glu Gln Phe Ser Phe Asp Gln Ile Asn Glu Ala
Ile Ala His Leu 305 310 315 320 Glu Ser Gly Lys Ala His Tyr Arg Val
Val Leu Ser His Ser Lys Asn 325 330 335 972781DNAE. coli K12
97atgaattctg ctgttactaa tgtcgctgaa cttaacgcac tcgtagagcg tgtaaaaaaa
60gcccagcgtg aatatgccag tttcactcaa gagcaagtag acaaaatctt ccgcgccgcc
120gctctggctg ctgcagatgc tcgaatccca ctcgcgaaaa tggccgttgc
cgaatccggc 180atgggtatcg tcgaagataa agtgatcaaa aaccactttg
cttctgaata tatctacaac 240gcctataaag atgaaaaaac ctgtggtgtt
ctgtctgaag acgacacttt tggtaccatc 300actatcgctg aaccaatcgg
tattatttgc ggtatcgttc cgaccactaa cccgacttca 360actgctatct
tcaaatcgct gatcagtctg aagacccgta acgccattat cttctccccg
420cacccgcgtg caaaagatgc caccaacaaa gcggctgata tcgttctgca
ggctgctatc 480gctgccggtg ctccgaaaga tctgatcggc tggatcgatc
aaccttctgt tgaactgtct 540aacgcactga tgcaccaccc agacatcaac
ctgatcctcg cgactggtgg tccgggcatg 600gttaaagccg catacagctc
cggtaaacca gctatcggtg taggcgcggg caacactcca 660gttgttatcg
atgaaactgc tgatatcaaa cgtgcagttg catctgtact gatgtccaaa
720accttcgaca acggcgtaat ctgtgcttct gaacagtctg ttgttgttgt
tgactctgtt 780tatgacgctg tacgtgaacg ttttgcaacc cacggcggct
atctgttgca gggtaaagag 840ctgaaagctg ttcaggatgt tatcctgaaa
aacggtgcgc tgaacgcggc tatcgttggt 900cagccagcct ataaaattgc
tgaactggca ggcttctctg taccagaaaa caccaagatt 960ctgatcggtg
aagtgaccgt tgttgatgaa agcgaaccgt tcgcacatga aaaactgtcc
1020ccgactctgg caatgtaccg cgctaaagat ttcgaagacg cggtagaaaa
agcagagaaa 1080ctggttgcta tgggcggtat cggtcatacc tcttgcctgt
acactgacca ggataaccaa 1140ccggctcgcg tttcttactt cggtcagaaa
atgaaaacgg cgcgtatcct gattaacacc 1200ccagcgtctc agggtggtat
cggtgacctg tataacttca aactcgcacc ttccctgact 1260ctgggttgtg
gttcttgggg tggtaactcc atctctgaaa acgttggtcc gaaacacctg
1320atcaacaaga aaaccgttgc taagcgagct gaaaacatgt tgtggcacaa
acttccgaaa 1380tctatctact tccgccgtgg ctccctgcca atcgcgctgg
atgaagtgat tactgatggc 1440cacaaacgtg cgctcatcgt gactgaccgc
ttcctgttca acaatggtta tgctgatcag 1500atcacttccg tactgaaagc
agcaggcgtt gaaactgaag tcttcttcga agtagaagcg 1560gacccgaccc
tgagcatcgt tcgtaaaggt gcagaactgg caaactcctt caaaccagac
1620gtgattatcg cgctgggtgg tggttccccg atggacgccg cgaagatcat
gtgggttatg 1680tacgaacatc cggaaactca cttcgaagag ctggcgctgc
gctttatgga tatccgtaaa 1740cgtatctaca agttcccgaa aatgggcgtg
aaagcgaaaa tgatcgctgt caccaccact 1800tctggtacag gttctgaagt
cactccgttt gcggttgtaa ctgacgacgc tactggtcag 1860aaatatccgc
tggcagacta tgcgctgact ccggatatgg cgattgtcga cgccaacctg
1920gttatggaca tgccgaagtc cctgtgtgct ttcggtggtc tggacgcagt
aactcacgcc 1980atggaagctt atgtttctgt actggcatct gagttctctg
atggtcaggc tctgcaggca 2040ctgaaactgc tgaaagaata tctgccagcg
tcctaccacg aagggtctaa aaatccggta 2100gcgcgtgaac gtgttcacag
tgcagcgact atcgcgggta tcgcgtttgc gaacgccttc 2160ctgggtgtat
gtcactcaat ggcgcacaaa ctgggttccc agttccatat tccgcacggt
2220ctggcaaacg ccctgctgat ttgtaacgtt attcgctaca atgcgaacga
caacccgacc 2280aagcagactg cattcagcca gtatgaccgt ccgcaggctc
gccgtcgtta tgctgaaatt 2340gccgaccact tgggtctgag cgcaccgggc
gaccgtactg ctgctaagat cgagaaactg 2400ctggcatggc tggaaacgct
gaaagctgaa ctgggtattc cgaaatctat ccgtgaagct 2460ggcgttcagg
aagcagactt cctggcgaac gtggataaac tgtctgaaga tgcattcgat
2520gaccagtgca ccggcgctaa cccgcgttac ccgctgatct ccgagctgaa
acagattctg 2580ctggatacct actacggtcg tgattatgta gaaggtgaaa
ctgcagcgaa gaaagaagct 2640gctccggcta aagctgagaa aaaagcgaaa
aaatccgctt aatcagtagc gctgtctggc 2700aacataaacg gccccttctg
ggcaatgccg atcagttaag gattagttga ccgatcctta 2760aactgaggca
ctataggatc c 278198893PRTE. coli K12 98Met Asn Ser Ala Val Thr Asn
Val Ala Glu Leu Asn Ala Leu Val Glu 1 5 10 15 Arg Val Lys Lys Ala
Gln Arg Glu Tyr Ala Ser Phe Thr Gln Glu Gln 20 25 30 Val Asp Lys
Ile Phe Arg Ala Ala Ala Leu Ala Ala Ala Asp Ala Arg 35 40 45 Ile
Pro Leu Ala Lys Met Ala Val Ala Glu Ser Gly Met Gly Ile Val 50 55
60 Glu Asp Lys Val Ile Lys Asn His Phe Ala Ser Glu Tyr Ile Tyr Asn
65 70 75 80 Ala Tyr Lys Asp Glu Lys Thr Cys Gly Val Leu Ser Glu Asp
Asp Thr 85 90 95 Phe Gly Thr Ile Thr Ile Ala Glu Pro Ile Gly Ile
Ile Cys Gly Ile 100 105 110 Val Pro Thr Thr Asn Pro Thr Ser Thr Ala
Ile Phe Lys Ser Leu Ile 115 120 125 Ser Leu Lys Thr Arg Asn Ala Ile
Ile Phe Ser Pro His Pro Arg Ala 130 135 140 Lys Asp Ala Thr Asn Lys
Ala Ala Asp Ile Val Leu Gln Ala Ala Ile 145 150 155 160 Ala Ala Gly
Ala Pro Lys Asp Leu Ile Gly Trp Ile Asp Gln Pro Ser 165 170 175 Val
Glu Leu Ser Asn Ala Leu Met His His Pro Asp Ile Asn Leu Ile 180 185
190 Leu Ala Thr Gly Gly Pro Gly Met Val Lys Ala Ala Tyr Ser Ser Gly
195 200 205 Lys Pro Ala Ile Gly Val Gly Ala Gly Asn Thr Pro Val Val
Ile Asp 210 215 220 Glu Thr Ala Asp Ile Lys Arg Ala Val Ala Ser Val
Leu Met Ser Lys 225 230 235 240 Thr Phe Asp Asn Gly Val Ile Cys Ala
Ser Glu Gln Ser Val Val Val 245 250 255 Val Asp Ser Val Tyr Asp Ala
Val Arg Glu Arg Phe Ala Thr His Gly 260 265 270 Gly Tyr Leu Leu Gln
Gly Lys Glu Leu Lys Ala Val Gln Asp Val Ile 275 280 285 Leu Lys Asn
Gly Ala Leu Asn Ala Ala Ile Val Gly Gln Pro Ala Tyr 290 295 300 Lys
Ile Ala Glu Leu Ala Gly Phe Ser Val Pro Glu Asn Thr Lys Ile 305 310
315 320 Leu Ile Gly Glu Val Thr Val Val Asp Glu Ser Glu Pro Phe Ala
His 325 330 335 Glu Lys Leu Ser Pro Thr Leu Ala Met Tyr Arg Ala Lys
Asp Phe Glu 340 345
350 Asp Ala Val Glu Lys Ala Glu Lys Leu Val Ala Met Gly Gly Ile Gly
355 360 365 His Thr Ser Cys Leu Tyr Thr Asp Gln Asp Asn Gln Pro Ala
Arg Val 370 375 380 Ser Tyr Phe Gly Gln Lys Met Lys Thr Ala Arg Ile
Leu Ile Asn Thr 385 390 395 400 Pro Ala Ser Gln Gly Gly Ile Gly Asp
Leu Tyr Asn Phe Lys Leu Ala 405 410 415 Pro Ser Leu Thr Leu Gly Cys
Gly Ser Trp Gly Gly Asn Ser Ile Ser 420 425 430 Glu Asn Val Gly Pro
Lys His Leu Ile Asn Lys Lys Thr Val Ala Lys 435 440 445 Arg Ala Glu
Asn Met Leu Trp His Lys Leu Pro Lys Ser Ile Tyr Phe 450 455 460 Arg
Arg Gly Ser Leu Pro Ile Ala Leu Asp Glu Val Ile Thr Asp Gly 465 470
475 480 His Lys Arg Ala Leu Ile Val Thr Asp Arg Phe Leu Phe Asn Asn
Gly 485 490 495 Tyr Ala Asp Gln Ile Thr Ser Val Leu Lys Ala Ala Gly
Val Glu Thr 500 505 510 Glu Val Phe Phe Glu Val Glu Ala Asp Pro Thr
Leu Ser Ile Val Arg 515 520 525 Lys Gly Ala Glu Leu Ala Asn Ser Phe
Lys Pro Asp Val Ile Ile Ala 530 535 540 Leu Gly Gly Gly Ser Pro Met
Asp Ala Ala Lys Ile Met Trp Val Met 545 550 555 560 Tyr Glu His Pro
Glu Thr His Phe Glu Glu Leu Ala Leu Arg Phe Met 565 570 575 Asp Ile
Arg Lys Arg Ile Tyr Lys Phe Pro Lys Met Gly Val Lys Ala 580 585 590
Lys Met Ile Ala Val Thr Thr Thr Ser Gly Thr Gly Ser Glu Val Thr 595
600 605 Pro Phe Ala Val Val Thr Asp Asp Ala Thr Gly Gln Lys Tyr Pro
Leu 610 615 620 Ala Asp Tyr Ala Leu Thr Pro Asp Met Ala Ile Val Asp
Ala Asn Leu 625 630 635 640 Val Met Asp Met Pro Lys Ser Leu Cys Ala
Phe Gly Gly Leu Asp Ala 645 650 655 Val Thr His Ala Met Glu Ala Tyr
Val Ser Val Leu Ala Ser Glu Phe 660 665 670 Ser Asp Gly Gln Ala Leu
Gln Ala Leu Lys Leu Leu Lys Glu Tyr Leu 675 680 685 Pro Ala Ser Tyr
His Glu Gly Ser Lys Asn Pro Val Ala Arg Glu Arg 690 695 700 Val His
Ser Ala Ala Thr Ile Ala Gly Ile Ala Phe Ala Asn Ala Phe 705 710 715
720 Leu Gly Val Cys His Ser Met Ala His Lys Leu Gly Ser Gln Phe His
725 730 735 Ile Pro His Gly Leu Ala Asn Ala Leu Leu Ile Cys Asn Val
Ile Arg 740 745 750 Tyr Asn Ala Asn Asp Asn Pro Thr Lys Gln Thr Ala
Phe Ser Gln Tyr 755 760 765 Asp Arg Pro Gln Ala Arg Arg Arg Tyr Ala
Glu Ile Ala Asp His Leu 770 775 780 Gly Leu Ser Ala Pro Gly Asp Arg
Thr Ala Ala Lys Ile Glu Lys Leu 785 790 795 800 Leu Ala Trp Leu Glu
Thr Leu Lys Ala Glu Leu Gly Ile Pro Lys Ser 805 810 815 Ile Arg Glu
Ala Gly Val Gln Glu Ala Asp Phe Leu Ala Asn Val Asp 820 825 830 Lys
Leu Ser Glu Asp Ala Phe Asp Asp Gln Cys Thr Gly Ala Asn Pro 835 840
845 Arg Tyr Pro Leu Ile Ser Glu Leu Lys Gln Ile Leu Leu Asp Thr Tyr
850 855 860 Tyr Gly Arg Asp Tyr Val Glu Gly Glu Thr Ala Ala Lys Lys
Glu Ala 865 870 875 880 Ala Pro Ala Lys Ala Glu Lys Lys Ala Lys Lys
Ser Ala 885 890 992747DNAThermosynechococcus elongatus BP-1
99atgaattccc caaccttgac cagtgacccc cccgttcaaa gccttgccga tctggaaggg
60ctgattgagc gcgtccaacg ggcgcagagt cagtacgccc aatttaccca agagcaagtg
120gatcacattt tccacgaagc agccatggcg gccaaccaag cccggattcc
cctggccaaa 180caagccgtag ccgaaacggg catgggggtt gtcgaagata
aagttattaa aaatcacttt 240gcttcggaat acatctacaa caagtacaaa
aatgagaaaa cctgcggcgt cattgaggat 300gaccccatct ttggtatcca
aaaaattgct gaaccggtgg ggatcattgc cggtgtggtg 360ccggtcacga
accccacttc aacgaccatc tttaaggcac tgattgccct gaagactcgc
420aatggcatta tcttttcgcc ccacccccgg gcaaaggcct gtacggttgc
agcggccaag 480gtagtgttgg atgcagcggt cgctgccggc gcaccccccg
atattattgg ctggattgat 540gagccgacga ttgaactctc ccaagccctg
atgcagcacc cgcagatcaa gctgattttg 600gccacggggg gaccaggtat
ggtcaaggca gcctattcct ctggccatcc ggcgatcggg 660gtcggggccg
ggaatacccc cgtgctcatt gatgccacag ccgatattcc cacggcagtg
720agttcgattc tcctcagtaa ggcctttgac aatggcatga tctgtgcctc
ggagcaggca 780gtgattgttg tggatgagat ttatgacgca cttaaagctg
agtttcaacg gcgaggggcc 840taccttctct cccctgagga acggcagcag
gtggcacaac tactgctgaa ggatggtcgc 900ctcaatgccg ccattgttgg
tcaatcggcc gccaccattg ccgcaatggc caatatccaa 960gtaccgccag
aaacccgggt actcattggc gaggtgagtg aagtggggcc gcaggagcca
1020ttttcctatg agaaactctg tccggtattg gcgttatatc gggcacccca
gttccataaa 1080ggggtggaga ttgcggccca gttggtgaat tttgggggca
aggggcatac atctgtgctc 1140tataccgatc cccgcaatca agatgatatt
gcctatttca aataccgcat gcaaacggcg 1200cgggttctga ttaacacccc
ttcttcccag ggggcaattg gcgatctcta caacttcaag 1260ttagatccgt
cgctaaccct tggttgtggt acgtggggcg gcaacgtcac atcggaaaat
1320gttggtcccc gtcacttgct gaatattaaa acggtgagcg atcgccggga
aaatatgctt 1380tggtttcggg tgccgcccaa gatctacttc aaacccggct
gtttgcccat tgccctgcgg 1440gagctggcgg ggaaaaaacg cgccttcctc
gtgacggata aacccctctt tgacttgggg 1500atcactgaac cgattgtcca
taccctcgaa gaactgggca tcaagtatga catcttccat 1560gaagtggaac
cagatccaac cctcagtacc gttaaccgcg gtctagggtt gctgcggcaa
1620tatcagccgg atgtgattgt tgctgtgggg ggtggctcac ctatggatgc
agccaaggtg 1680atgtggctgt tgtatgagca tccggaggtg gagtttgacg
gccttgcgat gcgcttcatg 1740gatattcgca agcgggtgta tcaactgcct
cccttgggtc aaaaggcaat cctggtggct 1800attcccacca cctcggggac
gggttcagag gtgaccccct ttgccgtggt taccgacgat 1860cgcgtgggga
ttaaatatcc cttggcagac tatgccctta cgccaacgat ggcgattgtg
1920gatcccgact tggtgctgca catgcccaag aaactgacgg cctacggtgg
cattgatgcg 1980ctgacccatg ccctggaggc ctatgtgtcg gtgctctcga
cggagtttac ggagggactg 2040gctctagagg ccattaaact gctctttacc
tacctacccc gtgcctatcg cttgggggcg 2100gcggatccgg aggcacggga
gaaggtccac tatgcggcga cgatcgctgg catggccttt 2160gcgaatgcct
tcttgggggt ctgccactcg ctggcccaca aactaggctc caccttccac
2220gtgccccacg gcttggcgaa tgcactcatg atttcccatg tgattcgcta
caatgccacg 2280gatgctcccc tgaagcaggc gattttcccg cagtacaagt
atccccaagc gaaggagcgc 2340tatgcccaaa ttgccgactt cctcgaattg
gggggcacga ccccagagga aaaagtggag 2400cgtctcattg cggcaattga
ggatttgaaa gcccaattag aaattcccgc cacgattaag 2460gaggccctca
acagtgagga tcaagcgttc tatgagcagg tggagagcat ggccgaactg
2520gcctttgacg atcagtgcac gggggccaat ccccgctatc cgctgatcca
agacctcaag 2580gagttgtata tcctggccta tatggggtgt cggcgggatg
cggcagccta ctatgggggg 2640gaggcaacgg ggagttgatg tggcgttata
ttcccccctt tgcagctcca gcgaaggtgc 2700aaatggcggt ggattcctgg
ctctggcagc ggagcgatcg cctgcag 2747100885PRTThermosynechococcus
elongatus BP-1 100Met Asn Ser Pro Thr Leu Thr Ser Asp Pro Pro Val
Gln Ser Leu Ala 1 5 10 15 Asp Leu Glu Gly Leu Ile Glu Arg Val Gln
Arg Ala Gln Ser Gln Tyr 20 25 30 Ala Gln Phe Thr Gln Glu Gln Val
Asp His Ile Phe His Glu Ala Ala 35 40 45 Met Ala Ala Asn Gln Ala
Arg Ile Pro Leu Ala Lys Gln Ala Val Ala 50 55 60 Glu Thr Gly Met
Gly Val Val Glu Asp Lys Val Ile Lys Asn His Phe 65 70 75 80 Ala Ser
Glu Tyr Ile Tyr Asn Lys Tyr Lys Asn Glu Lys Thr Cys Gly 85 90 95
Val Ile Glu Asp Asp Pro Ile Phe Gly Ile Gln Lys Ile Ala Glu Pro 100
105 110 Val Gly Ile Ile Ala Gly Val Val Pro Val Thr Asn Pro Thr Ser
Thr 115 120 125 Thr Ile Phe Lys Ala Leu Ile Ala Leu Lys Thr Arg Asn
Gly Ile Ile 130 135 140 Phe Ser Pro His Pro Arg Ala Lys Ala Cys Thr
Val Ala Ala Ala Lys 145 150 155 160 Val Val Leu Asp Ala Ala Val Ala
Ala Gly Ala Pro Pro Asp Ile Ile 165 170 175 Gly Trp Ile Asp Glu Pro
Thr Ile Glu Leu Ser Gln Ala Leu Met Gln 180 185 190 His Pro Gln Ile
Lys Leu Ile Leu Ala Thr Gly Gly Pro Gly Met Val 195 200 205 Lys Ala
Ala Tyr Ser Ser Gly His Pro Ala Ile Gly Val Gly Ala Gly 210 215 220
Asn Thr Pro Val Leu Ile Asp Ala Thr Ala Asp Ile Pro Thr Ala Val 225
230 235 240 Ser Ser Ile Leu Leu Ser Lys Ala Phe Asp Asn Gly Met Ile
Cys Ala 245 250 255 Ser Glu Gln Ala Val Ile Val Val Asp Glu Ile Tyr
Asp Ala Leu Lys 260 265 270 Ala Glu Phe Gln Arg Arg Gly Ala Tyr Leu
Leu Ser Pro Glu Glu Arg 275 280 285 Gln Gln Val Ala Gln Leu Leu Leu
Lys Asp Gly Arg Leu Asn Ala Ala 290 295 300 Ile Val Gly Gln Ser Ala
Ala Thr Ile Ala Ala Met Ala Asn Ile Gln 305 310 315 320 Val Pro Pro
Glu Thr Arg Val Leu Ile Gly Glu Val Ser Glu Val Gly 325 330 335 Pro
Gln Glu Pro Phe Ser Tyr Glu Lys Leu Cys Pro Val Leu Ala Leu 340 345
350 Tyr Arg Ala Pro Gln Phe His Lys Gly Val Glu Ile Ala Ala Gln Leu
355 360 365 Val Asn Phe Gly Gly Lys Gly His Thr Ser Val Leu Tyr Thr
Asp Pro 370 375 380 Arg Asn Gln Asp Asp Ile Ala Tyr Phe Lys Tyr Arg
Met Gln Thr Ala 385 390 395 400 Arg Val Leu Ile Asn Thr Pro Ser Ser
Gln Gly Ala Ile Gly Asp Leu 405 410 415 Tyr Asn Phe Lys Leu Asp Pro
Ser Leu Thr Leu Gly Cys Gly Thr Trp 420 425 430 Gly Gly Asn Val Thr
Ser Glu Asn Val Gly Pro Arg His Leu Leu Asn 435 440 445 Ile Lys Thr
Val Ser Asp Arg Arg Glu Asn Met Leu Trp Phe Arg Val 450 455 460 Pro
Pro Lys Ile Tyr Phe Lys Pro Gly Cys Leu Pro Ile Ala Leu Arg 465 470
475 480 Glu Leu Ala Gly Lys Lys Arg Ala Phe Leu Val Thr Asp Lys Pro
Leu 485 490 495 Phe Asp Leu Gly Ile Thr Glu Pro Ile Val His Thr Leu
Glu Glu Leu 500 505 510 Gly Ile Lys Tyr Asp Ile Phe His Glu Val Glu
Pro Asp Pro Thr Leu 515 520 525 Ser Thr Val Asn Arg Gly Leu Gly Leu
Leu Arg Gln Tyr Gln Pro Asp 530 535 540 Val Ile Val Ala Val Gly Gly
Gly Ser Pro Met Asp Ala Ala Lys Val 545 550 555 560 Met Trp Leu Leu
Tyr Glu His Pro Glu Val Glu Phe Asp Gly Leu Ala 565 570 575 Met Arg
Phe Met Asp Ile Arg Lys Arg Val Tyr Gln Leu Pro Pro Leu 580 585 590
Gly Gln Lys Ala Ile Leu Val Ala Ile Pro Thr Thr Ser Gly Thr Gly 595
600 605 Ser Glu Val Thr Pro Phe Ala Val Val Thr Asp Asp Arg Val Gly
Ile 610 615 620 Lys Tyr Pro Leu Ala Asp Tyr Ala Leu Thr Pro Thr Met
Ala Ile Val 625 630 635 640 Asp Pro Asp Leu Val Leu His Met Pro Lys
Lys Leu Thr Ala Tyr Gly 645 650 655 Gly Ile Asp Ala Leu Thr His Ala
Leu Glu Ala Tyr Val Ser Val Leu 660 665 670 Ser Thr Glu Phe Thr Glu
Gly Leu Ala Leu Glu Ala Ile Lys Leu Leu 675 680 685 Phe Thr Tyr Leu
Pro Arg Ala Tyr Arg Leu Gly Ala Ala Asp Pro Glu 690 695 700 Ala Arg
Glu Lys Val His Tyr Ala Ala Thr Ile Ala Gly Met Ala Phe 705 710 715
720 Ala Asn Ala Phe Leu Gly Val Cys His Ser Leu Ala His Lys Leu Gly
725 730 735 Ser Thr Phe His Val Pro His Gly Leu Ala Asn Ala Leu Met
Ile Ser 740 745 750 His Val Ile Arg Tyr Asn Ala Thr Asp Ala Pro Leu
Lys Gln Ala Ile 755 760 765 Phe Pro Gln Tyr Lys Tyr Pro Gln Ala Lys
Glu Arg Tyr Ala Gln Ile 770 775 780 Ala Asp Phe Leu Glu Leu Gly Gly
Thr Thr Pro Glu Glu Lys Val Glu 785 790 795 800 Arg Leu Ile Ala Ala
Ile Glu Asp Leu Lys Ala Gln Leu Glu Ile Pro 805 810 815 Ala Thr Ile
Lys Glu Ala Leu Asn Ser Glu Asp Gln Ala Phe Tyr Glu 820 825 830 Gln
Val Glu Ser Met Ala Glu Leu Ala Phe Asp Asp Gln Cys Thr Gly 835 840
845 Ala Asn Pro Arg Tyr Pro Leu Ile Gln Asp Leu Lys Glu Leu Tyr Ile
850 855 860 Leu Ala Tyr Met Gly Cys Arg Arg Asp Ala Ala Ala Tyr Tyr
Gly Gly 865 870 875 880 Glu Ala Thr Gly Ser 885
1011680DNAZymobacter palmae 101atgaattccg ttggtatgta cttggcagaa
cgcctagccc agatcggcct gaaacaccac 60tttgccgtgg ccggtgacta caacctggtg
ttgcttgatc agctcctgct gaacaaagac 120atggagcagg tctactgctg
taacgaactt aactgcggct ttagcgccga aggttacgct 180cgtgcacgtg
gtgccgccgc tgccatcgtc acgttcagcg taggtgctat ctctgcaatg
240aacgccatcg gtggcgccta tgcagaaaac ctgccggtca tcctgatctc
tggctcaccg 300aacaccaatg actacggcac aggccacatc ctgcaccaca
ccattggtac tactgactat 360aactatcagc tggaaatggt aaaacacgtt
acctgcgcac gtgaaagcat cgtttctgcc 420gaagaagcac cggcaaaaat
cgaccacgtc atccgtacgg ctctacgtga acgcaaaccg 480gcttatctgg
aaatcgcatg caacgtcgct ggcgctgaat gtgttcgtcc gggcccgatc
540aatagcctgc tgcgtgaact cgaagttgac cagaccagtg tcactgccgc
tgtagatgcc 600gccgtagaat ggctgcagga ccgccagaac gtcgtcatgc
tggtcggtag caaactgcgt 660gccgctgccg ctgaaaaaca ggctgttgcc
ctagcggacc gcctgggctg cgctgtcacg 720atcatggctg ccgaaaaagg
cttcttcccg gaagatcatc cgaacttccg cggcctgtac 780tggggtgaag
tcagctccga aggtgcacag gaactggttg aaaacgccga tgccatcctg
840tgtctggcac cggtattcaa cgactatgct accgttggct ggaactcctg
gccgaaaggc 900gacaatgtca tggtcatgga caccgaccgc gtcactttcg
caggacagtc cttcgaaggt 960ctgtcattga gcaccttcgc cgcagcactg
gctgagaaag caccttctcg cccggcaacg 1020actcaaggca ctcaagcacc
ggtactgggt attgaggccg cagagcccaa tgcaccgctg 1080accaatgacg
aaatgacgcg tcagatccag tcgctgatca cttccgacac tactctgaca
1140gcagaaacag gtgactcttg gttcaacgct tctcgcatgc cgattcctgg
cggtgctcgt 1200gtcgaactgg aaatgcaatg gggtcatatc ggttggtccg
taccttctgc attcggtaac 1260gccgttggtt ctccggagcg tcgccacatc
atgatggtcg gtgatggctc tttccagctg 1320actgctcaag aagttgctca
gatgatccgc tatgaaatcc cggtcatcat cttcctgatc 1380aacaaccgcg
gttacgtcat cgaaatcgct atccatgacg gcccttacaa ctacatcaaa
1440aactggaact acgctggcct gatcgacgtc ttcaatgacg aagatggtca
tggcctgggt 1500ctgaaagctt ctactggtgc agaactagaa ggcgctatca
agaaagcact cgacaatcgt 1560cgcggtccga cgctgatcga atgtaacatc
gctcaggacg actgcactga aaccctgatt 1620gcttggggta aacgtgtagc
agctaccaac tctcgcaaac cacaagcgta agttgagctc 1680102556PRTZymobacter
palmae 102Met Asn Ser Val Gly Met Tyr Leu Ala Glu Arg Leu Ala Gln
Ile Gly 1 5 10 15 Leu Lys His His Phe Ala Val Ala Gly Asp Tyr Asn
Leu Val Leu Leu 20 25 30 Asp Gln Leu Leu Leu Asn Lys Asp Met Glu
Gln Val Tyr Cys Cys Asn 35 40 45 Glu Leu Asn Cys Gly Phe Ser Ala
Glu Gly Tyr Ala Arg Ala Arg Gly 50 55 60 Ala Ala Ala Ala Ile Val
Thr Phe Ser Val Gly Ala Ile Ser Ala Met 65 70 75 80 Asn Ala Ile Gly
Gly Ala Tyr Ala Glu Asn Leu Pro Val Ile Leu Ile 85 90 95 Ser Gly
Ser Pro Asn Thr Asn Asp Tyr Gly Thr Gly His Ile Leu His 100 105 110
His Thr Ile Gly Thr Thr Asp Tyr Asn Tyr Gln Leu Glu Met Val Lys 115
120 125 His Val Thr Cys Ala Arg Glu Ser Ile Val Ser Ala Glu Glu Ala
Pro 130 135 140 Ala Lys Ile Asp His Val Ile Arg Thr Ala Leu Arg Glu
Arg Lys Pro 145 150 155 160 Ala Tyr Leu Glu Ile Ala Cys Asn Val Ala
Gly Ala Glu Cys Val Arg 165 170
175 Pro Gly Pro Ile Asn Ser Leu Leu Arg Glu Leu Glu Val Asp Gln Thr
180 185 190 Ser Val Thr Ala Ala Val Asp Ala Ala Val Glu Trp Leu Gln
Asp Arg 195 200 205 Gln Asn Val Val Met Leu Val Gly Ser Lys Leu Arg
Ala Ala Ala Ala 210 215 220 Glu Lys Gln Ala Val Ala Leu Ala Asp Arg
Leu Gly Cys Ala Val Thr 225 230 235 240 Ile Met Ala Ala Glu Lys Gly
Phe Phe Pro Glu Asp His Pro Asn Phe 245 250 255 Arg Gly Leu Tyr Trp
Gly Glu Val Ser Ser Glu Gly Ala Gln Glu Leu 260 265 270 Val Glu Asn
Ala Asp Ala Ile Leu Cys Leu Ala Pro Val Phe Asn Asp 275 280 285 Tyr
Ala Thr Val Gly Trp Asn Ser Trp Pro Lys Gly Asp Asn Val Met 290 295
300 Val Met Asp Thr Asp Arg Val Thr Phe Ala Gly Gln Ser Phe Glu Gly
305 310 315 320 Leu Ser Leu Ser Thr Phe Ala Ala Ala Leu Ala Glu Lys
Ala Pro Ser 325 330 335 Arg Pro Ala Thr Thr Gln Gly Thr Gln Ala Pro
Val Leu Gly Ile Glu 340 345 350 Ala Ala Glu Pro Asn Ala Pro Leu Thr
Asn Asp Glu Met Thr Arg Gln 355 360 365 Ile Gln Ser Leu Ile Thr Ser
Asp Thr Thr Leu Thr Ala Glu Thr Gly 370 375 380 Asp Ser Trp Phe Asn
Ala Ser Arg Met Pro Ile Pro Gly Gly Ala Arg 385 390 395 400 Val Glu
Leu Glu Met Gln Trp Gly His Ile Gly Trp Ser Val Pro Ser 405 410 415
Ala Phe Gly Asn Ala Val Gly Ser Pro Glu Arg Arg His Ile Met Met 420
425 430 Val Gly Asp Gly Ser Phe Gln Leu Thr Ala Gln Glu Val Ala Gln
Met 435 440 445 Ile Arg Tyr Glu Ile Pro Val Ile Ile Phe Leu Ile Asn
Asn Arg Gly 450 455 460 Tyr Val Ile Glu Ile Ala Ile His Asp Gly Pro
Tyr Asn Tyr Ile Lys 465 470 475 480 Asn Trp Asn Tyr Ala Gly Leu Ile
Asp Val Phe Asn Asp Glu Asp Gly 485 490 495 His Gly Leu Gly Leu Lys
Ala Ser Thr Gly Ala Glu Leu Glu Gly Ala 500 505 510 Ile Lys Lys Ala
Leu Asp Asn Arg Arg Gly Pro Thr Leu Ile Glu Cys 515 520 525 Asn Ile
Ala Gln Asp Asp Cys Thr Glu Thr Leu Ile Ala Trp Gly Lys 530 535 540
Arg Val Ala Ala Thr Asn Ser Arg Lys Pro Gln Ala 545 550 555
1035711DNAartificialpSK10 cloning vector (derivate of pSK9 [V. V.
