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

netically 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.

Description

CROSS-REFERENCE TO RELATED APPLICATION

[0001]This application is a continuation of 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; both 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. §§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⁺/NADH-cofactor-specific enzyme that has been genetically engineered to become NADP⁺/NADPH-cofactor specific enzyme. In embodiments thereof, the NAD⁺/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)⁺/NAD(P)H-cofactor-specific enzyme, and the host cell comprises an enhanced level of NAD(P)⁺/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)⁺ 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⁺, 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 Gregory 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) Yinl, Y., Chory, J., and Baulcombe, 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 Zn²+-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, lrtA, 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 3' 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 Zn²+ 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 Zn²+ dependent alcohol dehydrogenase enzyme converting acetaldehyde to ethanol.

[0048]In an embodiment thereof, the Zn²+ dependent alcohol dehydrogenase enzyme comprises AdhI from Zymomonas mobilis.

[0049]In another embodiment thereof, the Zn²+ 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 Zn²+ dependent alcohol dehydrogenase, wherein the heterologous nucleic acid sequence is flanked at its 5' and 3' 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 Zn²+ 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⁺/NADH-cofactor specific alcohol dehydrogenase enzyme, wherein the host cell comprises an enhanced level of NAD⁺/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⁺, 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)⁺ transhydrogenase converting NADPH to NADH, wherein this NAD(P)⁺ 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, lrtA, 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, (ii) 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° 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 CO₂ concentration, and after having reached stationary phase the light intensity is increased to a second light intensity and the CO₂ concentration is increased to a second CO₂ 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 CO₂, 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, 1B, 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. 6A 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 ldh-Kan-a.

[0122]FIG. 8C presents a schematic for the gene organization of the plasmid pBlue ldh-Kan-a.

[0123]FIG. 8D presents the nucleotide sequence of the insert contained in the construct pBlue ldh-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 sir2132 that encodes a phosphoacetyltransacetylase (EC 2.3.1.8) (Genbank No. P73662) 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 pIC20H/AphaC-KM.

[0134]FIG. 11C presents the nucleotide sequence of the insert of construct as pIC20H/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-adhll (SEQ ID NO:21).

[0138]FIG. 11G presents the amino acid sequence of Glucose-1-phosphate adenylyltransferase (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 β-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. 16B presents the nucleotide sequence of the vector pUC 19. (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 Q55863) 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. 21D 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) 24G presents the nucleotide sequence of the insert of construct pVZ322-PpetJ-pyk2-eno-pgm. (SEQ ID NO:52)

[0190]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)

[0191]FIG. 25B presents a schematic representation of the gene organization for the construct pVZ322 PpetJ-phk.

[0192]FIG. 25C presents the nucleotide sequence of the insert of the construct pVZ322 PpetJ-phk. (SEQ ID NO:54)

[0193]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)

[0194]FIG. 26B presents a schematic representation of gene organization in the construct pVZ322 PpetJ pta.

[0195]FIG. 26C presents the nucleotide sequence of the insert of construct pVZ322 PpetJ pta. (SEQ ID NO:56)

[0196]FIG. 26D presents a schematic representation of gene organization in construct pVZ322 PpetJ phk pta.

[0197]FIG. 26E presents the nucleotide sequence of the insert of construct pVZ322 PpetJ phk pta. (SEQ ID NO:57)

[0198]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) of Synechocystis sp. strain PCC6803. (SEQ ID NO:58)

[0199]FIG. 27B is a schematic representation of gene organization in construct pVZ 322 PpetJ aldh.

[0200]FIG. 27C presents the nucleotide sequence of the insert of construct pVZ 322 PpetJ aldh. (SEQ ID NO:59)

[0201]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)

[0202]FIG. 28B is a schematic representation of gene organization in pVZ321-PpetJ-ppc.

[0203]FIG. 28C presents the nucleotide sequence of the insert of construct pVZ321-PpetJ-ppc. (SEQ ID NO:61)

[0204]FIG. 28D presents the nucleotide sequence of primer SynRbc-BglII-fw (SEQ ID NO:62).

[0205]FIG. 28E presents the nucleotide sequence of primer SynRbc-PstI-rev (SEQ ID NO:63).

[0206]FIG. 28F presents the nucleotide sequence of primer SynRbc-SacI-fw (SEQ ID NO:64).

[0207]FIG. 28G presents the nucleotide sequence of the rbcLXS operon of Synechocystis PCC 6803 (SEQ ID NO:65).

[0208]FIG. 28H presents the amino acid sequence of the rbcL large subunit of Synechocystis PCC 6803 (SEQ ID NO:66).

[0209]FIG. 28I presents the amino acid sequence of the rbcX Rubisco chaperonin protein of Synechocystis PCC 6803 (SEQ ID NO:67).

[0210]FIG. 28J presents the amino acid sequence of the ribulose bisphosphate carboxylase small subunit (rbcS) of Synechocystis PCC 6803 (SEQ ID NO:68).