Zinchenko, Moscow, Russia; unpublished]) 103gtcgacatat gtttctcggc
aaaaattaat tatcgattgg ctggaacctg gtcaaaccag 60ggcttttcat ccattggaaa
agcgattttg atcatctagg gtcaggagca aagatctgat 120caaatattga
tcatttatta ggaaagctga actttcacca ctttattttt ggcttcctct
180actttgggca aagtcaaagt taggataccg gcatcgtaat tagctttaac
ttctgtgttt 240tggattgctc caggtacagg aataacccgg cggaaactgc
catagcggaa ctctgtgcgc 300cgcaccccat ctttttcggt gctatgggta
tcctggcgat cgccgctgac ggtcaccgca 360tccctggcgg cttggatgtc
caaattatcg gggtccatgc caggtaattc tagtttgagc 420acataggctt
cttcagtttc agttagttct gctttaggat taaacccttg gcgatcgccg
480tggcggtccg tagggacaaa aacttcttca aacagttggt tcatctgctg
ctggaaatta 540tccatttccc gcaggggatt gtaaagaatg agagacataa
tgttaactcc tgatgtgtgg 600aaggaattga ttacccttga atggttctat
cttaaaattt ccccttccag gttagattcg 660gttttcagga aagaaggtgg
ggggattgcc gaaattacat ttctagccgc aatttttagt 720aaaaaaaaga
tgagttttta cctcacctta agtaaatatt tgagtggcaa aacaaaatgg
780taaaaatagc taagcttcca ccgccctatg gatttttgga aggaagtctt
aggttgtgaa 840aaactataaa aaccaaccat aggaatggag acctttaccc
aacaagttga cccctaggta 900acaaatccaa accaccgtaa aaccgctggc
ggccaaaata gcgggcttgc ggccttgcca 960acctttggta atgcgggcat
ggagataggc ggcaaatact agccaggtga ttagggcccg 1020gtacccagct
tttgttccct ttagtgaggg ttaatttcga gcttggcgta atcatggtca
1080tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
acgagccgga 1140agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
aactcacatt aattgcgttg 1200cgctcactgc ccgctttcca gtcgggaaac
ctgtcgtgcc agctgcatta atgaatcggc 1260caacgcgcgg ggagaggcgg
tttgcgtatt gggcgctctt ccgcttcctc gctcactgac 1320tcgctgcgct
cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
1380cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
aggccagcaa 1440aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
tccataggct ccgcccccct 1500gacgagcatc acaaaaatcg acgctcaagt
cagaggtggc gaaacccgac aggactataa 1560agataccagg cgtttccccc
tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg 1620cttaccggat
acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
1680cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
tgtgcacgaa 1740ccccccgttc agcccgaccg ctgcgcctta tccggtaact
atcgtcttga gtccaacccg 1800gtaagacacg acttatcgcc actggcagca
gccactggta acaggattag cagagcgagg 1860tatgtaggcg gtgctacaga
gttcttgaag tggtggccta actacggcta cactagaagg 1920acagtatttg
gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
1980tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
caagcagcag 2040attacgcgca gaaaaaaagg atctcaagaa gatcctttga
tcttttctac ggggtctgac 2100gctcagtgga acgaaaactc acgttaaggg
attttggtca tgagattatc aaaaaggatc 2160ttcacctaga tccttttaaa
ttaaaaatga agttttaaat caatctaaag tatatatgag 2220taaacttggt
ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
2280ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
gatacgggag 2340ggcttaccat ctggccccag tgctgcaatg ataccgcgag
acccacgctc accggctcca 2400gatttatcag caataaacca gccagccgga
agggccgagc gcagaagtgg tcctgcaact 2460ttatccgcct ccatccagtc
tattaattgt tgccgggaag ctagagtaag tagttcgcca 2520gttaatagtt
tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
2580tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
atgatccccc 2640atgttgtgca aaaaagcggt tagctccttc ggtcctccga
tcgttgtcag aagtaagttg 2700gccgcagtgt tatcactcat ggttatggca
gcactgcata attctcttac tgtcatgcca 2760tccgtaagat gcttttctgt
gactggtgag tactcaacca agtcattctg agaatagtgt 2820atgcggcgac
cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
2880agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
ctcaaggatc 2940ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
cacccaactg atcttcagca 3000tcttttactt tcaccagcgt ttctgggtga
gcaaaaacag gaaggcaaaa tgccgcaaaa 3060aagggaataa gggcgacacg
gaaatgttga atactcatac tcttcctttt tcaatattat 3120tgaagcattt
atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
3180aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctaa
attgtaagcg 3240ttaatatttt gttaaaattc gcgttaaatt tttgttaaat
cagctcattt tttaaccaat 3300aggccgaaat cggcaaaatc ccttataaat
caaaagaata gaccgagata gggttgagtg 3360ttgttccagt ttggaacaag
agtccactat taaagaacgt ggactccaac gtcaaagggc 3420gaaaaaccgt
ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt
3480tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc
cgatttagag 3540cttgacgggg aaagccggcg aacgtggcga gaaaggaagg
gaagaaagcg aaaggagcgg 3600gcgctagggc gctggcaagt gtagcggtca
cgctgcgcgt aaccaccaca cccgccgcgc 3660ttaatgcgcc gctacagggc
gcgtcccatt cgccattcag gctgcgcaac tgttgggaag 3720ggcgatcggt
gcgggcctct tcgctattac gccagctggc gaaaggggga tgtgctgcaa
3780ggcgattaag ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa
acgacggcca 3840gtgaattgta atacgactca ctatagggcg aattggaggc
cagtgctgga ggaatatgat 3900tttgtcatcc tcgactgtgc ccctggttat
aatctgttga cccgcagtgg cattgcggcc 3960agcgactttt atctgttgcc
ggctcgtcct gaacccctat cggtggtggg gatgcagtta 4020ctggaaagaa
gaattgagaa actgaaggaa agccataagg cctccgatga tcccctgaat
4080atcaatctga tcggagtggt gtttattctg tccggcggcg gtttgatgag
tcgctactat 4140aaccaggtaa tgcggcgggt acaaacggat ttcaccccgg
gacaactttt tcagcagtcc 4200attcccatgg atgtcaatgt ggctaaggca
gtggatagct ttatgccggt ggttacctcc 4260atgcccaata cggcgggttc
aaaagctttt attaaattaa cccaggaatt tttacagaaa 4320gtagaagctt
ttggctaaag caaagccccc attgattaac aacgggaggg gtaccgaggt
4380gctgctgaag ttgcccgcaa cagagagtgg aaccaaccgg tagtgcatct
aacgcttgag 4440ttaagccgcg ccgcgaagcg gcgtcggctt gaacgaattg
ttagacatta tttgccgact 4500accttggtga tctcgccttt cacgtagtgg
acaaattctt ccaactgatc tgcgcgcgag 4560gccaagcgat cttcttcttg
tccaagataa gcctgtctag cttcaagtat gacgggctga 4620tactgggccg
gcaggcgctc cattgcccag tcggcagcga catccttcgg cgcgattttg
4680ccggttactg cgctgtacca aatgcgggac aacgtaagca ctacatttcg
ctcatcgcca 4740gcccagtcgg gcggcgagtt ccatagcgtt aaggtttcat
ttagcgcctc aaatagatcc 4800tgttcaggaa ccggatcaaa gagttcctcc
gccgctggac ctaccaaggc aacgctatgt 4860tctcttgctt ttgtcagcaa
gatagccaga tcaatgtcga tcgtggctgg ctcgaagata 4920cctgcaagaa
tgtcattgcg ctgccattct ccaaattgca gttcgcgctt agctggataa
4980cgccacggaa tgatgtcgtc gtgcacaaca atggtgactt ctacagcgcg
gagaatctcg 5040ctctctccag gggaagccga agtttccaaa aggtcgttga
tcaaagctcg ccgcgttgtt 5100tcatcaagcc ttacggtcac cgtaaccagc
aaatcaatat cactgtgtgg cttcaggccg 5160ccatccactg cggagccgta
caaatgtacg gccagcaacg tcggttcgag atggcgctcg 5220atgacgccaa
ctacctctga tagttgagtc gatacttcgg cgatcaccgc ttccctcatg
5280atgtttaact ttgttttagg gcgactgccc tgctgcgtaa catcgttgct
gctccataac 5340atcaaacatc gacccacggc gtaacgcgct tgctgcttgg
atgcccgagg catagactgt 5400accccaaaaa aacagtcata acaagccatg
aaaaccgcca ctgcgccgtt accaccgctg 5460cgttcggtca aggttctgga
ccagttgcgt gagcgcatac gctacttgca ttacagctta 5520cgaaccgaac
aggcttatgt ccactgggtt cgtgccttca tccgtttcca cggtgtgcgt
5580cacccggcaa ccttgggcag cagcgaagtc gaggcatttc tgtcctggct
ggcgaacgag 5640cgcaaggttt cggtctccac gcatcgtcag gcattggcgg
ccttgctgtt cttctagaca 5700aggctgcagt t 5711104397DNAAnabaena sp.
PCC7120 104gtcgactcta gaaagatgcc actagcacca gacgactagt tagcgatagt
ctatccacca 60ttgttcgttt tgtaggtttt gcttttatag cgatcggttt tgtattttgc
ggtaacttca 120tcaatttttt aggggctggt aatttttaac atatctcacg
gggtgcaatc ttcgcgcccc 180tactagtcca tcgaatcgtc atttccaact
attaatatta aagtttagag aaattggatt 240atatgtaacc tgtactctgt
taagattcac cattggggta ttcgctatca gtcttggcgc 300tactgcccat
cccgcccctc aaacctttgt ccgtccgcct aagactgata ccgctactgg
360tgacaggccg atgttatatc tggagttcta tgaattc 397105359DNAAnabaena
sp. PCC7120 105gtcgactttt ttgctgaggt actgagtaca cagctaataa
aattgggcaa tctccgcgcc 60tctatgactt gaaggagagt gtaggggtat aggggaaaga
tatcttttat ctacatcaca 120taaataaaaa atttaatttg tcgctctggc
tgcatatatt gatgtatttt tagccataag 180ttttttagtg ccatgtaatt
atagtgattt ttagcgatcg cagagcattt ttccctggat 240ttatcgcgat
ctcaaaaaaa atttgcccga agtatgacag attgtcatat ttggtgtcga
300ttttatttaa aatgaaataa gaaaaataaa actacaggtt aggagaacgc catgaattc
35910613102DNAartificialplasmid pRL1049-PpetE-PDC-ADHII
106atcgataatt aatttttgcc gagaaacata tgtcgacttt tttgctgagg
tactgagtac 60acagctaata aaattgggca atctccgcgc ctctatgact tgaaggagag
tgtaggggta 120taggggaaag atatctttta tctacatcac ataaataaaa
aatttaattt gtcgctctgg 180ctgcatatat tgatgtattt ttagccataa
gttttttagt gccatgtaat tatagtgatt 240tttagcgatc gcagagcatt
tttccctgga tttatcgcga tctcaaaaaa aatttgcccg 300aagtatgaca
gattgtcata tttggtgtcg attttattta aaatgaaata agaaaaataa
360aactacaggt taggagaacg ccatgaattc ttatactgtc ggtacctatt
tagcggagcg 420gcttgtccag attggtctca agcatcactt cgcagtcgcg
ggcgactaca acctcgtcct 480tcttgacaac ctgcttttga acaaaaacat
ggagcaggtt tattgctgta acgaactgaa 540ctgcggtttc agtgcagaag
gttatgctcg tgccaaaggc gcagcagcag ccgtcgttac 600ctacagcgtc
ggtgcgcttt ccgcatttga tgctatcggt ggcgcctatg cagaaaacct
660tccggttatc ctgatctccg gtgctccgaa caacaatgat cacgctgctg
gtcacgtgtt 720gcatcacgct cttggcaaaa ccgactatca ctatcagttg
gaaatggcca agaacatcac 780ggccgcagct gaagcgattt acaccccaga
agaagctccg gctaaaatcg atcacgtgat 840taaaactgct cttcgtgaga
agaagccggt ttatctcgaa atcgcttgca acattgcttc 900catgccctgc
gccgctcctg gaccggcaag cgcattgttc aatgacgaag ccagcgacga
960agcttctttg aatgcagcgg ttgaagaaac cctgaaattc atcgccaacc
gcgacaaagt 1020tgccgtcctc gtcggcagca agctgcgcgc agctggtgct
gaagaagctg ctgtcaaatt 1080tgctgatgct ctcggtggcg cagttgctac
catggctgct gcaaaaagct tcttcccaga 1140agaaaacccg cattacatcg
gtacctcatg gggtgaagtc agctatccgg gcgttgaaaa 1200gacgatgaaa
gaagccgatg cggttatcgc tctggctcct gtcttcaacg actactccac
1260cactggttgg acggatattc ctgatcctaa gaaactggtt ctcgctgaac
cgcgttctgt 1320cgtcgttaac ggcgttcgct tccccagcgt tcatctgaaa
gactatctga cccgtttggc 1380tcagaaagtt tccaagaaaa ccggtgcttt
ggacttcttc aaatccctca atgcaggtga 1440actgaagaaa gccgctccgg
ctgatccgag tgctccgttg gtcaacgcag aaatcgcccg 1500tcaggtcgaa
gctcttctga ccccgaacac gacggttatt gctgaaaccg gtgactcttg
1560gttcaatgct cagcgcatga agctcccgaa cggtgctcgc gttgaatatg
aaatgcagtg 1620gggtcacatc ggttggtccg ttcctgccgc cttcggttat
gccgtcggtg ctccggaacg 1680tcgcaacatc ctcatggttg gtgatggttc
cttccagctg acggctcagg aagtcgctca 1740gatggttcgc ctgaaactgc
cggttatcat cttcttgatc aataactatg gttacaccat 1800cgaagttatg
atccatgatg gtccgtacaa caacatcaag aactgggatt atgccggtct
1860gatggaagtg ttcaacggta acggtggtta tgacagcggt gctggtaaag
gcctgaaggc 1920taaaaccggt ggcgaactgg cagaagctat caaggttgct
ctggcaaaca ccgacggccc 1980aaccctgatc gaatgcttca tcggtcgtga
agactgcact gaagaattgg tcaaatgggg 2040taagcgcgtt gctgccgcca
acagccgtaa gcctgttaac aagctcctct agtttttggg 2100gatcaattcg
agctcggtac ccaaactagt atgtagggtg aggttatagc tatggcttct
2160tcaacttttt atattccttt cgtcaacgaa atgggcgaag gttcgcttga
aaaagcaatc 2220aaggatctta acggcagcgg ctttaaaaat gcgctgatcg
tttctgatgc tttcatgaac 2280aaatccggtg ttgtgaagca ggttgctgac
ctgttgaaag cacagggtat taattctgct 2340gtttatgatg gcgttatgcc
gaacccgact gttaccgcag ttctggaagg ccttaagatc 2400ctgaaggata
acaattcaga cttcgtcatc tccctcggtg gtggttctcc ccatgactgc
2460gccaaagcca tcgctctggt cgcaaccaat ggtggtgaag tcaaagacta
cgaaggtatc 2520gacaaatcta agaaacctgc cctgcctttg atgtcaatca
acacgacggc tggtacggct 2580tctgaaatga cgcgtttctg catcatcact
gatgaagtcc gtcacgttaa gatggccatt 2640gttgaccgtc acgttacccc
gatggtttcc gtcaacgatc ctctgttgat ggttggtatg 2700ccaaaaggcc
tgaccgccgc caccggtatg gatgctctga cccacgcatt tgaagcttat
2760tcttcaacgg cagctactcc gatcaccgat gcttgcgcct tgaaggctgc
gtccatgatc 2820gctaagaatc tgaagaccgc ttgcgacaac ggtaaggata
tgccagctcg tgaagctatg 2880gcttatgccc aattcctcgc tggtatggcc
ttcaacaacg cttcgcttgg ttatgtccat 2940gctatggctc accagttggg
cggctactac aacctgccgc atggtgtctg caacgctgtt 3000ctgcttccgc
atgttctggc ttataacgcc tctgtcgttg ctggtcgtct gaaagacgtt
3060ggtgttgcta tgggtctcga tatcgccaat ctcggtgata aagaaggcgc
agaagccacc 3120attcaggctg ttcgcgatct ggctgcttcc attggtattc
cagcaaatct gaccgagctg 3180ggtgctaaga aagaagatgt gccgcttctt
gctgaccacg ctctgaaaga tgcttgtgct 3240ctgaccaacc cgcgtcaggg
tgatcagaaa gaagttgaag aactcttcct gagcgctttc 3300taatttcaaa
acaggaaaac ggttttccgt cctgtcttga ttttcaagca aacaatgcct
3360ccgatttcta atcggaggca tttgtttttg tttattgcaa aaacaaaaaa
tattgttaca 3420aatttttaca ggctattaag cctaccgtca taaataattt
gccatttggg gatcccggta 3480gagggaaacc gttgtggtct ccctatagtg
agtcgtatta atttcgcggg atcgagatcc 3540tttttgataa tctcatgacc
aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag 3600accccgtaga
aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
3660gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
caagagctac 3720caactctttt tccgaaggta actggcttca gcagagcgca
gataccaaat actgtccttc 3780tagtgtagcc gtagttaggc caccacttca
agaactctgt agcaccgcct acatacctcg 3840ctctgctaat cctgttacca
gtggctgctg ccagtggcga taagtcgtgt cttaccgggt 3900tggactcaag
acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
3960gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
cagcgtgagc 4020attgagaaag cgccacgctt cccgaaggga gaaaggcgga
caggtatccg gtaagcggca 4080gggtcggaac aggagagcgc acgagggagc
ttccaggggg aaacgcctgg tatctttata 4140gtcctgtcgg gtttcgccac
ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg 4200ggcggagcct
atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
4260ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
aaccgtatta 4320ccgcctttga gtgagctgat accgctcgcc gcagccgaac
gaccgagcgc agcgagtcag 4380tgagcgagga agcggaagag cgcctgatgc
ggtattttct ccttacgcat ctgtgcggta 4440tttcacaccg catatggtgc
actctcagta caatctgctc tgatgccgca tagttaagcc 4500agtatacact
ccgctatcgc tacgtgactg ggtcatggct gcgccccgac acccgccaac
4560acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
gacaagctgt 4620gaccgtctcc gggagctgca tgtgtcagag gttttcaccg
tcatcaccga aacgcgcgag 4680gcagctgcgg taaagctcat cagcgtggtc
gtgaagcgat tcacagatgt ctgcctgttc 4740atccgcgtcc agctcgttga
gtttctccag aagcgttaat gtctggcttc tgataaagcg 4800ggcctgccac
catacccacg ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat
4860cttccccatc ggtgatgtcg gcgatatcct cgtgatgatc agtgatggaa
aaagcactgt 4920aattcccttg gtttttggct gaaagtttcg gactcagtag
acctaagtac agagtgatgt 4980caacgccttc aagctagacg ggaggcggct
tttgccatgg ttcagcgatc gctcctcatc 5040ttcaataagc agggcatgag
ccagcgttaa gcaaatcaaa tcaaatctcg cttctgggct 5100tcaataaatg
gttccgattg atgataggtt gattcatgca agcttggagc acaggatgac
5160gcctaacaat tcattcaagc cgacaccgct tcgcggcgcg gcttaattca
ggagttaaac 5220atcatgaggg aagcggtgat cgccgaagta tcgactcaac
tatcagaggt agttggcgtc 5280atcgagcgcc atctcgaacc gacgttgctg
gccgtacatt tgtacggctc cgcagtggat 5340ggcggcctga agccacacag
tgatattgat ttgctggtta cggtgaccgt aaggcttgat 5400gaaacaacgc
ggcgagcttt gatcaacgac cttttggaaa cttcggcttc ccctggagag
5460agcgagattc tccgcgctgt agaagtcacc attgttgtgc acgacgacat
cattccgtgg 5520cgttatccag ctaagcgcga actgcaattt ggagaatggc
agcgcaatga cattcttgca 5580ggtatcttcg agccagccac gatcgacatt
gatctggcta tcttgctgac aaaagcaaga 5640gaacatagcg ttgccttggt
aggtccagcg gcggaggaac tctttgatcc ggttcctgaa 5700caggatctat
ttgaggcgct aaatgaaacc ttaacgctat ggaactcgcc gcccgactgg
5760gctggcgatg agcgaaatgt agtgcttacg ttgtcccgca tttggtacag
cgcagtaacc 5820ggcaaaatcg
cgccgaagga tgtcgctgcc gactgggcaa tggagcgcct gccggcccag
5880tatcagcccg tcatacttga agctaggcag gcttatcttg gacaagaaga
tcgcttggcc 5940tcgcgcgcag atcagttgga agaatttgtt cactacgtga
aaggcgagat caccaaggta 6000gtcggcaaat aatgtctaac aattcgttca
agccgacgcc gcttcgcggc gcggcttaac 6060tcaagcgtta gagagctggg
gaagactatg cgcgatctgt tgaaggtggt tctaagcctc 6120gtacttgcga
tggcatcggg gcaggcactt gctgacctgc caattgtttt agtggatgaa
6180gctcgtcttc cctatgacta ctccccatcc aactacgaca tttctccaag
caactacgac 6240aactccataa gcaattacga caatagtcca tcaaattacg
acaactctga gagcaactac 6300gataatagtt catccaatta cgacaatagt
cgcaacggaa atcgtaggct tatatatagc 6360gcaaatgggt ctcgcacttt
cgccggctac tacgtcattg ccaacaatgg gacaacgaac 6420ttcttttcca
catctggcaa aaggatgttc tacaccccaa aaggggggcg cggcgtctat
6480ggcggcaaag atgggagctt ctgcggggca ttggtcgtca taaatggcca
attttcgctt 6540gccctgacag ataacggcct gaagatcatg tatctaagca
actagcctgc tctctaataa 6600aatgttaggc ctcaacatct agtcgcaagc
tgaggggaac cactagcagc acgccatagt 6660gactggcgat gctgtcggaa
tggacgatat ctagacttat atagacacta atatagacaa 6720tagtttatac
tgctatctat acaagtatag acattatcta atcatggcag acaaaactct
6780agccactttt cgtattgact ccgaagaatg ggagtctttt aaaaaccttg
ctagttctga 6840aagttccaac gcctcagcac tgttaacaga atttgttcgt
tggtatttgg caggtaacag 6900gtttaatact cccacttctc acactcccac
ccatctagac acatccctcg aacagcgtat 6960agacaatatt gaacaacgtc
tagataaagt cacaactaat aatctagaca atatagatga 7020atttatagac
aagcgtatag aagataatct agcaacacgt ctagacaaac ttcaatcgca
7080actggaggaa ctgcggggaa aatcgaaagc ccggtagttc aggcagaagg
acaagctacc 7140gggcaagaca gaaagaatat agacaatagt atagacaatc
tagacaaatt ggaggcaacc 7200cgcgatcgca ccctcaataa gctaaaaatg
ggtaggcagt cagccgccgg gaaagccatc 7260gacgcgttta tcaaagagtt
gctttcttca ggagacaaca taagctgaag ttatcaaaat 7320tctgtcctta
cgtcgaaagc ctgattttac cgtgcaacga ttgataagct tggctaaact
7380agcactggct ttcaacagaa agcatacgaa gaatcaatag atatagccac
caattccaca 7440aaatgcagat aacgtgtaga gtattggaat gcttaatctg
taagggttat gaaggttaac 7500ggcaacggac gagccaaaat actcacctcc
gacgaactca ggcgactgtt tagcgacgga 7560ttcaccacac cgcgcgatcg
cgttttgttt ggcatctgtc tattcaccgg ttgccgcgtt 7620agtgaagctc
tagcactcca aacaacggac attaaaggcg aaacactaac ctttaggaag
7680tctaccacca aagggaaact caaaacccgc gtggttgaca tccagccagg
actagccgca 7740ctcatggctg actatcaccc caaaccggga accctgttcc
ctggcatgag gggagtcagc 7800gataggctca cgcgatacgc ggcggataaa
atcttgcgcg atgcagccaa aagaatcggg 7860ctagaaggca tcagtaccca
cagtttccgc cgtactgccc tcaaccaaat gtctagcgcc 7920ggtatcccgt
tgcgacacat tcaagagata tccggtcaca atgaccttgg cacactgcaa
7980cgctatcttg aagttacacc cgaacagcga cgcaaagctg tatccgtgat
tggcttctaa 8040tgtacgccaa cgctgtttag acccctatgg gtgctaaaaa
aagacgcagc ctaaacacac 8100gctctacact tgaggatact tttaaagtat
ccatcggttc tagaactctg cacacgttcc 8160ggactttgga aacgttatac
ctttccctgt gttgcagaat gctgcaatat ttcttcgaca 8220agttaacttg
tgactggttt aatattttct caaattgccc caaaacaaca cgcctaaatc
8280cttagacgtt tctgtggaaa cctattaggt ttttatcgcc gttgttttag
tggtaaaccc 8340aaagggtttg tatattcttg tatgaagttc gactctgagg
gttaagaaga atggctcgcc 8400gaatttttta caagtggaaa ccgattaaag
gttaagggtc aatcgggacg atgaatattt 8460tctaattgtg accttctcca
tctaataagc tttctttggg gttaaggtcg aagaaagtac 8520tacgcatgat
ctgcatacga tctctattgc caaaaagccg cgaccctata ggctctcggt
8580catgctgcac tagttcgtgt cgatcactat actggttgcc gcagcatttc
acgctaaaaa 8640aaaattctta aaaatgtcct tcatatctcg ccagagtggc
aacctattac aaaacggttg 8700cctacccgac cggctcgatt ttcgctgaag
tggcactgtg acagtttgaa atggtacttc 8760cgccgtgctg ctgacatcgt
tgttagggtg aattgttcgc ggtagatgtt gcaccgattc 8820atgaacacct
tgtcacccac tttgaataat cgaccgtcaa attcagtcgc gtcaatttgg
8880taagtgttgg gctgtctctt tttggctcca ggggcaatgc catcagaaaa
cacaaccgcg 8940tcacccataa cttgataacc gatatcagtt ttggttccag
tgaaagccca aaattcagac 9000gcgtcattat tccgagcgtg ccggagttga
ttgtactcaa ttttggcttg gcaaagttga 9060cggcgattca tgcccagctg
cttttgatgt cgtcgcactg tgcgcttgtg aatacccaac 9120tcacagctga
cagctttttg agatgtacca tagtggatga aactttttga gacgaatatc
9180cgcgacgaac taatgtgaag tacacaaggt acttccccct ctggcgattt
aagagaggat 9240tgccttgtgt ccttcactag ctcgttcggg tgtggcgctc
caaaaagttt tctgtactct 9300ggtttaagtt gtctgttggc cgcatagcgg
ctcttttgtt gaaagctttg tgtgactatg 9360ccagtggtca gtgagcgtaa
atcgcttaac acttggacta aaggcactac tgcaacatca 9420ccccatcttt
ttaaatttag gttgtaacaa acttgaaaca taccgcccaa gtagacggtt
9480atcattcctg ctttaatttt gtagcggcgg aatgctccta ttttttttcc
atcctgtaac 9540caacggtaaa cagacttatc actacaatct aagaacgtct
gtactacagg caatggcaat 9600gttaaatgac cagacccatc cttatcaagc
gctcgacaca aataccacaa ccgcgcacaa 9660ggttctcgac caatgcgagt
gtgtaccctg accgtgtaag tgccaagaat tatttcagtt 9720tgtagttccc
ttgtaagcag ggttagtgat acatttgtat ttaagctttc tgggctgatc
9780atttggaaat gtctcagtcc agtacctatt gaatgttatt tgcttaacct
gaagctaaat 9840aaaacttgtt aactacaccc attaattgat aaattcaaag
cacgtttttt ctgtttggtg 9900tttggtgtgg taacaattct gtgtatgtgt
gttttattta gcttcggtta agtagcataa 9960caacccccaa gcactgaact
ttttttaata ggtaatttaa actttgccta tcggcaaaat 10020tttcaatcaa
ttgtacgcca aagtgttgca tgatcaacgt ttgacttatt tttgtattta
10080ctaaatactg aatttcgccg tgacgctttt tacagatgga aattcacggc
aaaatgtttt 10140ttgctaactt tgctatgtaa aacaagaaac ttggcactcg
gttattacta aataaactgg 10200taaaaaataa ccattagaac caaaaagaac
gaaaaccagt acacccttgc cagttttcaa 10260gcttttgcta tgacgactct
aataatcggg tttaacacca ttccgctttg agaaaattat 10320ccttgtacag
caagtaacag tcaatgctaa accgcaccgc tacaaatcct taagtttttc
10380cagtagcgat ttaccttctt ggtaacgccc gccttgatag cccaaaattt
ctttaatcac 10440cttactttct gaaaaacccg cttccagaca ggcttttacc
acttttgcta gggtttcatc 10500tcttggttct gggagggatg aaacgggctg
taatgcttgt tctgaggtcg gttgagccgt 10560ttggagtggc tgaaaactgg
ttacagactg taaccggggc ataaccattt tgtaactgct 10620tacatctggt
aactgacacg gcatatcatc caccatgcag cgatatttcc ccgactttaa
10680ccactccaca agggcaaggt cttttaagga cttggcgtgg ctaactgcaa
acttacccag 10740gcgtaacatc ctaaaacact tacggacacc gccttcaccc
tcgataccta aggtcttgac 10800attatcatct tgagtcagcc caataacaaa
acgcttgggc ttgcggccgc gcctggcgtg 10860tttgatgagc cattcggttg
ctatctcgac ttcatctctc agcagtggca gttcttcagc 10920aattaaaacg
ctttcttttc ctgctagtgc cttatcccca gactcacccc gtagctcaat
10980ccggcgctgc aattcctcca ggtcagcagc catgcccgac tgtatagcct
caaagtcacc 11040acggcggcca atgacattta accccgtcca ctcgtccggt
gcagcgtcag cgtcatagac 11100tgtcacctca cccccgactt gataagcaag
ccattgggct atggtgcttt tgccagttcc 11160cgtatcccca actattaaac
agtgcttacc agacagagct tgcatcaagt cggtgatgat 11220tccctctggt
tcgaccgcaa gggtgacggc ggtagtgtca atgatagccg cgccgtaagt
11280gccagcatag ggcaattggt cgtaaacttt gaccaagttg tatacagact
gtctacacca 11340cttcaccact gttaacgctg tttgcaaagc gtaagacgtg
gcatcaaata aaaatatgct 11400ggcactaaaa gttaatcgcc ccaatcccca
cagtaaaaac ctgcctagct gttgacgact 11460aggcaagtgc atttcaatcc
agtcatttgc cataaatcac cccgtcttta aagccttgca 11520gttgagcgcg
acaggtattt aactgtgctt gtaactctgt ttgctggttt tgataccaca
11580gactgacggc ggcggccgcc agtcctaaaa atagaaactg gcgatcgctc
attattgact 11640tactccctgt tgattagcgt ggtagtgagt catagccgca
ttgaccgctt cttgggcttg 11700gggtgttctg ccaagattgg gttttgtagg
gtcatcgttg gctacgacta aggacgcttg 11760ttcggctatc gcttgcggga
caccaacttt agttaactct gtcaaggata cttggtaaag 11820tcgctcgttc
attagccgat tctccggtac ataaaactgt tgctggcagt cccttcattg
11880gcgacgagtt cttcagccgg agtatcagcg ataatgtcag cccagccggt
gacattatta 11940ttaataatgt tttgttcggc aattgcaccc aagccaggac
gcgccgtttc aaactcagag 12000atgacttgct gctctttctc ggtgagtggt
ctatctgtca tgataattat gtccttcatt 12060atgtaggcga ttccagtggg
tgtttacgag gcagtccaca ggaatcagtg cgattcacct 12120ttaaggtgaa
tcgtcatcaa aaaatcactc ggtagcaacg acccgaaccg accaggattg
12180atttcccggt tctcagttcg caggcttttg agcgcgtcac cttgaccatt
gggtaactgc 12240catcagccga taagctaaac gggctgtata gcggtaaagc
atcccacaca gtcgggctgg 12300catcaacttt gcaggaatag ctcacgtcac
tcatctcact cgcgcctggg ttggatggca 12360gcgaaggcag attacgacgc
agttttttac tggcactttt acccgcatta aaaacgggta 12420cagtgccatt
gttgacggtc tgtacttcgg tcatatactc ggtgtacact taatacactc
12480tatactatta ctgccgatta gtacatttgt caatcactct ttgcacaagg
tgtatgatat 12540ggactcagga gtacaccaaa cgtcatgcca accaataaag
ggagaatagc agtcactcta 12600gaagctgaaa tttaccaatg gattgctaac
cgagcgtctg aggaaggaag accgttggct 12660aatcttgccg ctttcttact
cacacgagtt gttaaagaac aaatggaaca agaagccaag 12720gacaaccaag
acaagcaggg ggcagcatga gcgaagacag actagccaga atagaagctg
12780cgttagacag ccaagttgca gtgaatgccg acctccgcac atcggttaca
gaactccgcg 12840caaccgcaga agcattgttg caaacagttc aaatccatca
gcagaacttt gaaattctta 12900ccgctaggca attacaaacc gaagcacggc
ttgatgagta ccaacgtacc actagcgcgg 12960cactcgacag aattggcgcg
gtcttagact acctcgttag gcagcaaaac ggttgaggtg 13020agggatgagc
gatgactatc tagacggata tcccgcaaga ggccctttcg tcttcaagaa
13080ttctcatgtt tgacagctta tc 1310210712472DNAartificialplasmid
pRL593-PisiA-PDC-ADHII 107ctagcgctat atgcgttgat gcaatttcta
tgcgcacccg ttctcggagc actgtccgac 60cgctttggcc gccgcccagt cctgctcgct
tcgctacttg gagccactat cgactacgcg 120atcatggcga ccacacccgt
cctgtggatc actaccgggc gtattttttg agttatcgag 180attttcagga
gctaaggaag ctaaaatgga gaaaaaaatc actggatata ccaccgttga
240tatatcccaa tggcatcgta aagaacattt tgaggcattt cagtcagttg
ctcaatgtac 300ctataaccag accgttcagc tggatattac ggccttttta
aagaccgtaa agaaaaataa 360gcacaagttt tatccggcct ttattcacat
tcttgcccgc ctgatgaatg ctcatccgga 420attccgtatg gcaatgaaag
acggtgagct ggtgatatgg gatagtgttc acccttgtta 480caccgttttc
catgagcaaa ctgaaacgtt ttcatcgctc tggagtgaat accacgacga
540tttccggcag tttctacaca tatattcgca agatgtggcg tgttacggtg
aaaacctggc 600ctatttccct aaagggttta ttgagaatat gtttttcgtc
tcagccaatc cctgggtgag 660tttcaccagt tttgatttaa acgtggccaa
tatggacaac ttcttcgccc ccgttttcac 720catgggcaaa tattatacgc
aaggcgacaa ggtgctgatg ccgctggcga ttcaggttca 780tcatgccgtt
tgtgatggct tccatgtcgg cagaatgctt aatgaattac aacagtactg
840cgatgagtgg cagggcgggg cgtaattttt ttaaggcagt tattggtgcc
cttaaacgcc 900tggtgctacg cctgaataag tgataataag cggatgaatg
gcagaaattc gaaagcaaat 960tcgacccggt cgtcggttca gggcagggtc
gttaaatagc cgcttatgtc tattgctggt 1020ttaccggttt attgactacc
ggaagcagtg tgaccgtgtg cttctcaaat gcctgaggcc 1080agtttgctca
ggctctcccc gtggaggtaa taattgacga tatgatcctc tacgccggac
1140gcatcgtggc cggcatcacc ggcgataagc ttcacgctgc cgcaagcact
cagggcgcaa 1200gggctgctaa aggaagcgga acacgtagaa agccagtccg
cagaaacggt gctgaccccg 1260gatgaatgtc agctactggg ctatctggac
aagggaaaac gcaagcgcaa agagaaagca 1320ggtagcttgc agtgggctta
catggcgata gctagactgg gcggttttat ggacagcaag 1380cgaaccggaa
ttgccagctg gggcgccctc tggtaaggtt gggaagccct gcaaagtaaa
1440ctggatggct ttcttgccgc caaggatctg atggcgcagg ggatcaagat
ctgatcaaga 1500gacaggatga ggatcgtttc gcatgattga acaagatgga
ttgcacgcag gttctccggc 1560cgcttgggtg gagaggctat tcggctatga
ctgggcacaa cagacaatcg gctgctctga 1620tgccgccgtg ttccggctgt
cagcgcaggg gcgcccggtt ctttttgtca agaccgacct 1680gtccggtgcc
ctgaatgaac tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac
1740gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg
actggctgct 1800attgggcgaa gtgccggggc aggatctcct gtcatctcac
cttgctcctg ccgagaaagt 1860atccatcatg gctgatgcaa tgcggcggct
gcatacgctt gatccggcta cctgcccatt 1920cgaccaccaa gcgaaacatc
gcatcgagcg agcacgtact cggatggaag ccggtcttgt 1980cgatcaggat
gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag
2040gctcaaggcg cgcatgcccg acggcgagga tctcgtcgtg acccatggcg
atgcctgctt 2100gccgaatatc atggtggaaa atggccgctt ttctggattc
atcgactgtg gccggctggg 2160tgtggcggac cgctatcagg acatagcgtt
ggctacccgt gatattgctg aagagcttgg 2220cggcgaatgg gctgaccgct
tcctcgtgct ttacggtatc gccgctcccg attcgcagcg 2280catcgccttc
tatcgccttc ttgacgagtt cttctgagcg ggactctggg gttcgaaatg
2340accgaccaag cgacgcccaa cctgccatca cgagatttcg attccaccgc
cgccttctat 2400gaaaggttgg gcttcggaat cgttttccgg gacgccggct
ggatgatcct ccagcgcggg 2460gatctcatgc tggagttctt cgcccacccc
aacgatctga tagagaaggg tttgctcggg 2520tcggtggctc tggtaacgac
cagtatcccg atcccggctg gccgtcctgg ccgccacatg 2580aggcatgttc
cgcgtccttg caatactgtg tttacataca gtctatcgct tagcggaaag
2640ttcttttacc ctcagccgaa atgcctgccg ttgctagaca ttgccagcca
gtgcccgtca 2700ctcccgtact aactgtcacg aacccctgca ataactgtca
cgcccccctg caataactgt 2760cacgaacccc tgcaataact gtcacgcccc
caaacctgca aacccagcag gggcgggggc 2820tggcggggtg ttggaaaaat
ccatccatga ttatctaaga ataatccact aggcgcggtt 2880atcagcgccc
ttgtggggcg ctgctgccct tgcccaatat gcccggccag aggccggata
2940gctggtctat tcgctgcgct aggctacaca ccgccccacc gctgcgcggc
agggggaaag 3000gcgggcaaag cccgctaaac cccacaccaa accccgcaga
aatacgctgg agcgctttta 3060gccgctttag cggcctttcc ccctacccga
agggtggggg cgcgtgtgca gccccgcagg 3120gcctgtctcg gtcgatcatt
cagcccggct catccttctg gcgtggcggc agaccgaaca 3180aggcgcggtc
gtggtcgcgt tcaaggtacg catccattgc cgccatgagc cgatcctccg
3240gccactcgct gctgttcacc ttggccaaaa tcatggcccc caccagcacc
ttgcgccttg 3300tttcgttctt gcgctcttgc tgctgttccc ttgcccgctc
ccgctgaatt tcggcattga 3360ttcgcgctcg ttgttcttcg agcttggcca
gccgatccgc cgccttgttg ctccccttaa 3420ccatcttgac accccattgt
taatgtgctg tctcgtaggc tatcatggag gcacagcggc 3480ggcaatcccg
accctacttt gtaggggagg gcgcacttac cggtttctct tcgagaaact
3540ggccctaacg gccacccttc gggcggtgcg ctctccgagg gccattgcat
ggagccgaaa 3600agcaaaagca acagcgaggc agcatggcga tttatcacct
tacggcgaaa accggcagca 3660ggtcgggcgg ccaatcggcc agggccaagg
ccgactacat ccagcgcgaa ggcaagtatg 3720cccgcgacat ggatgaagtc
ttgcacgccg aatccgggca catgccggag ttcgtcgagc 3780ggcccgccga
ctactgggat gctgccgacc tgtatgaacg cgccaatggg cggctgttca
3840aggaggtcga atttgccctg ccggtcgagc tgaccctcga ccagcagaag
gcgctggcgt 3900ccgagttcgc ccagcacctg accggtgccg agcgcctgcc
gtatacgctg gccatccatg 3960ccggtggcgg cgagaacccg cactgccacc
tgatgatctc cgagcggatc aatgacggca 4020tcgagcggcc cgccgctcag
tggttcaagc ggtacaacgg caagaccccg gagaagggcg 4080gggcacagaa
gaccgaagcg ctgaagccca aggcatggct tgagcagacc cgcgaggcat
4140gggccgacca tgccaaccgg gcattagagc gggctggcca cgacgcccgc
attgaccaca 4200gaacacttga ggcgcagggc atcgagcgcc tgcccggtgt
tcacctgggg ccgaacgtgg 4260tggagatgga aggccggggc atccgcaccg
accgggcaga cgtggccctg aacatcgaca 4320ccgccaacgc ccagatcatc
gacttacagg aataccggga ggcaatagac catgaacgca 4380atcgacagag
tgaagaaatc cagaggcatc aacgagttag cggagcagat cgaaccgctg
4440gcccagagca tggcgacact ggccgacgaa gcccggcagg tcatgagcca
gaccaagcag 4500gccagcgagg cgcaggcggc ggagtggctg aaagcccagc
gccagacagg ggcggcatgg 4560gtggagctgg ccaaagagtt gcgggaggta
gccgccgagg tgagcagcgc cgcgcagagc 4620gcccggagcg cgtcgcgggg
gtggcactgg aagctatggc taaccgtgat gctggcttcc 4680atgatgccta
cggtggtgct gctgatcgca tcgttgctct tgctcgacct gacgccactg
4740acaaccgagg acggctcgat ctggctgcgc ttggtggccc gatgaagaac
gacaggactt 4800tgcaggccat aggccgacag ctcaaggcca tgggctgtga
gcgcttcgat atcggcgtca 4860gggacgcacc caccggccag atgatgaacc
gggaatggtc agccgccgaa gtgctccaga 4920acacgccatg gctcaagcgg
atgaatgccc agggcaatga cgtgtatatc aggcccgccg 4980agcaggagcg
gcatggtctg gtgctggtgg acgacctcag cgagtttgac ctggatgaca
5040tgaaagccga gggccgggag cctgccctgg tagtggaaac cagcccgaag
aactatcagg 5100catgggtcaa ggtggccgac gccgcaggcg gtgaacttcg
ggggcagatt gcccggacgc 5160tggccagcga gtacgacgcc gacccggcca
gcgccgacag ccgccactat ggccgcttgg 5220cgggcttcac caaccgcaag
gacaagcaca ccacccgcgc cggttatcag ccgtgggtgc 5280tgctgcgtga
atccaagggc aagaccgcca ccgctggccc ggcgctggtg cagcaggctg
5340gccagcagat cgagcaggcc cagcggcagc aggagaaggc ccgcaggctg
gccagcctcg 5400aactgcccga gcggcagctt agccgccacc ggcgcacggc
gctggacgag taccgcagcg 5460agatggccgg gctggtcaag cgcttcggtc
atgacctcag caagtgcgac tttatcgccg 5520cgcagaagct ggccagccgg
ggccgcagtg ccgaggaaat cggcaaggcc atggccgagg 5580ccagcccagc
gctggcagag cgcaagcccg gccacgaagc ggattacatc gagcgcaccg
5640tcagcaaggt catgggtctg cccagcgtcc agcttgcgcg ggccgagctg
gcacgggcac 5700cggcaccccg ccagcgaggc atggacaggg gcgggccaga
tttcagcatg tagtgcttgc 5760gttggtactc acgcctgtta tactatgagt
actcacgcac agaagggggt tttatggaat 5820acgaaaaaag cgcttcaggg
tcggtctacc tgatcaaaag tgacaagggc tattggttgc 5880ccggtggctt
tggttatacg tcaaacaagg ccgaggctgg ccgcttttca gtcgctgata
5940tggccagcct taaccttgac ggctgcacct tgtccttgtt ccgcgaagac
aagcctttcg 6000gccccggcaa gtttctcggt gactgatatg aaagaccaaa
aggacaagca gaccggcgac 6060ctgctggcca gccctgacgc tgtacgccaa
gcgcgatatg ccgagcgcat gaaggccaaa 6120gggatgcgtc agcgcaagtt
ctggctgacc gacgacgaat acgaggcgct gcgcgagtgc 6180ctggaagaac
tcagagcggc gcagggcggg ggtagtgacc ccgccagcgc ctaaccacca
6240actgcctgca aaggaggcaa tcaatggcta cccataagcc tatcaatatt
ctggaggcgt 6300tcgcagcagc gccgccaccg ctggactacg ttttgcccaa
catggtggcc ggtacggtcg 6360gggcgctggt gtcgcccggt ggtgccggta
aatccatgct ggccctgcaa ctggccgcac 6420agattgcagg cgggccggat
ctgctggagg tgggcgaact gcccaccggc ccggtgatct 6480acctgcccgc
cgaagacccg cccaccgcca ttcatcaccg cctgcacgcc cttggggcgc
6540acctcagcgc cgaggaacgg caagccgtgg ctgacggcct gctgatccag
ccgctgatcg 6600gcagcctgcc caacatcatg gccccggagt ggttcgacgg
cctcaagcgc gccgccgagg 6660gccgccgcct gatggtgctg gacacgctgc
gccggttcca catcgaggaa gaaaacgcca 6720gcggccccat ggcccaggtc
atcggtcgca tggaggccat cgccgccgat accgggtgct 6780ctatcgtgtt
cctgcaccat gccagcaagg gcgcggccat gatgggcgca ggcgaccagc
6840agcaggccag ccggggcagc tcggtactgg tcgataacat ccgctggcag
tcctacctgt 6900cgagcatgac cagcgccgag gccgaggaat ggggtgtgga
cgacgaccag cgccggttct 6960tcgtccgctt cggtgtgagc aaggccaact
atggcgcacc gttcgctgat cggtggttca 7020ggcggcatga cggcggggtg
ctcaagcccg ccgtgctgga gaggcagcgc aagagcaagg 7080gggtgccccg
tggtgaagcc taagaacaag cacagcctca gccacgtccg gcacgacccg
7140gcgcactgtc tggcccccgg cctgttccgt gccctcaagc ggggcgagcg
caagcgcagc 7200aagctggacg tgacgtatga ctacggcgac ggcaagcgga
tcgagttcag cggcccggag 7260ccgctgggcg ctgatgatct gcgcatcctg
caagggctgg tggccatggc tgggcctaat 7320ggcctagtgc ttggcccgga
acccaagacc gaaggcggac ggcagctccg gctgttcctg 7380gaacccaagt