[0211]FIG. 28K is a schematic presentation of gene organization for plasmid pVZ321b-Prbc-SynRbcLXS.

[0212]FIG. 29A is a schematic representation of the structure of the vector pSK9.

[0213]FIG. 29B presents the nucleotide sequence of the vector pSK9. (SEQ ID NO:69)

[0214]FIG. 30A is a schematic representation of gene organization in construct pVZ321. (GenBank No. AF100176).

[0215]FIG. 30B presents the nucleotide sequence of the pVZ321 vector. (SEQ ID NO:70)

[0216]FIG. 31A is a schematic representation of gene organization for construct pVZ322.

[0217]FIG. 31B presents the nucleotide sequence of the pVZ322 vector (SEQ ID NO:71).

[0218]FIG. 32A is a schematic representation of gene organization of construct pIC PpetJ.

[0219]FIG. 32B presents the nucleotide sequence of the construct pIC PpetJ. (SEQ ID NO:72)

[0220]FIG. 32C is a graphic presentation demonstrating growth properties and extracellular pyruvate levels of the ΔglgA1/ΔglgA2 double mutant (M8) under nitrogen replete and nitrogen starved conditions.

[0221]FIG. 32D is a graphic presentation of pyruvate levels in wildtype and mutant (ΔglgA1/ΔglgA2) media/cells as determined enzymatically and by ion chromatography.

[0222]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.

[0223]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.

[0224]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.

[0225]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.

[0226]FIG. 32I is a graphic presentation of ethanol production in wildtype, ack and ack/pta double mutant cells.

[0227]FIG. 32J is a graphic presentation of ethanol production in wildtype, ack and ack/pta double mutant cells when normalized for optical density.

[0228]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.

[0229]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.

[0230]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.

[0231]FIG. 32N is a graphic presentation of ethanol production normalized to the OD₇₅₀ for the mutant Synechocystis PCC6803 harboring the pSK10-PisiA-PDC/ADHII plasmid and the mutant additionally containing the vector pVZ321b-Prbc-SynRbc

[0232]FIG. 33A is a schematic representation of gene organization for the construct pVZ-PisiA-pdc/adh.

[0233]FIG. 33B is a schematic representation of gene organization for the construct pVZ-PntcA-pdc/adh.

[0234]FIG. 33C is a schematic representation of gene organization for the construct pVZ-PnblA-pdc/adh.

[0235]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)

[0236]FIG. 33E is a schematic representation of restriction sites present in the Z. mobilis adhII and pdc fragment.

[0237]FIG. 33F presents the amino acid sequence of Z. mobilis pdc protein. (SEQ ID NO:74)

[0238]FIG. 33G presents the amino acid sequence of the Z. mobilis AdhII protein. (SEQ ID NO:75)

[0239]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)

[0240]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)

[0241]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)

[0242]FIG. 35A presents the nucleotide sequence of the cloning vector pVZ321b, a derivative of pVZ321. (SEQ ID NO:79)

[0243]FIG. 35B is a schematic representation of gene organization for the cloning vector pVZ321b.

[0244]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)

[0245]FIG. 36B is a schematic representation of gene organization for the construct pVZ321b-PpetJ-PDC-ADHII.

[0246]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)

[0247]FIG. 36D is a schematic representation of gene organization for the construct pVZ321b-PsigB-PDC-ADHII.

[0248]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)

[0249]FIG. 36F is a schematic representation of gene organization for the construct pVZ321b-PhtpG-PDC-ADHII.

[0250]FIG. 36G presents the nucleotide sequence of the lrtA 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)

[0251]FIG. 36H is a schematic representation of gene organization in the construct pVZ321b-PlrtA-PDC-ADHII.

[0252]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:84)

[0253]FIG. 36J is a schematic representation of gene organization for the construct pVZ321b-PpsbA2-PDC-ADHII.

[0254]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)

[0255]FIG. 36L is a schematic representation of gene organization for the construct pVZ321b-PrbcL-PDC-ADHII.

[0256]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)

[0257]FIG. 36N is a schematic representation of the gene organization of the construct pVZ321b-PpsaA-PDC-ADHII.

[0258]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)

[0259]FIG. 36P is a schematic representation of the gene organization of the construct pVZ321b-PggpS-PDC-ADHII.

[0260]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)

[0261]FIG. 36R is a schematic representation of the gene organization of the construct pVZ321c-PnirA-PDC-ADHII.

[0262]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)

[0263]FIG. 36T is a schematic representation of gene organization for the construct pVZ321c-PpetE-PDC-ADHII.

[0264]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)

[0265]FIG. 36V is s schematic representation of gene organization for the construct pVZ321c-PhspA-PDC-ADHII.

[0266]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)

[0267]FIG. 36X is a schematic representation of gene organization of the construct pVZ321c-PhliB-PDC-ADHII.