gggaggccgt caccgctgaa tgccatgtgg tcaaaggtag ctatcgggcg
7440ctggcaaagg aaatcggggc agaggtcgat agtggtgggg cgctcaagca
catacaggac 7500tgcatcgagc gcctttggaa ggtatccatc atcgcccaga
atggccgcaa gcggcagggg 7560tttcggctgc tgtcggagta cgccagcgac
gaggcggacg ggcgcctgta cgtggccctg 7620aaccccttga tcgcgcaggc
cgtcatgggt ggcggccagc atgtgcgcat cagcatggac 7680gaggtagcgg
gcgctggaca gcgaaaccgc ccgcctgctg caccagcggc tgtgtggctg
7740gatcgacccc ggcaaaaccg gcaaggcttc catagatacc ttgtgcggct
atgtctggcc 7800gtcagaggcc agtggttcga ccatgcgcaa gcgccgcaag
cgggtgcgcg agcgttgccg 7860gagctggtcg cgctgggctg gacggtaacc
gagttcgcgg cgggcaagta cgacatcacc 7920cggcccaagg cggcaggctg
acccccccca ctctattgta aacaacacat ttttatcttt 7980tatattcaat
ggcttatttt cctgctaatt ggtaatacca tgaaaaatac catgctcaga
8040aaaggcttaa caatattttg aaaaattgcc tactgagcgc tgccgcacag
ctccataggc 8100cgctttccag gctttgcttc cagatgtatg ctcttctgct
cctgcagttc attcagggca 8160ccggacaggt cggtcttgac aaaaagaacc
gggcgcccct gcgctgacag ccggaacacg 8220gcggcatcag agcagccgat
tgtctgttgt gcccagtcat agccgaatag cctctccacc 8280caagcggccg
gagaacctgc gtgcaatcca tcttgttcaa tcatgcgaaa cgatcctcat
8340cctgtctctt gatcattgat cccctgcgcc atcagatcct tggcggcaag
aaagccatcc 8400agtttacttt gcagggcttc ccaaccttac cagagggcgc
cccagctggc aattccggtt 8460cgcttgctgt ccataaaacc gcccagtcta
gctatcgcca tgtaagccca ctgcaagcta 8520cctgctttct ctttgcgctt
gcgttttccc ttgtccagat agcccagtag ctgacattca 8580tccggggtca
gcaccgtttc tgcggactgg ctttctacgt gttccgcttc ctttagcagc
8640ccttgcgccc tgagtgcttg cggcagcgtg aagcttatcg attcacaaaa
aataggcaca 8700cgaaaaacaa gttaagggat gcagtttatg cactagccta
ggctcgagaa gcttgtcgac 8760cttccagcac cacgtcaact ttgtttaact
gctcccggag ttgtctttcc gctttggcaa 8820tgtgcccggg ataccattgg
attaaagcca tgagttgttc acttttttac tgacgagggc 8880ttccggaggc
cacgctccca cccataacag cttgccacat ccccgtcgga agttacgtta
8940cccttgggcg atcgccaaaa atcagcatat atacaccaat tctaaataag
atcttttaca 9000ccgctactgc aatcaacctc atcaacaaaa ttcccctcta
gcatccctgg aggcaaatcc 9060tcacctggcc atgggttcaa ccctgcttaa
catttcttaa taattttagt tgctataaat 9120tctcatttat gcccctataa
taattcggga gtaagtgcta aagattctca actgctccat 9180cagtggtttg
agcttagtcc tagggaaaga ttggcgatcg ccgttgtggt taagccagaa
9240taggtctcgg gtggacagag aacgctttat tctttgcctc catggcggca
tcccacctag 9300gtttctcggc acttattgcc ataatttatt atttgtcgtc
tcaattaagg aggcaattct 9360gtgaattctt atactgtcgg tacctattta
gcggagcggc ttgtccagat tggtctcaag 9420catcacttcg cagtcgcggg
cgactacaac ctcgtccttc ttgacaacct gcttttgaac 9480aaaaacatgg
agcaggttta ttgctgtaac gaactgaact gcggtttcag tgcagaaggt
9540tatgctcgtg ccaaaggcgc agcagcagcc gtcgttacct acagcgtcgg
tgcgctttcc 9600gcatttgatg ctatcggtgg cgcctatgca gaaaaccttc
cggttatcct gatctccggt 9660gctccgaaca acaatgatca cgctgctggt
cacgtgttgc atcacgctct tggcaaaacc 9720gactatcact atcagttgga
aatggccaag aacatcacgg ccgcagctga agcgatttac 9780accccagaag
aagctccggc taaaatcgat cacgtgatta aaactgctct tcgtgagaag
9840aagccggttt atctcgaaat cgcttgcaac attgcttcca tgccctgcgc
cgctcctgga 9900ccggcaagcg cattgttcaa tgacgaagcc agcgacgaag
cttctttgaa tgcagcggtt 9960gaagaaaccc tgaaattcat cgccaaccgc
gacaaagttg ccgtcctcgt cggcagcaag 10020ctgcgcgcag ctggtgctga
agaagctgct gtcaaatttg ctgatgctct cggtggcgca 10080gttgctacca
tggctgctgc aaaaagcttc ttcccagaag aaaacccgca ttacatcggt
10140acctcatggg gtgaagtcag ctatccgggc gttgaaaaga cgatgaaaga
agccgatgcg 10200gttatcgctc tggctcctgt cttcaacgac tactccacca
ctggttggac ggatattcct 10260gatcctaaga aactggttct cgctgaaccg
cgttctgtcg tcgttaacgg cgttcgcttc 10320cccagcgttc atctgaaaga
ctatctgacc cgtttggctc agaaagtttc caagaaaacc 10380ggtgctttgg
acttcttcaa atccctcaat gcaggtgaac tgaagaaagc cgctccggct
10440gatccgagtg ctccgttggt caacgcagaa atcgcccgtc aggtcgaagc
tcttctgacc 10500ccgaacacga cggttattgc tgaaaccggt gactcttggt
tcaatgctca gcgcatgaag 10560ctcccgaacg gtgctcgcgt tgaatatgaa
atgcagtggg gtcacatcgg ttggtccgtt 10620cctgccgcct tcggttatgc
cgtcggtgct ccggaacgtc gcaacatcct catggttggt 10680gatggttcct
tccagctgac ggctcaggaa gtcgctcaga tggttcgcct gaaactgccg
10740gttatcatct tcttgatcaa taactatggt tacaccatcg aagttatgat
ccatgatggt 10800ccgtacaaca acatcaagaa ctgggattat gccggtctga
tggaagtgtt caacggtaac 10860ggtggttatg acagcggtgc tggtaaaggc
ctgaaggcta aaaccggtgg cgaactggca 10920gaagctatca aggttgctct
ggcaaacacc gacggcccaa ccctgatcga atgcttcatc 10980ggtcgtgaag
actgcactga agaattggtc aaatggggta agcgcgttgc tgccgccaac
11040agccgtaagc ctgttaacaa gctcctctag tttttgggga tcaattcgag
ctcggtaccc 11100aaactagtat gtagggtgag gttatagcta tggcttcttc
aactttttat attcctttcg 11160tcaacgaaat gggcgaaggt tcgcttgaaa
aagcaatcaa ggatcttaac ggcagcggct 11220ttaaaaatgc gctgatcgtt
tctgatgctt tcatgaacaa atccggtgtt gtgaagcagg 11280ttgctgacct
gttgaaagca cagggtatta attctgctgt ttatgatggc gttatgccga
11340acccgactgt taccgcagtt ctggaaggcc ttaagatcct gaaggataac
aattcagact 11400tcgtcatctc cctcggtggt ggttctcccc atgactgcgc
caaagccatc gctctggtcg 11460caaccaatgg tggtgaagtc aaagactacg
aaggtatcga caaatctaag aaacctgccc 11520tgcctttgat gtcaatcaac
acgacggctg gtacggcttc tgaaatgacg cgtttctgca 11580tcatcactga
tgaagtccgt cacgttaaga tggccattgt tgaccgtcac gttaccccga
11640tggtttccgt caacgatcct ctgttgatgg ttggtatgcc aaaaggcctg
accgccgcca 11700ccggtatgga tgctctgacc cacgcatttg aagcttattc
ttcaacggca gctactccga 11760tcaccgatgc ttgcgccttg aaggctgcgt
ccatgatcgc taagaatctg aagaccgctt 11820gcgacaacgg taaggatatg
ccagctcgtg aagctatggc ttatgcccaa ttcctcgctg 11880gtatggcctt
caacaacgct tcgcttggtt atgtccatgc tatggctcac cagttgggcg
11940gctactacaa cctgccgcat ggtgtctgca acgctgttct gcttccgcat
gttctggctt 12000ataacgcctc tgtcgttgct ggtcgtctga aagacgttgg
tgttgctatg ggtctcgata 12060tcgccaatct cggtgataaa gaaggcgcag
aagccaccat tcaggctgtt cgcgatctgg 12120ctgcttccat tggtattcca
gcaaatctga ccgagctggg tgctaagaaa gaagatgtgc 12180cgcttcttgc
tgaccacgct ctgaaagatg cttgtgctct gaccaacccg cgtcagggtg
12240atcagaaaga agttgaagaa ctcttcctga gcgctttcta atttcaaaac
aggaaaacgg 12300ttttccgtcc tgtcttgatt ttcaagcaaa caatgcctcc
gatttctaat cggaggcatt 12360tgtttttgtt tattgcaaaa acaaaaaata
ttgttacaaa tttttacagg ctattaagcc 12420taccgtcata aataatttgc
catttgggga tccgatacgt aacgcgtctg ca 12472108336PRTSynechocystis sp.
PCC 6803 108Met Ile Lys Ala Tyr Ala Ala Leu Glu Ala Asn Gly Lys Leu
Gln Pro 1 5 10 15 Phe Glu Tyr Asp Pro Gly Ala Leu Gly Ala Asn Glu
Val Glu Ile Glu 20 25 30 Val Gln Tyr Cys Gly Val Cys His Ser Asp
Leu Ser Met Ile Asn Asn 35 40 45 Glu Trp Gly Ile Ser Asn Tyr Pro
Leu Val Pro Gly His Glu Val Val 50 55 60 Gly Thr Val Ala Ala Met
Gly Glu Gly Val Asn His Val Glu Val Gly 65 70 75 80 Asp Leu Val Gly
Leu Gly Trp His Ser Gly Tyr Cys Met Thr Cys His 85 90 95 Ser Cys
Leu Ser Gly Tyr His Asn Leu Cys Ala Thr Ala Glu Ser Thr 100 105 110
Ile Val Gly His Tyr Gly Gly Phe Gly Asp Arg Val Arg Ala Lys Gly 115
120 125 Val Ser Val Val Lys Leu Pro Lys Gly Ile Asp Leu Ala Ser Ala
Gly 130 135 140 Pro Leu Phe Cys Gly Gly Ile Thr Val Phe Ser Pro Met
Val Glu Leu 145 150 155 160 Ser Leu Lys Pro Thr Ala Lys Val Ala Val
Ile Gly Ile Gly Gly Leu 165 170 175 Gly His Leu Ala Val Gln Phe Leu
Arg Ala Trp Gly Cys Glu Val Thr 180 185 190 Ala Phe Thr Ser Ser Ala
Arg Lys Gln Thr Glu Val Leu Glu Leu Gly 195 200 205 Ala His His Ile
Leu Asp Ser Thr Asn Pro Glu Ala Ile Ala Ser Ala 210 215 220 Glu Gly
Lys Phe Asp Tyr Ile Ile Ser Thr Val Asn Leu Lys Leu Asp 225 230 235
240 Trp Asn Leu Tyr Ile Ser Thr Leu Ala Pro Gln Gly His Phe His Phe
245 250 255 Val Gly Val Val Leu Glu Pro Leu Asp Leu Asn Leu Phe Pro
Leu Leu 260 265 270 Met Gly Gln Arg Ser Val Ser Ala Ser Pro Val Gly
Ser Pro Ala Thr 275 280 285 Ile Ala Thr Met Leu Asp Phe Ala Val Arg
His Asp Ile Lys Pro Val 290 295 300 Val Glu Gln Phe Ser Phe Asp Gln
Ile Asn Glu Ala Ile Ala His Leu 305 310 315 320 Glu Ser Gly Lys Ala
His Tyr Arg Val Val Leu Ser His Ser Lys Asn 325 330 335
109341PRTOceanobacter sp. RED65 109Met Ile Lys Ala Phe Ala Ala Asp
Thr Ala Lys Gly Glu Leu Lys Pro 1 5 10 15 Phe Glu Tyr Glu Val Gly
Glu Leu Gly Ser Gln Glu Val Glu Ile Glu 20 25 30 Val His Tyr Cys
Gly Val Cys His Ser Asp Ile Ser Met Leu Asp Asn 35 40 45 Glu Trp
Gly Met Thr Gln Tyr Pro Phe Val Pro Gly His Glu Val Ala 50 55 60
Gly Leu Ile Lys Gln Val Gly Ala Glu Val Asn His Leu Lys Val Gly 65
70 75 80 Asp Arg Val Gly Leu Gly Trp Gln Ser Gly Tyr Cys Asn His
Cys Glu 85 90 95 Asn Cys Met Ser Gly Asp His Asn Leu Cys Gly Thr
Ala Glu Met Thr 100 105 110 Ile Val Gly Arg His Gly Gly Phe Ala Asp
His Val Arg Ala Gln Ala 115 120 125 Ser Ser Val Val Lys Leu Pro Asp
Asp Ile His Met Ala Asp Ala Gly 130 135 140 Pro Leu Phe Cys Gly Gly
Val Thr Val Tyr Asn Pro Met Lys Gln Phe 145 150 155 160 Asp Leu Lys
Pro Thr Ala Lys Val Ala Val Ile Gly Ile Gly Gly Leu 165 170 175 Gly
His Met Ala Leu Gln Phe Leu Asn Ser Trp Gly Cys Glu Val Thr 180 185
190 Ala Phe Thr Ser Thr Glu Glu Lys Arg Lys Glu Ala Ile Ala Leu Gly
195 200 205 Ala His Lys Thr Leu Asn Ser Arg Asp Glu Gly Glu Leu Lys
Gly Ala 210 215 220 Ala Gly Ser Phe Asp Met Ile Ile Ser Thr Val Asn
Val Ser Leu Asn 225 230 235 240 Trp Glu Ala Tyr Ile Asn Thr Leu Lys
Ala Lys Gly Arg Leu His Phe 245 250 255 Val Gly Ala Val Leu Glu Pro
Ile Gln Val Gly Val Phe Pro Leu Met 260 265 270 Met Gly Gln Arg Ser
Ile Ser Ala Ser Pro Val Gly Ser Pro Ser Thr 275 280 285 Ile Ser Gln
Met Leu Glu Phe Thr Ala Arg His Gln Ile Lys Pro Gln 290 295 300 Val
Glu Leu Phe Gln Lys Asp Gln Ile Asn Asp Ala Ile Asn His Val 305 310
315 320 Arg Glu Gly Lys Ala Arg Tyr Arg Ala Val Ile Gln Phe Lys Ala
Thr 325 330 335 Ser Asp Asn Ser Ala 340 110337PRTLimnobacter sp.
MED105 110Met Glu Leu Ile Met Ile Asn Ala Tyr Ala Ala Phe Glu Ala
Lys Gly 1 5 10 15 Pro Leu Lys Pro Phe Gln Tyr Asp Pro Gly Glu Leu
Asn Ala Phe Asp 20 25 30 Ile Glu Ile Asp Val Asp His Cys Gly Ile
Cys His Ser Asp Val Ser 35 40 45 Met Leu Asp Asn Asp Trp Gly Arg
Ala Lys Tyr Pro Met Val Ala Gly 50 55 60 His Glu Ile Ile Gly Arg
Val Ser Gln Val Gly Ser His Val Ser His 65 70 75 80 Leu Ala Ile Gly
Asp Val Val Gly Leu Gly Trp His Ser Gly Tyr Cys 85 90 95 Glu Ser
Cys Arg Met Cys Met Gly Gly Asp His Asn Leu Cys Ser Thr 100 105 110
Ala Lys Gly Thr Ile Val Gly Arg His Gly Gly Phe Ala Asp Lys Val 115
120 125 Arg Ala Gln Ala Val Ser Ala Val Lys Ile Pro Ala Gly Val Asn
Pro 130 135 140 Ala Thr Ala Gly Pro Leu Leu Cys Gly Gly Ile Thr Val
Tyr Asn Pro 145 150 155 160 Leu Val Gln Phe Asn Ile Ser Pro Gln Ser
Lys Val Ala Val Ile Gly 165 170 175 Val Gly Gly Leu Gly His Met Ala
Val Met Phe Leu Lys Ala Trp Gly 180 185 190 Cys Glu Val Thr Ala Phe
Ser Ser Asn Val Ser Lys Thr Asp Glu Leu 195 200 205 Leu Gly Met Gly
Ala His His Val Leu Asn Ser Lys Asp Pro Asp Ala 210 215 220 Leu Lys
Lys Ala Ala Gly Ser Phe Asp Leu Ile Leu Ser Thr Val Asn 225 230 235
240 Val Lys Leu Asp Trp Asn Ala Tyr Ile Gly Thr Leu Ala Pro Lys Gly
245 250 255 Arg Leu His Phe Leu Gly Ala Val Leu Glu Pro Leu Asp Ile
Gly Val 260 265 270 Phe Gly Leu Met Gly Gln Gln Arg Ser Ile Ser Ser
Ser Pro Val Gly 275 280 285 Ser Pro Arg Val Ile Ala Asp Met Leu Lys
Phe Ala Ala Leu His Asn 290 295 300 Ile Gln Pro Ile Val Glu Thr Tyr
Ser Phe Asp Gln Ile Asn Glu Ala 305 310 315 320 Val Asp Lys Val Arg
Asn Gly Ser Pro Arg Phe Arg Val Val Leu Ser 325 330 335 Arg
111333PRTPsychrobacter cryohalolentis K5 111Met Ile Asn Ala Tyr Ala
Ala Lys Glu Lys Gly Gly Glu Phe Val Pro 1 5 10 15 Tyr Gln Tyr Asp
Pro Gly Thr Leu Gly Asp His Glu Val Glu Ile Glu 20 25 30 Val His
Ser Cys Gly Ile Cys His Ser Asp Leu Ser Met Trp Gln Asn 35 40 45
Glu Trp Gly Met Thr Gln Tyr Pro Phe Val Gly Gly His Glu Val Ala 50
55 60 Gly Lys Val Leu Ala Lys Gly Lys His Val Lys His Leu Glu Leu
Gly 65 70 75 80 Asp Lys Val Gly Leu Gly Trp His Lys Gly Tyr Cys Asn
Val Cys Asp 85 90 95 Leu Cys Ile Gly Gly Asp His Asn Leu Cys Pro
Glu Gln Glu Gly Thr 100 105 110 Ile Ile Gly Asn His Gly Gly Phe Ala
Asp Lys Val Arg Ala Lys Asp 115 120 125 Thr Ser Val Ile Lys Ile Pro
Glu Gly Leu Asp Phe Asn Ala Val Gly 130 135 140 Pro Leu Leu Cys Gly
Gly Val Thr Val Phe Asn Pro Leu Met Gln Tyr 145 150 155 160 Asp Ile
Thr Pro Thr Ser Arg Val Ala Val Ile Gly Ile Gly Gly Leu 165 170 175
Gly His Leu Ala Leu Gln Phe Ala Asn Ala Trp Gly Cys Glu Val Thr 180
185 190 Ala Phe Thr Ser Glu Ser Lys Met Glu Glu Ala Lys Glu Met Gly
Ala 195 200 205 His His Ser Leu Asn Ser Arg Glu Asp Ser Glu Ile Glu
Lys Ala Ala 210 215 220 Gly Ser Phe Asp Leu Ile Ile Ser Thr Val Asn
Val Asp Met Asn Trp 225 230 235 240 Asp Val Val Ile Lys Thr Leu Arg
Pro Lys Gly Lys Leu His Phe Val 245 250 255 Gly Leu Leu Glu Ala Pro
Leu Glu Ile Ser Ala Ala Pro Met Ile Met 260 265 270 Ala Gln Asn Ser
Leu Ser Gly Ser Pro Val Gly Ser Pro Ser Thr Leu 275 280 285 Arg Lys
Met Leu Asp Phe Ala Ala Arg His Asn Ile Gln Pro Val Thr 290 295 300
Glu Thr Tyr Lys Met Ser Glu Ile Asn Glu Ala Phe Glu Arg Leu Glu 305
310 315 320 Ser Gly Asn Ala Arg Tyr Arg Val Val Leu Glu Arg Asp 325
330 112333PRTVerrucomicrobiae bacterium DG1235 112Met Ile Lys Ala
Tyr Ala Thr His Thr Pro Gly Gly Lys Leu Glu Pro 1 5 10 15 Phe Glu
Tyr Asp Pro Gly Glu Leu Ala Pro Thr Asp Val Glu Ile Asn 20 25 30
Val Glu His Cys Gly Ile Cys His Ser Asp Leu Ser Met Leu Asn Asn 35
40 45 Glu Trp Gly Met Thr Thr Tyr Pro Phe Val Pro Gly His Glu Val
Val 50 55 60 Gly Thr Ile Gly Ala Ile Gly Ser Asp Val Lys Asn Leu
Ala Pro Gly 65 70 75 80 Gln Arg Val Gly Leu Gly Trp His Ser Ser Tyr
Cys Thr Thr Cys Pro 85 90 95 Ser Cys Leu Ser Gly Asp His Asn Leu
Cys Gln Ala Ala Ala Gly Thr 100 105 110 Ile Val Gly Arg His Gly Gly
Phe Ala Asp Lys Val Arg Ala Ser Ala 115 120 125 Leu Ser Val Ile Pro
Leu Pro Asp Ser Ile Asp Ala Ala Lys Ala Gly 130 135 140 Pro Leu Phe
Cys Gly Gly Ile Thr Val Phe Asn Pro Leu Ile Gln Tyr 145 150 155 160
Glu Val Ser Pro Thr Ala Lys Val Ala Val Ile Gly Ile Gly Gly Leu 165
170 175 Gly His Met Ala Leu Ala Phe Leu Asn Ala Trp Gly Cys Glu Val
Thr 180 185 190 Ala Phe Thr Thr Ser Glu Ala Lys
Arg Gln Glu Ala Leu Lys Leu Gly 195 200 205 Ala His His Thr Leu Asn
Ser Arg Asp Ala Ala Glu Ile Glu Ala Ala 210 215 220 Ala Gly Arg Phe
Asp Leu Ile Leu Ser Thr Val Asn Val Gly Leu Asp 225 230 235 240 Trp
Asn Gly Tyr Val Asn Thr Leu Lys Pro Lys Gly Arg Leu His Phe 245 250
255 Leu Gly Ala Ala Leu Glu Pro Ile Gln Ile Gly Ala Phe Ser Leu Ile
260 265 270 Met Ala Gln Arg Gln Ile Ser Gly Ser Pro Val Gly Ser Pro
Ala Thr 275 280 285 Ile Ala Lys Met Ile Glu Phe Ala Ala Leu His Lys
Ile Glu Pro Val 290 295 300 Thr Glu His Phe Lys Phe Asp Gln Ala Asn
Glu Ala Leu Ala His Leu 305 310 315 320 Glu Ser Gly Gln Ala Arg Tyr
Arg Ile Val Leu Ser His 325 330 113334PRTSaccharophagus degradans
2-40 113Met Ile Lys Ala Tyr Ala Ala Met Glu Pro Gly Ala Ala Leu Val
Pro 1 5 10 15 Phe Glu Tyr Glu Pro Gly Pro Leu Ala Asn Asn Glu Val
Glu Leu Lys 20 25 30 Val Glu Ser Cys Gly Ile Cys His Ser Asp Leu
Ser Met Leu Asp Asn 35 40 45 Glu Trp Gly Phe Thr Gln Tyr Pro Phe
Val Gly Gly His Glu Val Ile 50 55 60 Gly Ile Val Glu Ala Val Gly
Ser Ser Val Asn Asn Val Ala Val Gly 65 70 75 80 Gln Arg Val Gly Leu
Gly Trp His Ser Gly Tyr Cys Asn Thr Cys Ala 85 90 95 Ser Cys Gln
Ser Gly Asp Gln Asn Leu Cys Asn Ser Ala Gln Pro Thr 100 105 110 Ile
Ala Gly His His Gly Gly Phe Ala Asp Lys Val Arg Ala Asp Ala 115 120
125 Asn Ala Val Val Ala Leu Pro Glu Gly Val Asn Pro Asp Ser Ala Gly
130 135 140 Pro Leu Phe Cys Gly Gly Ile Thr Val Phe Asn Pro Leu Val
Gln Phe 145 150 155 160 Gly Ile Lys Pro Thr Ser Lys Val Gly Val Ile
Gly Ile Gly Gly Leu 165 170 175 Gly His Ile Ala Leu Gln Phe Leu Asn
Ala Trp Gly Cys Glu Val Thr 180 185 190 Ala Phe Thr Ser Ser Glu Ser
Lys Lys Glu Glu Ala Leu Lys Leu Gly 195 200 205 Ala His His Val Leu
Asn Ser Ser Asp Ala Ala Gln Leu Glu Ala Ala 210 215 220 Ala Gly Arg
Phe Asp Phe Ile Ile Ser Thr Val Asn Val Lys Leu Asp 225 230 235 240
Trp Asn Glu Tyr Leu Ala Thr Leu Ala Pro Lys Gly Arg Leu His Phe 245
250 255 Val Gly Ala Thr Leu Ala Pro Leu Asp Ile Asn Val Phe Gln Leu
Ile 260 265 270 Gly Ser Gln Arg Glu Ile Ser Gly Ser Pro Val Gly Ser
Pro Gly Thr 275 280 285 Ile Ser Gln Met Leu Asp Phe Ala Ala Leu His
Asn Ile Gln Pro Val 290 295 300 Thr Glu Tyr Phe Arg Phe Asp Gln Ile
Asn Glu Ala Leu Thr Lys Leu 305 310 315 320 Arg Glu Gly Lys Ala His
Tyr Arg Ile Val Leu Thr Asn Lys 325 330 114333PRTAlteromonas
macleodii 'Deep ecotype' 114Met Ile Tyr Ala Tyr Ala Ala Lys Glu Ala
Gly Gly Lys Leu Glu Lys 1 5 10 15 Phe Glu Tyr Asp Pro Gly Glu Leu
Gly Ala His Asp Val Glu Ile Asp 20 25 30 Val Glu Ser Cys Gly Ile
Cys His Ser Asp Leu Ser Met Leu Asp Asn 35 40 45 Glu Trp Gly Ile
Thr Glu Phe Pro Phe Val Pro Gly His Glu Val Val 50 55 60 Gly Thr
Val Ser Lys Ile Gly Asp His Val Thr Ser Leu Lys Val Gly 65 70 75 80
Gln Arg Val Gly Leu Gly Trp His Ala Ser Tyr Cys Asn Ser Cys Arg 85
90 95 Thr Cys Glu Ala Gly Asp His Asn Leu Cys Ala Gly Ala Thr Met
Thr 100 105 110 Ile Gly Gly Arg His Gly Gly Phe Ala Asp Lys Val Arg
Ala Gln Ala 115 120 125 Arg Ala Val Ile Pro Leu Pro Glu Ser Ile Asp
Ser Thr Lys Ala Gly 130 135 140 Pro Leu Phe Cys Gly Gly Ile Thr Val
Phe Asn Pro Leu Val Gln Phe 145 150 155 160 Asn Ile Ser Pro Thr Ser
Glu Val Gly Val Val Gly Ile Gly Gly Leu 165 170 175 Gly His Leu Ala
Leu Gln Phe Leu Asn Ala Trp Gly Cys Lys Val Val 180 185 190 Ala Phe
Thr Ser Ser Glu Ser Lys Glu Lys Glu Ala Leu Ser Leu Gly 195 200 205
Ala Ser Glu Thr Ile Asn Ser Arg Asp Glu Asp Glu Ile Lys Lys Ala 210
215 220 Gln Gly Arg Phe Asp Leu Ile Ile Ser Thr Val Asn Val Lys Leu
Asp 225 230 235 240 Trp Asn Leu Tyr Leu Ser Thr Leu Ala Pro Lys Gly
Arg Leu His Phe 245 250 255 Val Gly Ala Thr Leu Glu Pro Leu Asp Ile
Gly Ala Phe Asn Leu Ile 260 265 270 Gly Gly Gln Lys Ser Val Ser Gly
Ser Pro Val Gly Ser Pro Ala Thr 275 280 285 Ile Lys Thr Met Leu Asp
Phe Ala Ala His His Asp Ile Glu Pro Val 290 295 300 Thr Glu Thr Phe
Lys Phe Glu Asp Val Asn Lys Ala Ile Asp Arg Leu 305 310 315 320 Arg
Glu Gly Lys Ala His Tyr Arg Ile Val Leu Thr Arg 325 330
115332PRTAcaryochloris marina MBIC11017 115Met Val Asn Ala Tyr Ala
Ala Phe Glu Gln Gly Gly Val Leu Gln Pro 1 5 10 15 Phe Glu Tyr Asp
Pro Gly Pro Leu Gly Arg Gln Gln Val Asp Ile Gln 20 25 30 Val Glu
Tyr Cys Gly Ile Cys His Ser Asp Leu Ser Met Ile Lys Asn 35 40 45
Glu Trp Gly Met Thr Gln Tyr Pro Phe Val Pro Gly His Glu Ile Val 50
55 60 Gly Ile Val Ala Glu Ile Gly Ser Glu Val Thr Thr Leu Arg Val
Gly 65 70 75 80 Gln Arg Val Gly Leu Gly Trp Tyr Ser Ser Ser Cys Met
His Cys Glu 85 90 95 Trp Cys Met Gly Gly Asp His His Leu Cys Leu
Ser Ala Glu Gly Thr 100 105 110 Ile Val Gly Arg Pro Gly Gly Phe Ala
Asp Gln Val Arg Ala Asp Gln 115 120 125 Ser Trp Ile Val Pro Ile Pro
Glu Ser Ile Asp Ser Ala Val Ala Gly 130 135 140 Pro Leu Phe Cys Ala
Gly Ile Thr Val Phe Gln Pro Ile Ile Gln Cys 145 150 155 160 Gly Val
Gln Pro Thr Asp Arg Val Ala Val Ile Gly Ile Gly Gly Leu 165 170 175
Gly His Leu Ala Leu Gln Phe Leu Asn Ala Trp Gly Cys Glu Val Thr 180
185 190 Ala Leu Ser Thr Gln Pro Asp Lys Glu Ala Glu Ala Arg Arg Leu
Gly 195 200 205 Ala His His Phe Val Asn Thr Arg Asp Pro Ala Ala Leu
Gln Ala Ile 210 215 220 Ala Asn Ser Cys Asp Tyr Ile Ile Ser Thr Val
Asn Val Ser Leu Glu 225 230 235 240 Trp Ser Ile Tyr Leu Asn Ala Leu
Arg Pro Lys Gly Arg Leu His Leu 245 250 255 Val Gly Val Ala Pro Asp
Leu Ser Leu Pro Val Phe Pro Leu Leu Ala 260 265 270 Gly Gln Arg Ser
Ile Ser Gly Ser Pro Val Gly Ser Pro Ala Thr Ile 275 280 285 Thr Lys
Met Leu Asn Phe Val Ala Arg His Gly Leu Ala Pro Gln Thr 290 295 300
Glu Val Phe Pro Leu Ala Gln Val Asn Glu Ala Leu Glu Lys Leu Arg 305
310 315 320 Ser Gln His Pro Pro Tyr Arg Leu Ala Leu Lys Cys 325 330
116335PRTCyanothece sp. PCC 7424 116Met Ile Arg Ala Tyr Ala Ala His
Glu Pro Gly Gly Lys Leu Glu Pro 1 5 10 15 Phe Glu Tyr Glu Pro Gly
Ser Leu Gly Asp Glu Glu Val Asp Ile Lys 20 25 30 Val Glu Tyr Cys
Gly Ile Cys His Ser Asp Leu Ser Met Leu Lys Asn 35 40 45 Asp Trp
Gly Met Thr Gln Tyr Pro Phe Val Pro Gly His Glu Val Val 50 55 60
Gly Val Val Glu Ala Val Gly Ser Lys Val Lys Asn Leu Gln Ile Gly 65
70 75 80 Gln Lys Val Gly Leu Gly Trp Tyr Ser Arg Ser Cys Met Thr
Cys Glu 85 90 95 Phe Cys Met Ser Gly Asn His Asn Leu Cys Gln Asp
Ala Glu Gly Thr 100 105 110 Ile Val Gly Arg Tyr Gly Gly Phe Ala Glu
Lys Val Arg Ala His Gln 115 120 125 Gly Trp Val Ile Pro Leu Pro Glu
Gly Val Asn Pro Val Thr Ala Gly 130 135 140 Pro Leu Phe Cys Gly Gly
Ile Thr Val Phe Asn Pro Ile Val Gln Phe 145 150 155 160 Asn Ile Lys
Pro Thr Asp Gln Val Gly Val Ile Gly Ile Gly Gly Leu 165 170 175 Gly
His Met Ala Leu Gly Phe Leu Arg Ala Trp Gly Cys Glu Ile Thr 180 185
190 Ala Phe Ser Thr Ser Pro Asp Lys Glu Ala Glu Ala Lys Ala Leu Gly
195 200 205 Ala Thr His Phe Val Asn Ser Arg Asp Pro Glu Ala Leu Lys
Ala Leu 210 215 220 Thr Asn Ser Phe Asp Val Ile Leu Ser Thr Val Asn
Ala Asp Leu Asp 225 230 235 240 Trp Pro Thr Tyr Ile Lys Leu Leu Arg
Pro Gln Gly Arg Leu His Leu 245 250 255 Val Gly Val Ile Pro Asn Pro
Leu Ser Val Pro Ile Phe Pro Met Ile 260 265 270 Leu Gly Gln Lys Ser
Val Ser Ala Ser Pro Leu Gly Ser Pro Thr Thr 275 280 285 Ile Ala Gln
Met Leu Asn Phe Ala Gly Arg His His Leu Glu Pro Ile 290 295 300 Val
Glu Phe Phe Pro Leu Glu Gln Val Asn Glu Ala Leu Glu Arg Leu 305 310
315 320 Gln Ser Asn Lys Ala Arg Tyr Arg Ile Ile Leu Lys Met Asp His
325 330 335 117335PRTCyanothece sp. PCC 7424 117Met Ile Arg Ala Tyr
Ala Ala His Glu Pro Gly Gly Lys Leu Glu Pro 1 5 10 15 Phe Glu Tyr
Glu Pro Gly Ser Leu Gly Asp Glu Glu Val Asp Ile Lys 20 25 30 Val
Glu Tyr Cys Gly Ile Cys His Ser Asp Leu Ser Met Leu Lys Asn 35 40
45 Asp Trp Gly Met Thr Gln Tyr Pro Phe Val Pro Gly His Glu Val Val
50 55 60 Gly Val Val Glu Ala Val Gly Ser Lys Val Lys Asn Leu Gln
Ile Gly 65 70 75 80 Gln Lys Val Gly Leu Gly Trp Tyr Ser Arg Ser Cys
Met Thr Cys Glu 85 90 95 Phe Cys Met Ser Gly Asn His Asn Leu Cys
Gln Asp Ala Glu Gly Thr 100 105 110 Ile Val Gly Arg Tyr Gly Gly Phe
Ala Glu Lys Val Arg Ala His Gln 115 120 125 Gly Trp Val Ile Pro Leu
Pro Glu Gly Val Asn Pro Val Thr Ala Gly 130 135 140 Pro Leu Phe Cys
Gly Gly Ile Thr Val Phe Asn Pro Ile Val Gln Phe 145 150 155 160 Asn
Ile Lys Pro Thr Asp Gln Val Gly Val Ile Gly Ile Gly Gly Leu 165 170
175 Gly His Met Ala Leu Gly Phe Leu Arg Ala Trp Gly Cys Glu Ile Thr
180 185 190 Ala Phe Ser Thr Ser Pro Asp Lys Glu Ala Glu Ala Lys Ala
Leu Gly 195 200 205 Ala Thr His Phe Val Asn Ser Arg Asp Pro Glu Ala
Leu Lys Ala Leu 210 215 220 Thr Asn Ser Phe Asp Val Ile Leu Ser Thr
Val Asn Ala Asp Leu Asp 225 230 235 240 Trp Pro Thr Tyr Ile Lys Leu
Leu Arg Pro Gln Gly Arg Leu His Leu 245 250 255 Val Gly Val Ile Pro
Asn Pro Leu Ser Val Pro Ile Phe Pro Met Ile 260 265 270 Leu Gly Gln
Lys Ser Val Ser Ala Ser Pro Leu Gly Ser Pro Thr Thr 275 280 285 Ile
Ala Gln Met Leu Asn Phe Ala Gly Arg His His Leu Glu Pro Ile 290 295
300 Val Glu Phe Phe Pro Leu Glu Gln Val Asn Glu Ala Leu Glu Arg Leu
305 310 315 320 Gln Ser Asn Lys Ala Arg Tyr Arg Ile Ile Leu Lys Met
Asp His 325 330 335 118336PRTCyanothece sp. PCC 7822 118Met Ile Arg
Ala Tyr Ala Ala His Glu Pro Gly Gly Lys Leu Glu Pro 1 5 10 15 Phe
Glu Tyr Asp Pro Gly Ser Leu Gly Asp Glu Asp Val Glu Ile Gln 20 25
30 Val Glu Tyr Cys Gly Ile Cys His Ser Asp Leu Ser Met Leu Asn Asn
35 40 45 Glu Trp Gly Met Thr Arg Tyr Pro Phe Val Pro Gly His Glu
Val Val 50 55 60 Gly Thr Ile Asn Ala Val Gly Glu Arg Val Lys His
Leu Gln Val Gly 65 70 75 80 Gln Arg Val Gly Leu Gly Trp Tyr Ser Arg
Ser Cys Met Thr Cys Glu 85 90 95 Trp Cys Leu Ser Gly Asn Gln Asn
Leu Cys Pro Gln Ala Glu Gly Thr 100 105 110 Ile Val Gly Arg Tyr Gly
Gly Phe Ala Glu Lys Val Arg Ala His Gln 115 120 125 Gly Trp Val Leu
Pro Leu Pro Glu Lys Leu Asn Pro Leu Thr Ala Gly 130 135 140 Pro Leu
Phe Cys Gly Gly Ile Thr Val Phe Asn Pro Ile Val Gln Phe 145 150 155
160 Asp Val Lys Pro Thr Asp Arg Val Gly Val Ile Gly Ile Gly Gly Leu
165 170 175 Gly His Met Ala Leu Gly Phe Leu Ala Ala Trp Gly Cys Glu
Ile Thr 180 185 190 Ala Phe Ser Thr Ser Pro Asp Lys Glu Ile Glu Ala
Lys Asn Leu Gly 195 200 205 Ala Asn His Phe Val Asn Ser Arg Asp Pro
Gln Ala Leu Lys Ala Leu 210 215 220 Ala Asn Ser Leu Asp Leu Ile Leu
Ser Thr Val Asn Ala Asp Leu Asp 225 230 235 240 Trp Asp Thr Tyr Ile
Ser Leu Leu Arg Pro Lys Gly Arg Leu His Phe 245 250 255 Val Gly Val
Ile Pro Asn Pro Leu Ser Val Gln Leu Phe Pro Leu Ile 260 265 270 Gly
Gly Gln Lys Ser Val Ser Gly Ser Pro Leu Gly Ser Pro Val Thr 275 280
285 Leu Ala Gln Met Leu Asn Phe Ala Gly Arg His His Val Glu Pro Val
290 295 300 Val Glu Phe Tyr Pro Ile Glu Gln Val Asn Glu Ala Met Glu
Arg Leu 305 310 315 320 Lys Ala Asn Lys Ala Arg Tyr Arg Ile Val Leu
Thr Phe Lys Asn Ser 325 330 335 119333PRTCyanothece sp. PCC 8801
119Met Ile Lys Ala Tyr Ala Ala Ser Glu Pro Gly Lys Glu Leu Asn Ser
1 5 10 15 Phe Glu Tyr Asp Pro Gly Leu Leu Gly Glu Glu Asp Val Glu
Ile Asn 20 25 30 Val Gln Tyr Cys Gly Ile Cys His Ser Asp Leu Ser
Met Leu Asp Asn 35 40 45 Glu Trp Gly Ile Thr Gln Tyr Pro Phe Val
Pro Gly His Glu Val Val 50 55 60 Gly Thr Ile Gly Ala Val Gly Ser
Lys Val Thr Thr Phe Gln Val Gly 65 70 75 80 Gln Thr Val Gly Leu Gly
Trp Phe Ser Arg Ser Cys Phe Asp Cys Glu 85 90 95 Trp Cys Leu Ser
Gly Asp Gln Asn Leu Cys Gln Thr Ala Glu Gly Thr 100 105 110 Ile Val
Gly Arg Pro Gly Gly Phe Ala Asp Lys Val Arg Ala His His 115
120 125 Arg Trp Val Val Pro Leu Pro Ser Gly Val Asn Pro Glu Thr Ala
Gly 130 135 140 Pro Leu Phe Cys Gly Gly Ile Thr Val Phe Asn Pro Ile
Ile Gln Cys 145 150 155 160 Gly Val Lys Ser Thr Asp Arg Val Gly Val
Ile Gly Ile Gly Gly Leu 165 170 175 Gly His Leu Ala Ile Glu Phe Leu
His Ala Trp Gly Cys Glu Val Thr 180 185 190 Ala Phe Ser Ser Asn Pro
Glu Lys Glu Ser Glu Val Lys Gln Leu Gly 195 200 205 Ala Asp Tyr Phe
Val Asn Ser Arg Asp Pro Glu Ala Ile Lys Ala Val 210 215 220 Glu Asn
Ser Phe Asp Phe Ile Ile Ser Thr Val Asn Val Ser Leu Asp 225 230 235
240 Trp Asn Ser Tyr Ile Leu Ala Leu Arg Pro Arg Gly Thr Leu His Phe
245 250 255 Val Gly Ala Val Leu Asn Pro Ile Ser Thr Gln Ile Phe Pro
Leu Leu 260 265 270 Met Gly Gln Lys Thr Ile Ser Gly Ser Pro Thr Gly
Ser Pro Thr Thr 275 280 285 Ile Ala Gln Met Leu Asp Phe Ala Ala Arg
His Gln Ile Glu Pro Val 290 295 300 Thr Glu Ile Phe Pro Phe Glu Gln
Val Asn Glu Ala Ile Asp Lys Leu 305 310 315 320 Arg His Gly Gln Pro
Arg Tyr Arg Leu Val Leu Lys Met 325 330 120362PRTCyanothece sp. PCC
8801 120Met Arg Gly Glu Arg Ile Val Arg Ser Gly Val Lys Glu Asp Ile
Leu 1 5 10 15 Cys Asn Asn Ala Ile Asn Thr Thr Ile Glu Val Lys Val
Val Ile Lys 20 25 30 Ala Tyr Ala Ala Ser Glu Pro Gly Lys Glu Leu
Asn Ser Phe Glu Tyr 35 40 45 Asp Pro Gly Leu Leu Gly Glu Glu Asp
Val Glu Ile Asn Val Gln Tyr 50 55 60 Cys Gly Ile Cys His Ser Asp
Leu Ser Met Leu Asp Asn Glu Trp Gly 65 70 75 80 Ile Thr Gln Tyr Pro
Phe Val Pro Gly His Glu Val Val Gly Thr Ile 85 90 95 Gly Ala Val
Gly Ser Lys Val Thr Thr Phe Gln Val Gly Gln Thr Val 100 105 110 Gly
Leu Gly Trp Phe Ser Arg Ser Cys Phe Asp Cys Glu Trp Cys Leu 115 120
125 Ser Gly Asp Gln Asn Leu Cys Gln Thr Ala Glu Gly Thr Ile Val Gly
130 135 140 Arg Pro Gly Gly Phe Ala Asp Lys Val Arg Ala His His Arg
Trp Val 145 150 155 160 Val Pro Leu Pro Ser Gly Val Asn Pro Glu Thr
Ala Gly Pro Leu Phe 165 170 175 Cys Gly Gly Ile Thr Val Phe Asn Pro
Ile Ile Gln Cys Gly Val Lys 180 185 190 Ser Thr Asp Arg Val Gly Val
Ile Gly Ile Gly Gly Leu Gly His Leu 195 200 205 Ala Ile Glu Phe Leu
His Ala Trp Gly Cys Glu Val Thr Ala Phe Ser 210 215 220 Ser Asn Pro
Glu Lys Glu Ser Glu Val Lys Gln Leu Gly Ala Asp Tyr 225 230 235 240
Phe Val Asn Ser Arg Asp Pro Glu Ala Ile Lys Ala Val Glu Asn Ser 245
250 255 Phe Asp Phe Ile Ile Ser Thr Val Asn Val Ser Leu Asp Trp Asn
Ser 260 265 270 Tyr Ile Leu Ala Leu Arg Pro Arg Gly Thr Leu His Phe
Val Gly Ala 275 280 285 Val Leu Asn Pro Ile Ser Thr Gln Ile Phe Pro
Leu Leu Met Gly Gln 290 295 300 Lys Thr Ile Ser Gly Ser Pro Thr Gly
Ser Pro Thr Thr Ile Ala Gln 305 310 315 320 Met Leu Asp Phe Ala Ala
Arg His Gln Ile Glu Pro Val Thr Glu Ile 325 330 335 Phe Pro Phe Glu
Gln Val Asn Glu Ala Ile Asp Lys Leu Arg His Gly 340 345 350 Gln Pro
Arg Tyr Arg Leu Val Leu Lys Met 355 360 121362PRTCyanothece sp. PCC
8802 121Met Arg Gly Glu Arg Ile Val Arg Ser Gly Val Lys Glu Asp Ile
Leu 1 5 10 15 Cys Asn Asn Ala Ile Asn Thr Thr Ile Glu Val Lys Val
Val Ile Lys 20 25 30 Ala Tyr Ala Ala Ser Glu Pro Gly Lys Glu Leu
Asn Ser Phe Glu Tyr 35 40 45 Asp Pro Gly Leu Leu Gly Glu Glu Asp
Val Glu Ile Asn Val Gln Tyr 50 55 60 Cys Gly Ile Cys His Ser Asp
Leu Ser Met Leu Asp Asn Glu Trp Gly 65 70 75 80 Ile Thr Gln Tyr Pro
Phe Val Pro Gly His Glu Val Val Gly Thr Ile 85 90 95 Gly Ala Val
Gly Ser Lys Val Thr Thr Phe Gln Val Gly Gln Thr Val 100 105 110 Gly
Leu Gly Trp Phe Ser Arg Ser Cys Phe Asp Cys Glu Trp Cys Leu 115 120
125 Ser Gly Asp Gln Asn Leu Cys Gln Thr Ala Glu Gly Thr Ile Val Gly
130 135 140 Arg Pro Gly Gly Phe Ala Asp Lys Val Arg Ala His His Arg
Trp Val 145 150 155 160 Val Pro Leu Pro Ser Gly Val Asn Pro Glu Thr
Ala Gly Pro Leu Phe 165 170 175 Cys Gly Gly Ile Thr Val Phe Asn Pro
Ile Ile Gln Cys Gly Val Lys 180 185 190 Ser Thr Asp Arg Val Gly Val
Ile Gly Ile Gly Gly Leu Gly His Leu 195 200 205 Ala Ile Glu Phe Leu
His Ala Trp Gly Cys Glu Val Thr Ala Phe Ser 210 215 220 Ser Asn Pro
Glu Lys Glu Ser Glu Val Lys Gln Leu Gly Ala Asp Tyr 225 230 235 240
Phe Val Asn Ser Arg Asp Pro Glu Ala Ile Lys Ala Val Glu Asn Ser 245
250 255 Phe Asp Phe Ile Ile Ser Thr Val Asn Val Ser Leu Asp Trp Asn
Ser 260 265 270 Tyr Ile Leu Ala Leu Arg Pro Arg Gly Thr Leu His Phe
Val Gly Ala 275 280 285 Val Leu Asn Pro Ile Ser Thr Gln Ile Phe Pro
Leu Leu Met Gly Gln 290 295 300 Lys Thr Ile Ser Gly Ser Pro Thr Gly
Ser Pro Thr Thr Ile Ala Gln 305 310 315 320 Met Leu Asp Phe Ala Ala
Arg His Gln Ile Glu Pro Val Thr Glu Ile 325 330 335 Phe Pro Phe Glu
Gln Val Asn Glu Ala Ile Asp Lys Leu Arg His Gly 340 345 350 Gln Pro
Arg Tyr Arg Leu Val Leu Lys Met 355 360 122334PRTMicrocoleus
chthonoplastes PCC 7420 122Met Ile Lys Ala Tyr Ala Ala His Glu Pro
Gly Gly Gln Leu Gln Pro 1 5 10 15 Phe Glu Tyr Asp Pro Gly Thr Leu
Gly Asp Glu Glu Val Glu Ile Lys 20 25 30 Val Glu Tyr Cys Gly Ile
Cys His Ser Asp Leu Ser Met Leu Asp Asn 35 40 45 Glu Trp Gly Met
Thr Asp Tyr Pro Phe Val Pro Gly His Glu Val Val 50 55 60 Gly Thr
Ile Ala Ala Leu Gly Asp Lys Val Thr Thr Leu Asn Leu Gly 65 70 75 80
Gln Arg Val Gly Leu Gly Trp Phe Ser Gly Ser Cys Met Thr Cys Glu 85
90 95 Trp Cys Met Ser Gly Asn His Asn Leu Cys Ser Asn Ala Glu Gly
Thr 100 105 110 Ile Val Ser Arg His Gly Gly Phe Ala Asp Lys Val Arg
Ala Asp Tyr 115 120 125 Ser Trp Val Val Pro Leu Pro Asp Gly Ile Asn
Pro Ala Thr Ala Gly 130 135 140 Pro Leu Phe Cys Gly Gly Ile Thr Val
Phe Asn Pro Ile Val Gln Phe 145 150 155 160 Asp Ile Lys Pro Ser Asp
Arg Val Gly Val Ile Gly Ile Gly Gly Leu 165 170 175 Gly His Ile Ala
Leu Gly Phe Leu Gln Ala Trp Gly Cys Glu Ile Thr 180 185 190 Ala Phe
Ser Ser Ser Pro Asp Lys Glu Ala Glu Ala Arg Glu Leu Gly 195 200 205
Ala Thr His Phe Ile Asn Ser Gly Asp Val Asn Ala Leu Glu Ser Val 210
215 220 Gln Asn Ser Phe Asp Phe Ile Leu Ala Thr Ala Asn Ala Asp Leu
Asp 225 230 235 240 Trp Asn Ala Tyr Ile Ala Ala Leu Arg Pro Lys Gly
Arg Leu His Phe 245 250 255 Val Gly Val Ile Pro Asn Pro Leu Ser Thr
Pro Ile Phe Pro Leu Ile 260 265 270 Leu Gly Gln Lys Ser Ile Ser Ala
Ser Pro Val Gly Ser Pro Ala Thr 275 280 285 Ile Ser Gln Met Ile Asn
Phe Ala Ala Arg Gln Gly Val Glu Pro Ile 290 295 300 Thr Glu Thr Phe
Ser Phe Glu Gln Val Asn Glu Ala Met Glu Lys Leu 305 310 315 320 Arg
His Gly Lys Pro Arg Tyr Arg Leu Val Leu Lys His Ser 325 330
123334PRTMicrocystis aeruginosa NIES-843 123Met Ile Arg Ala Tyr Ala
Ala Arg Glu Lys Gly Gly Lys Leu Glu Pro 1 5 10 15 Phe Asp Tyr Asp
Pro Gly Ile Leu Ala Asp Glu Asp Val Glu Ile Ala 20 25 30 Val Glu
Tyr Cys Gly Ile Cys His Ser Asp Leu Ser Met Leu Asp Asn 35 40 45
Asp Trp Gly Leu Thr Thr Tyr Pro Phe Val Pro Gly His Glu Val Val 50
55 60 Gly Thr Ile Ala Ala Leu Gly Ala Lys Val Lys Glu Leu Lys Leu
Gly 65 70 75 80 Gln Arg Val Gly Leu Gly Trp Phe Ser Arg Ser Cys Ser
Thr Cys Glu 85 90 95 Thr Cys Met Ser Gly Asp Gln Asn Leu Cys Ala
Thr Ala Glu Gly Thr 100 105 110 Ile Val Gly Arg His Gly Gly Phe Ala
Asp Arg Val Arg Ala His His 115 120 125 Ser Trp Leu Val Pro Leu Gly
Asn Gln Leu Asp Ala Ala Lys Ala Gly 130 135 140 Pro Leu Phe Cys Gly
Gly Ile Thr Val Phe Asn Pro Ile Val Gln Phe 145 150 155 160 Asn Ile
Lys Pro Thr Ala Arg Val Gly Val Ile Gly Ile Gly Gly Leu 165 170 175
Gly His Ile Ala Leu Lys Phe Leu Lys Ala Trp Gly Cys Glu Val Thr 180
185 190 Ala Phe Ser Ser Ser Pro Asp Lys Glu Thr Glu Ala Lys Glu Leu
Gly 195 200 205 Ala Thr His Phe Ile Asn Ser Arg Asp Pro Glu Ala Leu
Gln Ser Val 210 215 220 Gln Asn Tyr Phe Asp Phe Ile Ile Ser Thr Val
Asn Val Asn Leu Asp 225 230 235 240 Trp Gly Leu Tyr Ile Ala Cys Leu
Arg Pro Lys Gly Arg Leu His Ile 245 250 255 Val Gly Ala Val Leu Glu
Pro Met Ala Thr Tyr Ala Phe Pro Leu Ile 260 265 270 Met Gly Gln Lys
Ser Ile Ser Gly Ser Pro Leu Gly Ser Pro Ser Thr 275 280 285 Ile Asn
Lys Met Ile Glu Phe Ala Ser Arg His Gly Ile Glu Pro Val 290 295 300
Thr Glu Ile Tyr Pro Ile Ser Gln Val Asn Glu Ala Met Glu Lys Leu 305
310 315 320 Arg Thr Gly Gln Pro Lys Tyr Arg Leu Val Leu Gln Ile Lys
325 330 124334PRTMicrocystis aeruginosa PCC 7806 124Met Ile Arg Ala
Tyr Ala Ala Gln Glu Lys Gly Gly Lys Leu Glu Pro 1 5 10 15 Phe Asp
Tyr Asp Pro Gly Ile Leu Ala Asp Glu Asp Val Glu Ile Ala 20 25 30
Val Glu Tyr Cys Gly Ile Cys His Ser Asp Leu Ser Met Leu Asp Asn 35
40 45 Asp Trp Gly Leu Thr Thr Tyr Pro Phe Val Pro Gly His Glu Val
Val 50 55 60 Gly Thr Ile Ala Ala Leu Gly Ala Lys Val Lys Glu Leu
Lys Leu Gly 65 70 75 80 Gln Arg Val Gly Leu Gly Trp Phe Ser Arg Ser
Cys Ser Thr Cys Glu 85 90 95 Thr Cys Met Ser Gly Asp Gln Asn Leu
Cys Ala Thr Ala Glu Gly Thr 100 105 110 Ile Val Gly Arg His Gly Gly
Phe Ala Glu Arg Val Arg Ala His His 115 120 125 Ser Trp Leu Val Pro
Leu Pro Asp Gln Leu Asp Ala Ala Lys Ala Gly 130 135 140 Pro Leu Phe
Cys Gly Gly Ile Thr Val Phe Asn Pro Ile Val Gln Phe 145 150 155 160
Asn Ile Lys Pro Thr Ala Arg Val Gly Val Ile Gly Ile Gly Gly Leu 165
170 175 Gly His Ile Ala Leu Lys Phe Leu Lys Ala Trp Gly Cys Glu Val
Thr 180 185 190 Ala Phe Ser Ser Ser Pro Asp Lys Glu Thr Glu Ala Lys
Glu Leu Gly 195 200 205 Ala Thr His Phe Ile Asn Ser Arg Asp Pro Glu
Ala Leu Gln Ser Val 210 215 220 Gln Asn Tyr Phe Asp Phe Ile Ile Ser
Thr Val Asn Val Asn Leu Asp 225 230 235 240 Trp Gly Leu Tyr Ile Ala
Cys Leu Arg Pro Lys Gly Arg Leu His Ile 245 250 255 Val Gly Ala Val
Leu Glu Pro Met Ala Thr Tyr Ala Phe Pro Leu Ile 260 265 270 Met Gly
Gln Lys Ser Ile Ser Gly Ser Pro Leu Gly Ser Pro Ser Thr 275 280 285
Val Ser Lys Met Ile Glu Phe Ala Ser Arg His Gly Ile Glu Pro Val 290
295 300 Thr Glu Thr Tyr Pro Ile Ser Arg Val Asn Glu Ala Met Glu Lys
Leu 305 310 315 320 Arg Thr Gly Gln Pro Lys Tyr Arg Leu Val Leu Gln
Ile Lys 325 330 125333PRTSynechococcus sp. WH 5701 125Met Gln Ile
Thr Val Trp Gln Ala Leu Ala Lys Gly Gly Arg Leu Glu 1 5 10 15 Arg
Ser Gln Ala Thr Leu Leu Asp Pro Gly Pro Asp Glu Val Leu Leu 20 25
30 Glu Val Leu His Cys Gly Leu Cys His Ser Asp Leu Ser Met Leu Asp
35 40 45 Asn Ser Trp Gly Ile Ser Thr Tyr Pro Leu Val Pro Gly His
Glu Val 50 55 60 Val Gly Arg Val Ala Ala Val Gly Ala Gly Val Asp
Ser Gly Leu Leu 65 70 75 80 Gly Ser Ile Gln Gly Leu Gly Trp Ile Ala
Gly Ser Cys Arg His Cys 85 90 95 Asp Trp Cys Leu Gly Gly Asn Ala
Asn Leu Cys Pro Ser Leu Glu Ala 100 105 110 Ser Val Val Gly Arg His
Gly Gly Phe Ala Ser His Val Met Ala His 115 120 125 Gln Asp Trp Ile
Val Ala Ile Pro Asp Gly Val Ser Ala Ala Asp Ala 130 135 140 Gly Pro
Leu Phe Cys Gly Gly Ile Thr Val Phe Ala Pro Leu Phe Asp 145 150 155
160 Glu Ala Val Ser Pro Thr Ser Arg Val Ala Val Ile Gly Ile Gly Gly
165 170 175 Leu Gly His Met Ala Leu Gln Phe Ala Arg Ala Trp Gly Cys
Glu Val 180 185 190 Thr Ala Val Thr Thr Ser Pro Ala Lys Ala Asp Glu
Ala Arg Arg Leu 195 200 205 Gly Ala His Arg Val Leu Ala Leu Ser Glu
Leu Gly Asp His Pro Gly 210 215 220 Val Phe Asp Leu Ile Ile Asn Thr
Ser Asn His Asp Leu Asp Trp Pro 225 230 235 240 Ala Leu Ile Gly Ser
Leu Ala Pro Leu Gly Arg Leu His Gln Leu Gly 245 250 255 Val Pro Leu
Ser Pro Leu Gln Ile Pro Ala Phe Pro Leu Ile Ala Gly 260 265 270 Arg
Arg Ser Val Thr Gly Ser Pro Thr Ser Ser Pro Ala Ser Leu Arg 275 280
285 Arg Met Val Glu Phe Cys Ala Arg His Gly Ile Ala Pro Leu Val Glu
290 295 300 His Leu Pro Met Ala Glu Ile Asn Thr Ala Ile Glu Arg Leu
Arg Gln 305 310 315 320 Gly Asp Val Arg Tyr Arg Phe Val Leu Asp Gly
Pro Ala 325 330
126336PRTSynechococcus sp. RS9917 126Met Val Val Thr Ile Thr Val
Trp Gln Ala Arg Glu Ala Gly Ala Pro 1 5 10 15 Leu Glu Arg Ala Glu
Arg Ala Met Leu Glu Pro Ala Ala Gly Glu Leu 20 25 30 Val Leu Glu
Val Leu His Cys Gly Leu Cys His Ser Asp Leu Ser Met 35 40 45 Leu
Asp Asn Asn Trp Gly Leu Ser Ala Tyr Pro Leu Val Pro Gly His 50 55
60 Glu Val Val Gly Arg Val Val Arg Val Gly Glu Gly Val Asp Pro Gly
65 70 75 80 Val Ile Gly Glu Leu Arg Gly Leu Gly Trp Ile Ser Gly Ser
Cys Met 85 90 95 His Cys Ala Leu Cys Leu Gly Gly Thr Ala Asn Leu
Cys Gly Ser Leu 100 105 110 Glu Ala Thr Ile Val Gly Arg Gln Gly Gly
Phe Ala Ser His Val Thr 115 120 125 Ala Arg Gln Asp Trp Ala Ile Arg
Leu Pro Glu Gly Met Asp Pro Ala 130 135 140 Ala Ala Gly Pro Leu Phe
Cys Gly Gly Ile Thr Val Phe Ala Pro Leu 145 150 155 160 Val Asp Glu
Val Val Ser Pro Thr Ala His Val Ala Val Ile Gly Ile 165 170 175 Gly
Gly Leu Gly His Met Ala Leu Gln Phe Ala Arg Ala Trp Gly Cys 180 185
190 Glu Val Thr Ala Leu Thr Thr His Leu Ala Lys Ala Glu Glu Ala Lys
195 200 205 Arg Phe Gly Ala His His Val Glu Ser Leu Glu Glu Leu Pro
Asp Leu 210 215 220 Ala Gly Arg Phe Asp Leu Val Ile Asn Thr Val Asn
His Ala Leu Asp 225 230 235 240 Trp Gly Ala Val Met Gly Ser Leu Ala
Pro Leu Gly Arg Leu His Gln 245 250 255 Leu Gly Ala Val Leu Glu Pro
Leu Gln Val Ser Ala Phe Asp Leu Ile 260 265 270 Met Ala Arg Arg Ser
Ile Thr Gly Ser Pro Thr Ser Ser Pro Ala Ser 275 280 285 Leu Met Lys
Met Val Glu Phe Cys Val Arg His Asn Ile Arg Pro Gln 290 295 300 Val
Glu His Leu Pro Met Asp Arg Leu Asn Glu Ala Ile Asp Arg Leu 305 310
315 320 Arg Arg Gly Asp Val Arg Tyr Arg Phe Val Leu Asp Ser Val Ala
Asp 325 330 335 127333PRTSynechococcus sp. WH 5701 127Met Gln Ile
Thr Val Trp Gln Ala Leu Ala Lys Gly Gly Arg Leu Glu 1 5 10 15 Arg
Ser Gln Ala Thr Leu Leu Asp Pro Gly Pro Asp Glu Val Leu Leu 20 25
30 Glu Val Leu His Cys Gly Leu Cys His Ser Asp Leu Ser Met Leu Asp
35 40 45 Asn Ser Trp Gly Ile Ser Thr Tyr Pro Leu Val Pro Gly His
Glu Val 50 55 60 Val Gly Arg Val Ala Ala Val Gly Ala Gly Val Asp
Ser Gly Leu Leu 65 70 75 80 Gly Ser Ile Gln Gly Leu Gly Trp Ile Ala
Gly Ser Cys Arg His Cys 85 90 95 Asp Trp Cys Leu Gly Gly Asn Ala
Asn Leu Cys Pro Ser Leu Glu Ala 100 105 110 Ser Val Val Gly Arg His
Gly Gly Phe Ala Ser His Val Met Ala His 115 120 125 Gln Asp Trp Ile
Val Ala Ile Pro Asp Gly Val Ser Ala Ala Asp Ala 130 135 140 Gly Pro
Leu Phe Cys Gly Gly Ile Thr Val Phe Ala Pro Leu Phe Asp 145 150 155
160 Glu Ala Val Ser Pro Thr Ser Arg Val Ala Val Ile Gly Ile Gly Gly
165 170 175 Leu Gly His Met Ala Leu Gln Phe Ala Arg Ala Trp Gly Cys
Glu Val 180 185 190 Thr Ala Val Thr Thr Ser Pro Ala Lys Ala Asp Glu
Ala Arg Arg Leu 195 200 205 Gly Ala His Arg Val Leu Ala Leu Ser Glu
Leu Gly Asp His Pro Gly 210 215 220 Val Phe Asp Leu Ile Ile Asn Thr
Ser Asn His Asp Leu Asp Trp Pro 225 230 235 240 Ala Leu Ile Gly Ser
Leu Ala Pro Leu Gly Arg Leu His Gln Leu Gly 245 250 255 Val Pro Leu
Ser Pro Leu Gln Ile Pro Ala Phe Pro Leu Ile Ala Gly 260 265 270 Arg
Arg Ser Val Thr Gly Ser Pro Thr Ser Ser Pro Ala Ser Leu Arg 275 280
285 Arg Met Val Glu Phe Cys Ala Arg His Gly Ile Ala Pro Leu Val Glu
290 295 300 His Leu Pro Met Ala Glu Ile Asn Thr Ala Ile Glu Arg Leu
Arg Gln 305 310 315 320 Gly Asp Val Arg Tyr Arg Phe Val Leu Asp Gly
Pro Ala 325 330 128330PRTSynechococcus sp. WH 7803 128Met Ile Ser
Val Trp Gln Ala Pro Ser Ala Gly Ala Pro Leu Glu Cys 1 5 10 15 Gly
Gln Arg Pro Ala Pro Glu Pro Ala Ala Asp Glu Leu Val Leu Glu 20 25
30 Val Met His Cys Gly Leu Cys His Ser Asp Leu Ser Met Ile Gly Asn
35 40 45 His Trp Gly Val Ser Arg Tyr Pro Leu Val Pro Gly His Glu
Val Ile 50 55 60 Gly Arg Val Thr Ala Val Gly Glu Gly Val Asp Pro
Gly Leu Ile Gly 65 70 75 80 Asp Val Arg Gly Leu Gly Trp Ile Ser Gly
Ser Cys Asn His Cys Ser 85 90 95 Leu Cys Leu Gly Gly Asp Gln Asn
Leu Cys Thr Ser Leu Glu Ala Thr 100 105 110 Ile Val Gly Arg Gln Gly
Gly Phe Ala Ser His Val Val Ala Arg Gln 115 120 125 Asp Trp Ala Ile
Pro Leu Pro Pro Gly Leu Asp Pro Ala Asp Ala Gly 130 135 140 Pro Leu
Phe Cys Gly Gly Ile Thr Val Phe Ala Pro Leu Val Asp Glu 145 150 155
160 Ala Val Ser Pro Thr Ala His Val Ala Val Val Gly Ile Gly Gly Leu
165 170 175 Gly His Ile Ala Leu Gln Phe Ala Arg Ala Trp Gly Cys Glu
Val Thr 180 185 190 Ala Ile Thr Thr Asn Leu Ala Lys Ala Glu Gln Ala
Arg Arg Phe Gly 195 200 205 Ala His His Val Glu Glu Leu Glu Met Leu
Pro Asp Leu Gln Ser Arg 210 215 220 Phe Asp Leu Val Ile Asn Thr Val
Asn His Pro Leu Asp Trp Ser Ala 225 230 235 240 Val Met Ala Ser Leu
Arg Pro Arg Gly Arg Leu His Gln Leu Gly Ala 245 250 255 Val Leu Glu
Pro Ile Gln Val Gly Ala Phe Asp Leu Ile Pro Ala Arg 260 265 270 Arg
Ser Ile Thr Gly Ser Pro Thr Ser Ser Pro Ala Ser Leu Gln Lys 275 280
285 Met Val Glu Phe Cys Val Arg His Asn Ile Leu Pro Leu Val Glu His
290 295 300 Leu Pro Met Asp Gln Val Asn Val Ala Ile Gln Arg Leu Ala
Lys Gly 305 310 315 320 Asp Val Arg Tyr Arg Phe Val Leu Asp Ala 325
330 129330PRTSynechococcus sp. WH 7805 129Met Ile Ser Val Trp Gln
Ala Pro Ser Ala Gly Ala Pro Leu Glu Cys 1 5 10 15 Ala Gln Arg Pro
Ala Leu Gln Pro Val Ala Asp Glu Leu Val Leu Glu 20 25 30 Val Met
His Cys Gly Leu Cys His Ser Asp Leu Ser Met Ile Gly Asn 35 40 45
His Trp Gly Val Ser Arg Tyr Pro Leu Val Pro Gly His Glu Val Ile 50
55 60 Gly Arg Val Thr Ala Val Gly Glu Gly Val Asp Pro Gly Val Ile
Gly 65 70 75 80 Glu Val Arg Gly Leu Gly Trp Ile Ser Gly Ser Cys Asn
His Cys Ser 85 90 95 Leu Cys Leu Gly Gly Asp Gln Asn Leu Cys Ser
Ser Leu Glu Ala Thr 100 105 110 Ile Val Gly Arg Gln Gly Gly Phe Ala
Ser His Val Val Ala Arg Gln 115 120 125 Asp Trp Thr Ile Pro Leu Pro
Thr Gly Leu Asp Pro Ala Glu Ala Gly 130 135 140 Pro Leu Phe Cys Gly
Gly Val Thr Val Phe Ala Pro Leu Val Asp Glu 145 150 155 160 Ala Val
Ser Pro Thr Ala His Val Ala Val Val Gly Ile Gly Gly Leu 165 170 175
Gly His Ile Ala Leu Gln Phe Ala Arg Ala Trp Gly Cys Glu Val Thr 180
185 190 Ala Ile Thr Thr Asn Pro Ala Lys Thr Glu Gln Ala Arg Arg Phe
Gly 195 200 205 Ala His His Val Glu Glu Leu Glu Ala Leu Ser Asp Leu
Gln Arg Arg 210 215 220 Phe Asp Leu Val Ile Asn Thr Val Asn His Pro
Leu Asp Trp Ser Ala 225 230 235 240 Val Met Ala Ser Leu Lys Pro Arg
Gly Arg Leu His Gln Leu Gly Ala 245 250 255 Val Leu Glu Pro Ile Gln
Val Gly Ala Phe Asp Leu Ile Ser Ala Arg 260 265 270 Arg Ser Ile Thr
Gly Ser Pro Thr Ser Ser Pro Ala Ser Leu Leu Lys 275 280 285 Met Val
Glu Phe Cys Val Arg His Asn Ile Leu Pro Leu Val Glu His 290 295 300
Leu Pro Met Asp Gln Val Asn Val Ala Ile Glu Arg Leu Ala Lys Gly 305
310 315 320 Asp Val Arg Tyr Arg Phe Val Leu Asp Ala 325 330
1301692DNASaccharomyces cerevisiae 130atgtctgaaa ttactttggg
taaatatttg ttcgaaagat taaagcaagt caacgttaac 60accgttttcg gtttgccagg
tgacttcaac ttgtccttgt tggacaagat ctacgaagtt 120gaaggtatga
gatgggctgg taacgccaac gaattgaacg ctgcttacgc cgctgatggt
180tacgctcgta tcaagggtat gtcttgtatc atcaccacct tcggtgtcgg
tgaattgtct 240gctttgaacg gtattgccgg ttcttacgct gaacacgtcg
gtgttttgca cgttgttggt 300gtcccatcca tctctgctca agctaagcaa
ttgttgttgc accacacctt gggtaacggt 360gacttcactg ttttccacag
aatgtctgcc aacatttctg aaaccactgc tatgatcact 420gacattgcta
ccgccccagc tgaaattgac agatgtatca gaaccactta cgtcacccaa
480agaccagtct acttaggttt gccagctaac ttggtcgact tgaacgtccc
agctaagttg 540ttgcaaactc caattgacat gtctttgaag ccaaacgatg
ctgaatccga aaaggaagtc 600attgacacca tcttggcttt ggtcaaggat
gctaagaacc cagttatctt ggctgatgct 660tgttgttcca gacacgacgt
caaggctgaa actaagaagt tgattgactt gactcaattc 720ccagctttcg
tcaccccaat gggtaagggt tccattgacg aacaacaccc aagatacggt
780ggtgtttacg tcggtacctt gtccaagcca gaagttaagg aagccgttga
atctgctgac 840ttgattttgt ctgtcggtgc tttgttgtct gatttcaaca
ccggttcttt ctcttactct 900tacaagacca agaacattgt cgaattccac
tccgaccaca tgaagatcag aaacgccact 960ttcccaggtg tccaaatgaa
attcgttttg caaaagttgt tgaccactat tgctgacgcc 1020gctaagggtt
acaagccagt tgctgtccca gctagaactc cagctaacgc tgctgtccca
1080gcttctaccc cattgaagca agaatggatg tggaaccaat tgggtaactt
cttgcaagaa 1140ggtgatgttg tcattgctga aaccggtacc tccgctttcg
gtatcaacca aaccactttc 1200ccaaacaaca cctacggtat ctctcaagtc
ttatggggtt ccattggttt caccactggt 1260gctaccttgg gtgctgcttt
cgctgctgaa gaaattgatc caaagaagag agttatctta 1320ttcattggtg
acggttcttt gcaattgact gttcaagaaa tctccaccat gatcagatgg
1380ggcttgaagc catacttgtt cgtcttgaac aacgatggtt acaccattga
aaagttgatt 1440cacggtccaa aggctcaata caacgaaatt caaggttggg
accacctatc cttgttgcca 1500actttcggtg ctaaggacta tgaaacccac
agagtcgcta ccaccggtga atgggacaag 1560ttgacccaag acaagtcttt
caacgacaac tctaagatca gaatgattga aatcatgttg 1620ccagtcttcg
atgctccaca aaacttggtt gaacaagcta agttgactgc tgctaccaac
1680gctaagcaat aa 1692131563PRTSaccharomyces cerevisiae 131Met Ser
Glu Ile Thr Leu Gly Lys Tyr Leu Phe Glu Arg Leu Lys Gln 1 5 10 15
Val Asn Val Asn Thr Val Phe Gly Leu Pro Gly Asp Phe Asn Leu Ser 20
25 30 Leu Leu Asp Lys Ile Tyr Glu Val Glu Gly Met Arg Trp Ala Gly
Asn 35 40 45 Ala Asn Glu Leu Asn Ala Ala Tyr Ala Ala Asp Gly Tyr
Ala Arg Ile 50 55 60 Lys Gly Met Ser Cys Ile Ile Thr Thr Phe Gly
Val Gly Glu Leu Ser 65 70 75 80 Ala Leu Asn Gly Ile Ala Gly Ser Tyr
Ala Glu His Val Gly Val Leu 85 90 95 His Val Val Gly Val Pro Ser
Ile Ser Ala Gln Ala Lys Gln Leu Leu 100 105 110 Leu His His Thr Leu
Gly Asn Gly Asp Phe Thr Val Phe His Arg Met 115 120 125 Ser Ala Asn
Ile Ser Glu Thr Thr Ala Met Ile Thr Asp Ile Ala Thr 130 135 140 Ala
Pro Ala Glu Ile Asp Arg Cys Ile Arg Thr Thr Tyr Val Thr Gln 145 150
155 160 Arg Pro Val Tyr Leu Gly Leu Pro Ala Asn Leu Val Asp Leu Asn
Val 165 170 175 Pro Ala Lys Leu Leu Gln Thr Pro Ile Asp Met Ser Leu
Lys Pro Asn 180 185 190 Asp Ala Glu Ser Glu Lys Glu Val Ile Asp Thr
Ile Leu Ala Leu Val 195 200 205 Lys Asp Ala Lys Asn Pro Val Ile Leu
Ala Asp Ala Cys Cys Ser Arg 210 215 220 His Asp Val Lys Ala Glu Thr
Lys Lys Leu Ile Asp Leu Thr Gln Phe 225 230 235 240 Pro Ala Phe Val
Thr Pro Met Gly Lys Gly Ser Ile Asp Glu Gln His 245 250 255 Pro Arg
Tyr Gly Gly Val Tyr Val Gly Thr Leu Ser Lys Pro Glu Val 260 265 270
Lys Glu Ala Val Glu Ser Ala Asp Leu Ile Leu Ser Val Gly Ala Leu 275
280 285 Leu Ser Asp Phe Asn Thr Gly Ser Phe Ser Tyr Ser Tyr Lys Thr
Lys 290 295 300 Asn Ile Val Glu Phe His Ser Asp His Met Lys Ile Arg
Asn Ala Thr 305 310 315 320 Phe Pro Gly Val Gln Met Lys Phe Val Leu
Gln Lys Leu Leu Thr Thr 325 330 335 Ile Ala Asp Ala Ala Lys Gly Tyr
Lys Pro Val Ala Val Pro Ala Arg 340 345 350 Thr Pro Ala Asn Ala Ala
Val Pro Ala Ser Thr Pro Leu Lys Gln Glu 355 360 365 Trp Met Trp Asn
Gln Leu Gly Asn Phe Leu Gln Glu Gly Asp Val Val 370 375 380 Ile Ala
Glu Thr Gly Thr Ser Ala Phe Gly Ile Asn Gln Thr Thr Phe 385 390 395
400 Pro Asn Asn Thr Tyr Gly Ile Ser Gln Val Leu Trp Gly Ser Ile Gly
405 410 415 Phe Thr Thr Gly Ala Thr Leu Gly Ala Ala Phe Ala Ala Glu
Glu Ile 420 425 430 Asp Pro Lys Lys Arg Val Ile Leu Phe Ile Gly Asp
Gly Ser Leu Gln 435 440 445 Leu Thr Val Gln Glu Ile Ser Thr Met Ile
Arg Trp Gly Leu Lys Pro 450 455 460 Tyr Leu Phe Val Leu Asn Asn Asp
Gly Tyr Thr Ile Glu Lys Leu Ile 465 470 475 480 His Gly Pro Lys Ala
Gln Tyr Asn Glu Ile Gln Gly Trp Asp His Leu 485 490 495 Ser Leu Leu
Pro Thr Phe Gly Ala Lys Asp Tyr Glu Thr His Arg Val 500 505 510 Ala
Thr Thr Gly Glu Trp Asp Lys Leu Thr Gln Asp Lys Ser Phe Asn 515 520
525 Asp Asn Ser Lys Ile Arg Met Ile Glu Ile Met Leu Pro Val Phe Asp
530 535 540 Ala Pro Gln Asn Leu Val Glu Gln Ala Lys Leu Thr Ala Ala
Thr Asn 545 550 555 560 Ala Lys Gln 1321047DNASaccharomyces
cerevisiae 132atgtctatcc cagaaactca aaaaggtgtt atcttctacg
aatcccacgg taagttggaa 60tacaaagata ttccagttcc aaagccaaag gccaacgaat
tgttgatcaa cgttaaatac 120tctggtgtct gtcacactga cttgcacgct
tggcacggtg actggccatt gccagttaag 180ctaccattag tcggtggtca
cgaaggtgcc ggtgtcgttg tcggcatggg tgaaaacgtt 240aagggctgga
agatcggtga ctacgccggt atcaaatggt tgaacggttc ttgtatggcc
300tgtgaatact gtgaattggg taacgaatcc aactgtcctc acgctgactt
gtctggttac 360acccacgacg gttctttcca acaatacgct accgctgacg
ctgttcaagc cgctcacatt 420cctcaaggta ccgacttggc ccaagtcgcc
cccatcttgt gtgctggtat caccgtctac 480aaggctttga agtctgctaa
cttgatggcc ggtcactggg ttgctatctc cggtgctgct 540ggtggtctag
gttctttggc tgttcaatac gccaaggcta tgggttacag agtcttgggt
600attgacggtg gtgaaggtaa ggaagaatta ttcagatcca tcggtggtga
agtcttcatt 660gacttcacta aggaaaagga cattgtcggt gctgttctaa
aggccactga cggtggtgct 720cacggtgtca tcaacgtttc cgtttccgaa
gccgctattg aagcttctac cagatacgtt 780agagctaacg gtaccaccgt
tttggtcggt atgccagctg gtgccaagtg ttgttctgat 840gtcttcaacc
aagtcgtcaa gtccatctct attgttggtt cttacgtcgg taacagagct
900gacaccagag aagctttgga cttcttcgcc agaggtttgg tcaagtctcc
aatcaaggtt 960gtcggcttgt ctaccttgcc agaaatttac gaaaagatgg
aaaagggtca aatcgttggt 1020agatacgttg ttgacacttc taaataa
1047133348PRTSaccharomyces cerevisiae 133Met Ser Ile Pro Glu Thr
Gln Lys Gly Val Ile Phe Tyr Glu Ser His 1 5 10 15 Gly Lys Leu Glu
Tyr Lys Asp Ile Pro Val Pro Lys Pro Lys Ala Asn 20 25 30 Glu Leu
Leu Ile Asn Val Lys Tyr Ser Gly Val Cys His Thr Asp Leu 35 40 45
His Ala Trp His Gly Asp Trp Pro Leu Pro Val Lys Leu Pro Leu Val 50
55 60 Gly Gly His Glu Gly Ala Gly Val Val Val Gly Met Gly Glu Asn
Val 65 70 75 80 Lys Gly Trp Lys Ile Gly Asp Tyr Ala Gly Ile Lys Trp
Leu Asn Gly 85 90 95 Ser Cys Met Ala Cys Glu Tyr Cys Glu Leu Gly
Asn Glu Ser Asn Cys 100 105 110 Pro His Ala Asp Leu Ser Gly Tyr Thr
His Asp Gly Ser Phe Gln Gln 115 120 125 Tyr Ala Thr Ala Asp Ala Val
Gln Ala Ala His Ile Pro Gln Gly Thr 130 135 140 Asp Leu Ala Gln Val
Ala Pro Ile Leu Cys Ala Gly Ile Thr Val Tyr 145 150 155 160 Lys Ala
Leu Lys Ser Ala Asn Leu Met Ala Gly His Trp Val Ala Ile 165 170 175
Ser Gly Ala Ala Gly Gly Leu Gly Ser Leu Ala Val Gln Tyr Ala Lys 180
185 190 Ala Met Gly Tyr Arg Val Leu Gly Ile Asp Gly Gly Glu Gly Lys
Glu 195 200 205 Glu Leu Phe Arg Ser Ile Gly Gly Glu Val Phe Ile Asp
Phe Thr Lys 210 215 220 Glu Lys Asp Ile Val Gly Ala Val Leu Lys Ala
Thr Asp Gly Gly Ala 225 230 235 240 His Gly Val Ile Asn Val Ser Val
Ser Glu Ala Ala Ile Glu Ala Ser 245 250 255 Thr Arg Tyr Val Arg Ala
Asn Gly Thr Thr Val Leu Val Gly Met Pro 260 265 270 Ala Gly Ala Lys
Cys Cys Ser Asp Val Phe Asn Gln Val Val Lys Ser 275 280 285 Ile Ser
Ile Val Gly Ser Tyr Val Gly Asn Arg Ala Asp Thr Arg Glu 290 295 300
Ala Leu Asp Phe Phe Ala Arg Gly Leu Val Lys Ser Pro Ile Lys Val 305
310 315 320 Val Gly Leu Ser Thr Leu Pro Glu Ile Tyr Glu Lys Met Glu
Lys Gly 325 330 335 Gln Ile Val Gly Arg Tyr Val Val Asp Thr Ser Lys
340 345 134232DNAChlamydomonas 134cactgaagac tgggatgagc gcacctgtac
ctgccagtat ggtaccggcg cgctaccgat 60gcgtgtagta gagcttgctg ccatacagta
actctggtac ccccagccac cgggcgtagc 120gagcagactc aataagtatg
atgggttctt attgcagccg ctgttacagt ttacagcgca 180agggaacacg
cccctcattc acagaactaa ctcaacctac tccataaaca tg
2321351202DNAChlamydomonas 135aggacagagt gcgtgtggcc agggcacagg
cgcccatcca gcagctcgcc gtctaagtag 60gccgtccatg cagtgccggt cgggtccgga
accacgaacc agtggtgagg gaaaacatcg 120ttacgctctg ggtgagcact
acacgatggg tattcctcaa ttagttccgg gtaagcgaca 180accgagcgag
tcgccgcgag tgcaagcagt gcaattgaca ggctgaacgc ggccatcggc
240aatccgcagc ggaactgtct caatttactt cgtgacctat gtatgttgaa
tatgctgtcg 300ggtcgaccag cggccagtag gagtggccac tcggtgtgga
agagtgggcc gcgctggact 360gctggcgcga cctttgaacg cggacaactt
gcaaaagtat ttgattatca tcaacgcaaa 420agtgatgctg gcgaattgga
gggggcgccg cgaggcacgc gccaggctgc tgcgcgcttg 480ccatgcgcgt
gccgggtctg tccgagagtc gagccaagtc gctgctttat gacacaacaa
540tatatcgtta gttgctctga aggcgaccaa gaacctcgcg gggcgtgcta
atgtaggaga 600aacaagcaag caaccgacac gaaccagctt gctttcccgc
cgtgcagtta atgcatgtgc 660gcatggatgc atgaaattcc tatggaagct
gcgcatttcc cacattgaaa aacgagcgcg 720aaaaacgcgc gtaggagtgc
atcgtgcgtg ccttttaagc gatgtgtgcg tgcaaagtat 780tgcattataa
ttgcatgata actcttgtat gtttagagtc agtagggcag ggccggccgg
840cagtcagacc tggattggcg acacaagtct gccaggacga ctgcggtggc
aaagttggtg 900gagattttcg agttggagct cctctgtgtc tgtgtcagac
actctaactt tcttgtctcc 960tgttttctgc tttgcctttc agcggccggt
tgcattggat gtacaagtgt ggcgtgtgga 1020aagcgcgcac gacacgcgcg
cgcgacgccc gccgcggctg gcaccagcct ggcgctggat 1080ccaatctgct
gcacgccgcg cataaatgca agtgtctact gtgatattgg cataatttaa
1140acactcccca ctggctcact aggactattg ctgctcgcaa gcccgtcgca
cagttaacca 1200tg 1202136680DNAartificialsynthetic ble marker gene
136atggccaggt gagtcgacga gcaagcccgg cggatcaggc agcgtgcttg
cagatttgac 60ttgcaacgcc cgcattgtgt cgacgaaggc ttttggctcc tctgtcgctg
tctcaagcag 120catctaaccc tgcgtcgccg tttccatttg caggatggcc
aagctgacca gcgccgttcc 180ggtgctcacc gcgcgcgacg tcgccggagc
ggtcgagttc tggaccgacc ggctcgggtt 240ctcccgggac ttcgtggagg
acgacttcgc cggtgtggtc cgggacgacg tgaccctgtt 300catcagcgcg
gtccaggacc aggtgagtcg acgagcaagc ccggcggatc aggcagcgtg
360cttgcagatt tgacttgcaa cgcccgcatt gtgtcgacga aggcttttgg
ctcctctgtc 420gctgtctcaa gcagcatcta accctgcgtc gccgtttcca
tttgcaggac caggtggtgc 480cggacaacac cctggcctgg gtgtgggtgc
gcggcctgga cgagctgtac gccgagtggt 540cggaggtcgt gtccacgaac
ttccgggacg cctccgggcc ggccatgacc gagatcggcg 600agcagccgtg
ggggcgggag ttcgccctgc gcgacccggc cggcaactgc gtgcacttcg
660tggccgagga gcaggactaa 6801372579DNAChlamydomonas 137gtgtctgacc
gcgactttgt gatcgagacg gtgtttgcgg ccagcctgct gtgcgtgcac 60ctgtcgcgct
gggcggagga cctcatcatc tacagctccg gccccttcgg ctacgtgcag
120tgcagcgacg cctacgccac cggctcctcg ctcatgccgc agaagaagaa
ccccgacgcc 180ctggagctca tcaggtgcgg gagggatggg gtgggggtgg
gggggttaca ttcatggtta 240gttaagaagt gaaggcgtag ggggtggatg
gggtgggtta cattcatgaa catttaagaa 300gtgaaggcgt agccaggaac
agtagtagag cagacgcgtt gtagtgtgtg ggtttgggtg 360ggagggatgg
ttgggtaaag cggtacagga tgtactgagg actgcagacc gaaggagcgg
420gggaggggga gcaggcaggc ggggcgaggg gcgtgggggc gggggttact
ggcaccgtgc 480cgggtaagca acacgtgaca cggagatgca ccacacaaag
agggacgtgg ggagtggcag 540gcgggggcca gggctgagag gcgcgtgtgg
aggggtgcgg ggttgggcgg ggggctgttt 600catgataccg ctgcctccac
ctcctccacc gcctcctgcc acctccacct cccccactgc 660ccctccccgc
ctcctcctgc tgcaggggca agggcggtcg tgtgcagggc aacctgatgg
720gcgtcatggc ggtgctcaag ggcacgccca ccacatacaa caaggacttc
caggcgagag 780agcgagagcg agggagggag ggagagcgag ggagagggag
ggagagggag ggagagggag 840acagagggac agggacaggg acagggacag
ggacagggac agggacaggg gcaggggcag 900gggcaggggc aggggcaggg
gcaggggagg cgcccccggg ggcggcgggc ccggggcatg 960aggtcagaca
taggggcgct gcactgaggc cgcgaggcgg gcgggaggca gggggcgggg
1020ggcggggggc gggagcggac atgcgccgca aacacagacg ggttgagaaa
gcacaacgac 1080tggaacgcag tgggcttact gacaattcat cattgtgcgc
atatgtgtgt atgtgtatgt 1140gtgtgtttgt ttgtgcagga gtgttgggag
ctgctgtttg acacggtgga cacggtgcac 1200gacgtggtgc gcatcgccac
cggcgtgctg tccaccctgc ggatcaagcc cgaccgcatg 1260aaggccggtg
agcgtagccg agcagggctg gagcagcagc cgggcagcag tagcagcagg
1320gcaggggagc agcgggagcg ggagcagcag gaggggtggt tgggaagcgg
tgggggtagg 1380gtgggagcgg aggaagggaa ggaggagcag gagcaggagg
aagaggagga ggaagggcgg 1440tggggggtgg ggggtcgtgt ccttggccgc
atgggcggag gcggggaggc ggggaggagg 1500cggggaagca gagcctgcac
ccacgctccg cgggtcccta ccgtcttgcg cctaaccccg 1560tgcgcctagc
ctcttgcgcc caccccctta gtgcatcctg tacccctctt tccaaacatc
1620cttgcaactc cctgacctcc tcgccaaacc tcccccgccc ccaggcctgt
ccgccgacat 1680gctcgccacg gacttggccg agtacctggt gcgcaagggc
gtgccgttcc gggagacaca 1740ccaccacagg tgcggccggg cgggagggcg
tgagggcgtg ggtggggcat gcccggggtt 1800gtgagagcta tcgaacgttg
tgccgcgcct gtttcacaat gtcgggccac agggtatgca 1860gtttcctctc
catatgtata acaaactgac caccaatcat gcacgctcac acgctctccc
1920acacacacgc gcaccacgcc accacagcgg cgccgccgtg aagatggccg
aggaccgcgg 1980ctgcacgctg ttcgacctca ccgtggacga cctcaagacc
atccacccgc tcttcaccga 2040cgacgtggcg gcggtgagcg gcggcgcgga
gcagcagcag cagcagcagc agcagcagca 2100gcagcagtag cctggggggg
agcgtgtggg aggaacggcg ggggagggga ggcggggggt 2160gtcgtttgca
gccgagcgca cgtggtgctt tgccccattc catgccagca gggtgacaca
2220cctgaccatg ctggtgtgct gctaggtggt tcacacctac gtgtgaattt
gtgctggcgt 2280gcgcacacct tactgtggcc atgtgaacgg catcctcatg
tcctcgtgat tgcgcccggc 2340acattgccca caaccccgca ccacccagct
cctcaatcca gtgcaaggaa aggaaatgca 2400cgcccgccgc accaacaaca
cgacgcatgt gtttgccacg tgcgcgcaca cacgcgcagg 2460tgtgggactt
caaccgcagc gccgagatgc gcgacacgga gggcggcacc agcaagcgct
2520cggtgctgga gcaggtgcag aagatgcgca cctacctggc ggcggaggga
cagcactga 25791383454DNAartificialplasmid pSP124S 138gtggcacttt
tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt 60caaatatgta
tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
120ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt
gcggcatttt 180gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
aaaagatgct gaagatcagt 240tgggtgcacg agtgggttac atcgaactgg
atctcaacag cggtaagatc cttgagagtt 300ttcgccccga agaacgtttt
ccaatgatga gcacttttaa agttctgcta tgtggcgcgg 360tattatcccg
tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
420atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc
atgacagtaa 480gagaattatg cagtgctgcc ataaccatga gtgataacac
tgcggccaac ttacttctga 540caacgatcgg aggaccgaag gagctaaccg
cttttttgca caacatgggg gatcatgtaa 600ctcgccttga tcgttgggaa
ccggagctga atgaagccat accaaacgac gagcgtgaca 660ccacgatgcc
tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
720ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt
gcaggaccac 780ttctgcgctc ggcccttccg gctggctggt ttattgctga
taaatctgga gccggtgagc 840gtgggtctcg cggtatcatt gcagcactgg
ggccagatgg taagccctcc cgtatcgtag 900ttatctacac gacggggagt
caggcaacta tggatgaacg aaatagacag atcgctgaga 960taggtgcctc
actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
1020agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
ctttttgata 1080atctcatgac caaaatccct taacgtgagt tttcgttcca
ctgagcgtca gaccccgtag 1140aaaagatcaa aggatcttct tgagatcctt
tttttctgcg cgtaatctgc tgcttgcaaa 1200caaaaaaacc accgctacca
gcggtggttt gtttgccgga tcaagagcta ccaactcttt 1260ttccgaaggt
aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc
1320cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
gctctgctaa 1380tcctgttacc agtggctgct gccagtggcg ataagtcgtg
tcttaccggg ttggactcaa 1440gacgatagtt accggataag gcgcagcggt
cgggctgaac ggggggttcg tgcacacagc 1500ccagcttgga gcgaacgacc
tacaccgaac tgagatacct acagcgtgag ctatgagaaa 1560gcgccacgct
tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
1620caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
agtcctgtcg 1680ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
ctcgtcaggg gggcggagcc 1740tatggaaaaa cgccagcaac gcggcctttt
tacggttcct ggccttttgc tggccttttg 1800ctcacatgtt ctttcctgcg
ttatcccctg attctgtgga taaccgtatt accgcctttg 1860agtgagctga
taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
1920aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg
attcattaat 1980gcagctggca cgacaggttt cccgactgga aagcgggcag
tgagcgcaac gcaattaatg 2040tgagttagct cactcattag gcaccccagg
ctttacactt tatgcttccg gctcgtatgt 2100tgtgtggaat tgtgagcgga
taacaatttc acacaggaaa cagctatgac catgattacg 2160ccaagcgcgc
aattaaccct cactaaaggg aacaaaagct ggagctccac cgcggtggcg
2220gccgctctag aactagtgga tcccccgggc tgcaggaatt cgatatcaag
cttatcgata 2280ccgtcgacct cgagatttaa atgccagaag gagcgcagcc
aaaccaggat gatgtttgat 2340ggggtatttg agcacttgca acccttatcc
ggaagccccc tggcccacaa aggctaggcg 2400ccaatgcaag cagttcgcat
gcagcccctg gagcggtgcc ctcctgataa accggccagg 2460gggcctatgt
tctttacttt tttacaagag aagtcactca acatcttaaa ccgacgtcga
2520cccactctag aggatcgatc cccgctccgt gtaaatggag gcgctcgttg
atctgagcct 2580tgccccctga cgaacggcgg tggatggaag atactgctct
caagtgctga agcggtagct 2640tagctccccg tttcgtgctg atcagtcttt
ttcaacacgt aaaaagcgga ggagttttgc 2700aattttgttg gttgtaacga
tcctccgttg attttggcct ctttctccat gggcgggctg 2760ggcgtatttg
aagcttaatt aactcgaggt cggggggccc ggtacccaat tcgccctata
2820gtgagtcgta ttacaattca ctggccgtcg ttttacaacg tcgtgactgg
gaaaaccctg 2880gcgttaccca acttaatcgc cttgcagcac atcccccttt
cgccagctgg cgtaatagcg 2940aagaggcccg caccgatcgc ccttcccaac
agttgcgcag cctgaatggc gaatggaaat 3000tgtaagcgtt aatattttgt
taaaattcgc gttaaatttt tgttaaatca gctcattttt 3060taaccaatag
gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg
3120gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg
actccaacgt 3180caaagggcga aaaaccgtct atcagggcga tggcccacta
cgtgaaccat caccctaatc 3240aagttttttg gggtcgaggt gccgtaaagc
actaaatcgg aaccctaaag ggagcccccg 3300atttagagct tgacggggaa
agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa 3360aggagcgggc
gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc
3420cgccgcgctt aatgcgccgc tacagggcgc gtca
345413911223DNAartificialplasmid pXX311 139ataacttcgt ataatgtatg
ctatacgaag ttatgaattc tagagtcgac gaggaggagg 60tgcaaggggg ataccagcgc
gtgtttctca gggcctgtgt gggacaccga aacgtggtaa 120aagagacccg
cccgcgaact gtgtatgtgg agtagcgtgg cgtgtgcggc cggaccgaca
180aggcagcttg tggactgccc cacgttgcag agtcagctga caacgacacg
tgcgccttcc 240tgtcattgcc cgtgcgcacg cacgtcctcc gcactcccaa
caaattgaca gcgacacgtg 300cgccttccta taagcctatg cccgcacacg
ctcccgcgcc ctcaggtgtc gggccagacc 360acagaccggt tggtccacga
gtgcgaggag gatgaggcgg gcggctgcgg cggcgccggc 420ggggcggcgg
gcggcgagga ggacggcctg ggactgggca tcacaggtgg gtggcaggct
480ggcagggact cacgcatggg ccttgtacgt gactgcggtt ctgcatggct
agtggctcac 540gcgctgcgca cgttcacgta cggcttgtgg gcatgcagtg
ccttgacgtg aggctgcgct 600gccttgctgc tgccgccttg ccccgctccc
tgcacacact gcagccggct tcgggcgcta 660cttcaccgcg ggctacgagt
gcgagaacgc gcagcagctc aacaggctgc tggggtacaa 720ggcgctgtga
gagcgcgccg cagggggagt gtgttcatat tgtggttgtt tgggccgtgg
780gcgcgggctg catgtgcgta ttgcacgcgt acagcattgg tgactggtca
ggtgtaagcg 840gccggcagtg cgccgcgagg cgctgcagcg agttgtgggg
catgcgtcat gcgcagacgg 900cccctggacg acaaggcgtt gagttggcgt
ttggaggtgt gggacgacgt ggggtttgtg 960ccgtcaaagc acagaacaga
aggcgtgacc gttttacgag ctcgtatgat gtagcatgga 1020ttgaataatg
acatgtgatt tttgttacaa gcgacgaatg cgtggggttt tggatggcag
1080gggtttcagt cgcccgattg cgcatgcaca cgtgaccaaa tttatgctca
acgacgtgac 1140cattgcttta tacatacttg tgtatcggtt ggcacttata
acaattggct cgtcaaattg 1200acgcgaggct gcacttcgat cctgaaagcc
ccagttcaac aagtcggata gccaaatggc 1260cccgctcgct ctccagcatc
aaggggcctc taagtgcctc gcggcaaccc agcgcaagtg 1320tgctcgcgtt
gcggtgagct ggactcgtgc acttgtcgac gccgtcggca ccgcaatcga
1380aagacgcgtg cgtcgagcaa ttgtggaagc cgctgacgaa ttgtccgcat
gtgacattgc 1440aggctcgcgt ccccgctcgt ctcagcgtca tggcccaggt
gcggacgttg ggactgcact 1500tgcacgaatg tgatggggcc gcaccgagtc
tgcgcggacg tctcgctgac gtttcgcgtt 1560gaatgcatct cgcaataggc
agctgctgcg cctgctgaca acactaagaa gctgtggggc 1620ggtcgcttca
cgggcaagac ggacccgctc atggagaagt tcaacgagtc gctgcccttt
1680gacaagcgcc tgtgggctga ggacatcaag gtgcggcaca gggagggggg
cgagtggtgg 1740ggtggggctg ggggggacgc gggtttggtg gccagggcag
ggagggaaga cgtgcggggc 1800taggcaagag gctgcgaggg cccagggtaa
caccagaccg tgccgtgtcg cgtgcccggc 1860ttgctgccca ccttgcccgg
ccatccccac cgccctcccc accagcaatg acacgtacac 1920attcacacac
tcccccacac ccacataccc acacacccac gcattcccca acagggcagc
1980caggcgtacg ccaaggctct tgccaaggcc ggcattctgg cacatgacga
ggccgtgacc 2040attgtggagg ggctggccaa ggtgcgcaca cccggcagca
gggcgggtgg gtgggtgggt 2100ggggtggggg ggcagagaga ggcgcgggct
gagagggggc tgagaggggg gtcagcgagg 2160cgcaggctca gggggaggcg
tctgaggggg gctgagatgg tggtggggga gctgcgggtg 2220ctggggctgc
tgcggtggcg ggcgggcggg cgggcgggcg acgtgtacgt gagtagccgc
2280tgaccgggcg ctgggccttt gcgcacgcca cagcccacat gacaccgccg
caaggcccgc 2340cgcgccccac ccacgttcac acactcccca cacccacgcg
tgcgcgcgcc tccttcccct 2400caatacacgc gcctccttcc cctggccccc
gcctgctccc cccatccggc cgccccgcct 2460gcaggtggct gaggagtgga
aggcgggtgc ctttgtgatc aaggcgggtg acgaggacat 2520ccacacggcc
aacgagcggc gcctcacgga gctggtgggg gcggtgggcg gcaagctgca
2580caccggccgc tcgcgcaacg accaggtgag ggtgggtggg tgggggtggg
gtgggtgggt 2640gggtgggtgg gtgggtgggt gggtgggtgg gtgggtgggt
gggtgggggt ttgagatacc 2700ggtaccaggc caaactaaac cgaacccaag
ggggtggcgt aggggcgtgg gaggggggga 2760gtgcggaagc cgggaggcag
gagtaagggc gggaggaggg ggccggagga gaagcaggga 2820cgaagtcgat
gacaggcgca gtcggtggcg gcggtggcgg gtgtgccgtt gtgcagtggc
2880tgtggaggcc atgtgcaggg cggcggcggg gccgggccgg gggtgggaga
cttgtccaga 2940ccccgtggcc ctcttccagc cccgtccgcc actgccgcca
ccaccaccgc cgccgccgta 3000gccaccaccc ctcacgtcga ggcacttcac
agatgcgaag caaccacacc gttctccaca 3060tgaacagcta ccctcccaaa
cccaactttc ccttcccgcc ttacctaacc atgacccgct 3120accccccccc
cctttatttc ttaactaacc atgaatgccc ccccccggct gtacctggct
3180acgacttcac ttcgtaaact taatgtgtgt aacccccctt acacacacac
acacacccct 3240ccccgcccct ccaaaggttg ccaccgacta ccggctgtgg
ctggtgggtc aggtggaggt 3300gatgcggtcc gaggtgggcg agctgatgcg
cgtggcggcg gaccgctccg aggcagaggt 3360ggaggtgctc atgccgggtg
agggggcagg gaggggggga gggggagggg gaggtgctca 3420tgccggtgag
ggtagggagg ggaggggcag aggagggagg gggaggaggg ggcggctgag
3480tgcgggagag gcagggatga gggcgataga aagttgcgta ttgtcggtaa
actcaaagga 3540ctagacgaag agaacaaacc taaacaaggg agctggagcg
aggccaaatc tgaacgtgac 3600atcgcccgcc tcctcccgct gcctgctccc
ccacctcctc ccccatctcg cccccccccc 3660cacacacaca caggcttcac
gcaccttcag aatgccatga ctgtgcgctg gagccactgg 3720ctgatgagcc
acgccgcggc ctggcagcgc gacgacatgc ggctgcggga cctgctgccg
3780cgggtggcca cactgccgct gggctcgggt gggtgaggga ggggagggga
ggggaggggg 3840ggagggggag ggagaggagg ggagaagggg gggggagacg
aggagggtgg aagggtgggg 3900gcggggcggt ggaggctaga
gggtggggct gggtgggtgg acggagtgca ctggtagagg 3960agggataggg
tacattgaga cgggaggagg gatgcagggg cgaaggtggg gaggagggga
4020ggggaggagg cgtggagctg gagtgggccg acgagtgtgc ggacggggca
ggcggcaacg 4080gggattaaac ggcggggggc cggggcgtgt gcacgacagg
ggcttgcgcg tctgcgattg 4140tgggggcaca cagggacagg agcacgacgt
gggacacgca tagatacgcc gcattgacaa 4200cacacacaca cacacacaca
cacacacaca cacacacaca cacacaaaca caaacacaca 4260caaacacaaa
cacacacacg cccccccccc tacacacacg ccccctcccc aggcgccctg
4320gccggcaacc cctttctggt ggaccgccag ttcatcgcca aggagttggg
tttcggcggc 4380ggcgtgtgcc ccaactccat ggacgcggtg aggggaggag
gagggggagg agggcggggg 4440ggggcaggag gggggaggag gaggggggga
gggggttaac tttgaagcgt aaggaaacag 4500tcgggaggag ggggggaagg
agggggcctg gaggaggggg ggaggaggag ggtggctgga 4560gggggctggg
ggaggaggag ggggaggatt gggagggggc tgggggaggg tgcccgcagc
4620tgggggaggt ggggagggag ggggttgctg ctggtgtaaa gggcctgtag
gcactgagag 4680cactgtgggg agccggggta ctgcctgggg ccccgcgctg
cagaggtgtc gcgcagtgtg 4740gcggcgcatc ccccgcatcc ccacacgcgg
gccgctgccg ctgcccgcca cacccttgcc 4800actttgtgtg ctttcctagg
atatacacac acacacacac acacacacac acacacacac 4860acacaaacac
aaacacacac gggcgcgggc tttcgtttcg ttttttaaca caaacacaca
4920ctccccctgt gctcctcaac acactccatc tttctcacac aaacacacac
gcacacacac 4980atgcgcaggc gggtcggggg agggggggcg ggtgtgtatg
tgtgtgtgtg tgcgtgtgta 5040agtctcggtg gaggggtggt cctctatatg
gcggcggggc cacaggggga cgggtgtgac 5100agagttacgg ccggagccag
cggagtcccg ggatggatta aggatcgacg aagatatcgt 5160accgatcccc
gggaattcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc
5220gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
ggggtgccta 5280atgagtgagc taactcacat taattgcgtt gcgctcactg
cccgctttcc agtcgggaaa 5340cctgtcgtgc cagctgcatt aatgaatcgg
ccaacgcgcg gggagaggcg gtttgcgtat 5400tgggcgctct tccgcttcct
cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg 5460agcggtatca
gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
5520aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
aggccgcgtt 5580gctggcgttt ttccataggc tccgcccccc tgacgagcat
cacaaaaatc gacgctcaag 5640tcagaggtgg cgaaacccga caggactata
aagataccag gcgtttcccc ctggaagctc 5700cctcgtgcgc tctcctgttc
cgaccctgcc gcttaccgga tacctgtccg cctttctccc 5760ttcgggaagc
gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
5820cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
gctgcgcctt 5880atccggtaac tatcgtcttg agtccaaccc ggtaagacac
gacttatcgc cactggcagc 5940agccactggt aacaggatta gcagagcgag
gtatgtaggc ggtgctacag agttcttgaa 6000gtggtggcct aactacggct
acactagaag gacagtattt ggtatctgcg ctctgctgaa 6060gccagttacc
ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
6120tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
gatctcaaga 6180agatcctttg atcttttcta cggggtctga cgctcagtgg
aacgaaaact cacgttaagg 6240gattttggtc atgagattat caaaaaggat
cttcacctag atccttttaa attaaaaatg 6300aagttttaaa tcaatctaaa
gtatatatga gtaaacttgg tctgacagtt accaatgctt 6360aatcagtgag
gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
6420ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
gtgctgcaat 6480gataccgcga gacccacgct cacgggctcc agatttatca
gcaataaacc agccagccgg 6540aagggccgag cgcagaagtg gtcctgcaac
tttatccgcc tccatccagt ctattaattg 6600ttgccgggaa gctagagtaa
gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat 6660tgctacaggc
atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
6720ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
ttagctcctt 6780cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
ttatcactca tggttatggc 6840agcactgcat aattctctta ctgtcatgcc
atccgtaaga tgcttttctg tgactggtga 6900gtactcaacc aagtcattct
gagaatagtg tatgcggcga ccgagttgct cttgcccggc 6960gtcaatacgg
gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
7020acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
gttcgatgta 7080acccactcgt gcacccaact gatcttcagc atcttttact
ttcaccagcg tttctgggtg 7140agcaaaaaca ggaaggcaaa atgccgcaaa
aaagggaata agggcgacac ggaaatgttg 7200aatactcata ctcttccttt
ttcaatatta ttgaagcatt tatcagggtt attgtctcat 7260gagcggatac
atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
7320tccccgaaaa gtgccacctg acgtctaaga aaccattatt atcatgacat
taacctataa 7380aaataggcgt atcacgaggc cctttcgtct cgcgcgtttc
ggtgatgacg gtgaaaacct 7440ctgacacatg cagctcccgg agacggtcac
agcttgtctg taagcggatg ccgggagcag 7500acaagcccgt cagggcgcgt
cagcgggtgt tggcgggtgt cggggctggc ttaactatgc 7560ggcatcagag
cagattgtac tgagagtgca ccatatgcgg tgtgaaatac cgcacagatg
7620cgtaaggaga aaataccgca tcaggcgcca ttcgccattc aggctgcgca
actgttggga 7680agggcgatcg gtgcgggcct cttcgctatt acgccagctg
gcgaaagggg gatgtgctgc 7740aaggcgatta agttgggtaa cgccagggtt
ttcccagtca cgacgttgta aaacgacggc 7800cagtgccaag cttccatggg
atatcgcatg cctgcagagc tctagaattc ataacttcgt 7860ataatgtatg
ctatacgaag ttatggtacc gcggccgcgt agaggatctg ttgatcagca
7920gttcaacctg ttgatagtac gtactaagct ctcatgtttc acgtactaag
ctctcatgtt 7980taacgtacta agctctcatg tttaacgaac taaaccctca
tggctaacgt actaagctct 8040catggctaac gtactaagct ctcatgtttc
acgtactaag ctctcatgtt tgaacaataa 8100aattaatata aatcagcaac
ttaaatagcc tctaaggttt taagttttat aagaaaaaaa 8160agaatatata
aggcttttaa agcttttaag gtttaacggt tgtggacaac aagccaggga
8220tgtaacgcac tgagaagccc ttagagcctc tcaaagcaat tttgagtgac
acaggaacac 8280ttaacggctg acatgggaat tagcttcacg ctgccgcaag
cactcagggc gcaagggctg 8340ctaaaggaag cggaacacgt agaaagccag
tccgcagaaa cggtgctgac cccggatgaa 8400tgtcagctac tgggctatct
ggacaaggga aaacgcaagc gcaaagagaa agcaggtagc 8460ttgcagtggg
cttacatggc gatagctaga ctgggcggtt ttatggacag caagcgaacc
8520ggaattgcca gctggggcgc cctctggtaa ggttgggaag ccctgcaaag
taaactggat 8580ggctttcttg ccgccaagga tctgatggcg caggggatca
agatctgatc aagagacagg 8640atgaggatcg tttcgcatga ttgaacaaga
tggattgcac gcaggttctc cggccgcttg 8700ggtggagagg ctattcggct
atgactgggc acaacagaca atcggctgct ctgatgccgc 8760cgtgttccgg
ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg
8820tgccctgaat gaactgcagg acgaggcagc gcggctatcg tggctggcca
cgacgggcgt 8880tccttgcgca gctgtgctcg acgttgtcac tgaagcggga
agggactggc tgctattggg 8940cgaagtgccg gggcaggatc tcctgtcatc
tcaccttgct cctgccgaga aagtatccat 9000catggctgat gcaatgcggc
ggctgcatac gcttgatccg gctacctgcc cattcgacca 9060ccaagcgaaa
catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca
9120ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg
ccaggctcaa 9180ggcgcgcatg cccgacggcg aggatctcgt cgtgacacat
ggcgatgcct gcttgccgaa 9240tatcatggtg gaaaatggcc gcttttctgg
attcatcgac tgtggccggc tgggtgtggc 9300ggaccgctat caggacatag
cgttggctac ccgtgatatt gctgaagagc ttggcggcga 9360atgggctgac
cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc
9420cttctatcgc cttcttgacg agttcttctg agcgggactc tggggttcga
aatgaccgac 9480caagcgacgc ccaacctgcc atcacgagat ttcgattcca
ccgccgcctt ctatgaaagg 9540ttgggcttcg gaatcgtttt ccgggacgcc
ggctggatga tcctccagcg cggggatctc 9600atgctggagt tcttcgccca
ccccgggata tccggatata gttcctcctt tcagcaaaaa 9660acccctcaag
acccgtttag aggccccaag gggttatgct agttattgct cagcggtggc
9720agcagccaac tcagcttcct ttcgggcttt gttagcagcc ggatcttcta
gaatccccag 9780catgcctgct attgtcttcc caatcctccc ccttgctgtc
ctgccccacc ccacccccca 9840gaatagaatg acacctactc agacaatgcg
atgcaatttc ctcattttat taggaaagga 9900cagtgggagt ggcaccttcc
agggtcaagg aaggcacggg ggaggggcaa acaacagatg 9960gctggcaact
agaaggcaca gtcgaggctg atagcgagct cgagatggcc agcaaggtgt
10020acgaccccga gcagcgcaag cgcatgatca ccggccctca gtggtgggct
cgctgcaagc 10080agatgaacgt gctggacagc ttcatcaact actacgacag
cgagaagcac gccgagaacg 10140ccgtgatctt cctgcacggc aacgccgcca
gcagctacct gtggcgccac gtggtgcccc 10200acatcgagcc cgtggcccgc
tgcatcatcc ccgacctgat cggcatgggc aagagcggca 10260agagcggcaa
cggcagctac cgcctgctgg accactacaa gtacctgacc gcctggttcg
10320agctgctgaa cctgcccaag aagatcatct tcgtgggcca cgactggggc
gcctgcctgg 10380ccttccacta cagctacgag caccaggaca agatcaaggc
catcgtgcac gccgagagcg 10440tggtggacgt gatcgagagc tgggacgagt
ggcccgacat cgaggaggac atcgccctga 10500tcaagagcga ggagggcgag
aagatggtgc tggagaacaa cttcttcgtg gagaccatgc 10560tgcccagcaa
gatcatgcgc aagctggagc ccgaggagtt cgccgcctac ctggagccct
10620tcaaggagaa gggcgaggtg cgccgtccca ccctgagctg gcctcgcgag
atccccctgg 10680tgaagggcgg caagcccgac gtggtgcaga tcgtgcgcaa
ctacaacgcc tacctgcgcg 10740ccagcgacga cctgcccaag atgttcatcg
agagcgaccc cggcttcttc agcaacgcca 10800tcgtggaggg cgccaagaag
ttccccaaca ccgagttcgt gaaggtgaag ggcctgcact 10860tcagccagga
ggacgctccc gacgagatgg gcaagtacat caagagcttc gtggagcgcg
10920tgctgaagaa cgatacggcc agccagccgg agctggcccc ggaggatacg
taaggatccc 10980cgctccgtgt aaatggaggc gctcgttgat ctgagccttg
ccccctgacg aacggcggtg 11040gatggaagat actgctctca agtgctgaag
cggtagctta gctccccgtt tcgtgctgat 11100cagtcttttt caacacgtaa
aaagcggagg agttttgcaa ttttgttggt tgtaacgatc 11160ctccgttgat
tttggcctct ttctccatgg gcgggctggg cgtatttgaa gcgggtaccc 11220gac
1122314023DNAartificialprimer glgA-1fw 140cgacggtatg aagcttttat ttg
2314119DNAartificialprimer glgA-1rv 141ccggcggaac ggtaccaac
1914222DNAartificialprimer glgA-2fw 142ggccagggga attctcctcc ag
2214325DNAartificialprimer glgA-2rv 143gcggataata ctgaacgaag ctttg
2514420DNAartificialprimer Ald50.fw 144ggctgacccc cagtagtgta
2014520DNAartificialprimer Ald1042.rv 145attttccggc ttgaacattg
2014620DNAartificialprimer GlgC5.fw 146gttgttggca atcgagaggt
2014720DNAartificialprimer GlgCiR.rv 147gtctgccggt ttgaaacaat
2014810DNAartificialprimer BsaBI 148gatnnnnatc
1014920DNAartificialprimer GlgCiR.fw 149accccatcat catacgaagc
2015020DNAartificialprimer GlgC1233.rv 150agcctcctgg acattttcct
2015120DNAartificialprimer PpsA547.fw 151ttcactgacc gggctatttc
2015220DNAartificialprimer PpsA2329.rv 152cttggccaca gataccgatt
2015323DNAartificialprimer ldh-1fw 153gcgaactacc caacgctgac cgg
2315426DNAartificialprimer ldh-2rv 154gcatcaagtg ttgggggata tccctg
2615523DNAartificialprimer ack-1 fw 155ccgggacgtg acagaacggg tgg
2315623DNAartificialprimer ack-2 rv 156gcgttggcga tcgccgtcac tag
2315721DNAartificialprimer pta-1fw 157gccattgtgg gggtgggtca g
2115822DNAartificialprimer pta-2rv 158cagtttatgc cccgctaccg gg
2215924DNAartificialprimer phaC-25_XbaI.fw 159ccgatgtcta gataattcac
catc 2416021DNAartificialprimer phaC404_BamHI.rv 160tctaggggga
tccaacgatc g 2116122DNAartificialprimer phaC711_BamHI.fw
161ccaggggatc ctcttaacct ag 2216221DNAartificialprimer
phaC1133_ClaI.rv 162tgtcgtatcg atagccaatg g
2116323DNAartificialprimer agp1.1 163catccatcat gagctctgtt aac
2316423DNAartificialprimer agp2.1 164gtatctcgag cgatgcctac agg
2316522DNAartificialprimer agp3.1 165cgcattggtt tctagatggc gc
2216623DNAartificialprimer agp4.1 166cgataactct agacgagtca ttg
2316723DNAartificialprimer agp1.2 167gaggcaatga gctccactgg acg
2316822DNAartificialprimer agp2.2 168ctggcgttcc actcgagctt gg
2216922DNAartificialprimer agp3.1 169cgcattggtt tctagatggc gc
2217023DNAartificialprimer agp4.2 170cgataactct agacgagtca tcg
2317126DNAartificialprimer PdhBantiClaI.fw 171atcgatataa tttccgggtc
gtagcc 2617256DNAartificialprimer PdhBantioopBglII.rv 172gatctggaat
aaaaaacgcc cggcggcaac cgagcggcag ccattcggga taataa
5617324DNAartificialprimer PdhBNdeI.fw 173catatggctg agaccctact
gttt 2417426DNAartificialprimer PdhB1061ClaI.rv 174atcgatctta
caagctcccg gacaaa 2617520DNAartificialprimer PdhB.fw 175aatcgacatc
cacccttgtc 2017620DNAartificialprimer PdhB.rv 176gccttaactg
cgtccacaat 2017724DNAartificialprimer Mae-NdeI.fw 177catatggtta
gcctcacccc caat 2417825DNAartificialprimer MeLongClaI.rv
178atcgatcggg atggcctatt tatgg 2517926DNAartificialprimer
Mdh-NdeI.fw 179catatgaata ttttggagta tgctcc
2618023DNAartificialprimer Mdh-ClaI.rv 180atcgataagc cctaacctcg gtg
2318124DNAartificialprimer Mae-NdeI.fw 181catatggtta gcctcacccc
caat 2418230DNAartificialprimer MeShortClaI.rv 182atcgatacaa
ttcccgatta actattgacc 3018326DNAartificialprimer MdhRBSClaI.fw
183atcgattttt ctccaccatc aacacc 2618423DNAartificialprimer
MdhBglII.rv 184agatctaagc cctaacctcg gtg 2318523DNAartificialprimer
pykA-5fw 185cctgttattg gccacgggca gta 2318627DNAartificialprimer
pykA-2rv 186ggtttaccct gggctcgaga atttagg
2718724DNAartificialprimer pykA-3fw 187cccggtgaag catatgagac ccct
2418827DNAartificialprimer pykA-2rv 188ggtttaccct gggctcgaga
atttagg 2718925DNAartificialprimer pykB-1fw 189cctaaattca
ggtcgaccgg caaac 2519021DNAartificialprimer pykB-2rv 190caccaaccag
gctcgagtgg g 2119126DNAartificialprimer pykB-3fw 191cctaatttca
gccccatatg caaacg 2619221DNAartificialprimer pykB-2rv 192caccaaccag
gctcgagtgg g 2119331DNAartificialprimer Eno-SacI-ATG 193tagagctctt
aagtaaagtc cccgccacca t 3119432DNAartificialprimer Eno-XhoI-rev
194tactcgaggt cattgcttcc ttggcttaga ac 3219532DNAartificialprimer
Pgm-SacI-ATG 195tagagctcac caaagacgat gtggcccacc aa
3219630DNAartificialprimer Pgm-XhoI-rev 196tactcgagta tgaccccgct
gttgcagttc 3019724DNAartificialprimer pykA-3fw 197cccggtgaag
catatgagac ccct 2419830DNAartificialprimer Pyk1-SacI-rev
198tagagctctt aagaaatacg gtgaatcttg 3019926DNAartificialprimer
pykB-3fw 199cctaatttca gccccatatg caaacg 2620025DNAartificialprimer
Pyk2-SacI-rev 200tagagctccc tatcctttgg acacc
2520133DNAartificialprimer Eno-SacI-fw 201tagagctcgt gtttggagca
ttacacaccg atg 3320231DNAartificialprimer Eno-BglII-rev
202taagatcttt ttaagaatgt ttgggaccca g 3120331DNAartificialprimer
Pgm-BglII-fw 203tcagatctgc ccctctggga aaaaatgacc a
3120430DNAartificialprimer Pgm-XhoI-rev 204tactcgagta tgaccccgct
gttgcagttc 3020533DNAartificialprimer phk1-NdeI 205gtgtctcata
tggttacatc ccccttttcc ctt 3320620DNAartificialprimer phk2-XhoI
206cgagccctgc tcgagcaggc 2020735DNAartificialprimer pta_pPETJ1-NdeI
207gtgcctcata tgacgagttc cctttattta agcac
3520834DNAartificialprimer pta_pPETJ2-XhoI 208cggttgctcg agcatctgga
acggttgggt aaat 3420933DNAartificialprimer phk1 209gtgtctcata
tggttacatc ccccttttcc ctt 3321034DNAartificialprimer phk-BglII-rev
210ggtcacagat ctgttgtccc ccatggccta gcta 3421140DNAartificialprimer
pta-BglII-fw 211ccttgcagat ctggatacgt tgaggttatt taaattatga
4021234DNAartificialprimer pta_pPETJ2-XhoI 212cggttgctcg agcatctgga
acggttgggt aaat 3421335DNAartificialprimer aldh1-NdeI-fw
213gtgcctcata tgaatactgc taaaactgtt gttgc
3521435DNAartificialprimer aldh2-XhoI-rev 214gatctcctcg aggtaaagaa
tcagcatagg tctgg 3521523DNAartificialprimer ppc.NdeI.fw
215ctagaggttc atatgaactt ggc 2321620DNAartificialprimer ppc.XhoI.rv
216gtaagcaggc tcgaggcaag 2021725DNAartificialprimer isiA-fw-SalI
217gtcgaccttc cagcaccacg tcaac 2521829DNAartificialprimer
isiA-rev-EcoRI 218gaattcacag aattgcctcc
ttaattgag 2921929DNAartificialprimer nblA-fw-SalI 219acgcgtcgac
ttatggttga ttcgcattg 2922027DNAartificialprimer nblA-rev-EcoRI
220cggaattcat agctgttgcc ctccaag 2722125DNAartificialprimer
ntcA-fw-SalI 221gtcgacaacg acggaggttt aaggg
2522226DNAartificialprimer ntcA-rev-EcoRI 222gaattcatgc acgttcacgg
taatgg 2622327DNAartificialprimer for ORF slr1192 amplification
223ctctaggatc catgattaaa gcctacg 2722428DNAartificialprimer for ORF
slr1192 amplification 224cacggaccca gcggccgcct ttgcagag
2822533DNAartificialprimer ScPDC1-XhoI-F 225catgctcgag atgtctgaaa
ttactttggg taa 3322630DNAartificialprimer ScPDC1-BamHI-R
226catgggatcc ttattgctta gcgttggtag 3022732DNAartificialprimer
ScADH1-XhoI-F 227catgctcgag atgtctatcc cagaaactca aa
3222833DNAartificialprimer ScADH1-BamHI-R 228catgggatcc ttatttagaa
gtgtcaacaa cgt 3322928DNAartificialprimer Pcyc6-NotI-F
229gcggccgcca ctgaagactg ggatgagc 2823034DNAartificialprimer
Pcyc6-NotI-SpeI-F 230gcggccgcac tagtcactga agactgggat gagc
3423123DNAartificialprimer Pcyc6-XhoI-R 231ctcgagcatg tttatggagt
agg 2323226DNAartificialprimer Pfea1-NotI-F 232gcggccgcag
gacagagtgc gtgtgg 2623332DNAartificialprimer Pfea1-NotI-SpeI-F
233gcggccgcac tagtaggaca gagtgcgtgt gg 3223420DNAartificialprimer
Pfea1-XhoI-R 234ctcgagcatg gttaactgtg 2023522DNAartificialprimer
3'UTR-BamHI-F 235catgggatcc ccgctccgtg ta
2223634DNAartificialprimer 3'UTR-XbaI-KpnI-R 236catgggtacc
tctagacgct tcaaatacgc ccag 34
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