[0268]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)

[0269]FIG. 36Z is a schematic representation of the gene organization for the construct pVZ321c-PclpB1-PDC-ADHII.

[0270]FIG. 37A presents the nucleotide sequence of the adhA gene from Zymomonas mobilis ZM4. (SEQ ID NO:93)

[0271]FIG. 37B presents the amino acid sequence for the ZmAdhI protein sequence (AAV89860). (SEQ ID NO:94)

[0272]FIG. 37C is a schematic presentation of the gene organization for construct pVZ321b-PisiA-PDC-ADHI.

[0273]FIG. 37D is a schematic presentation of the gene organization for construct pVZ321b-PntcA-PDC-ZmADHI.

[0274]FIG. 37E is a schematic presentation of the gene organization for construct pVZ321b-PnblA-PDC-ZmADHI.

[0275]FIG. 38A presents the nucleotide sequence of SynAdh, the adh gene (slr1192) of Synechocystis sp. PCC 6803. (SEQ ID NO:95)

[0276]FIG. 38B presents the amino acid sequence of SynAdh (protein sequence BAA18840) of Synechocystis sp. PCC 6803. (SEQ ID NO:96)

[0277]FIG. 38C is a schematic representation of the gene organization for construct pVZ321b-PisiA-PDC-SynADH.

[0278]FIG. 38D is a schematic representation of the gene organization for construct pVZ321b-PntcA-PDC-SynADH.

[0279]FIG. 38E is a schematic representation of the gene organization for construct pVZ321b-PnblA-PDC-SynADH.

[0280]FIG. 39A presents the nucleotide sequence of EcAdhE, the AdhE gene from E. coli K12. (SEQ ID NO:97)

[0281]FIG. 39B presents the amino acid sequence of EcAdhE (protein sequence NP 415757). (SEQ ID NO:98)

[0282]FIG. 39C is a schematic representation of the gene organization for construct pVZ321b-PisiA-PDC-EcAdhE.

[0283]FIG. 39D is a schematic representation of the gene organization for construct pVZ321b-PntcA-PDC-EcAdhE.

[0284]FIG. 39E is a schematic representation of the gene organization for construct pVZ321b-PnblA-PDC-EcAdhE.

[0285]FIG. 40A presents the nucleotide sequence of ThAdhE, the adhE gene (tlr0227) from Thermosynechococcus elongatus BP-1. (SEQ ID NO:99)

[0286]FIG. 40B presents the amino acid sequence of ThAdhE (protein sequence BAC07780). (SEQ ID NO:100)

[0287]FIG. 40C is a schematic representation of the gene organization for the construct pVZ321b-PisiA-ThAdhE.

[0288]FIG. 40D is a schematic representation of the gene organization for the construct pVZ321b-PntcA-ThAdhE.

[0289]FIG. 40E is a schematic representation of the gene organization for the construct pVZ321b-PnblA-ThAdhE.

[0290]FIG. 41A presents the nucleotide sequence of ZpPdcpdc gene from Zymobacter palmae ATCC 51623 (SEQ ID NO:101)

[0291]FIG. 41B presents the amino acid sequence of ZpPdc (protein sequence AAM49566). (SEQ ID NO:102)

[0292]FIG. 42A presents the nucleotide sequence of pSK10 cloning vector (derivate of pSK9 [V. V. Zinchenko, Moscow, Russia; unpublished]). (SEQ ID NO:103)

[0293]FIG. 42B is a schematic representation of the gene organization for the plasmid pSK10.

[0294]FIG. 42C is a schematic representation of the gene organization of the construct pSK10-PisiA-PDC-ADHII.

[0295]FIG. 42D is a schematic representation of the gene organization of the construct pSK10-PnblA-PDC-ADHII.

[0296]FIG. 42E is a schematic representation of the gene organization of the construct pSK10-PntcA-PDC-ADHII.

[0297]FIG. 42F is a schematic representation of the gene organization of the construct pSK10-PisiA-PDC-ADHI.

[0298]FIG. 42G is a schematic representation of the gene organization of the construct pSK10-PnblA-PDC-ADHI.

[0299]FIG. 42H is a schematic representation of the gene organization of the construct pSK10-PntcA-PDC-ADHI.

[0300]FIG. 42I is a schematic representation of the gene organization of the construct pSK10-PisiA-PDC-SynADH.

[0301]FIG. 42J is a schematic representation of the gene organization of the construct pSK10-PnblA-PDC-SynADH.

[0302]FIG. 42K is a schematic representation of the gene organization of the construct pSK10-PntcA-PDC-SynADH.

[0303]FIG. 42L is a schematic representation of the gene organization of the construct pSK10-PisiA-PDC-EcAdhE.

[0304]FIG. 42M is a schematic representation of the gene organization of the construct pSK10-PnblA-PDC-EcAdhE.

[0305]FIG. 42N is a schematic representation of the gene organization of the construct pSK10-PntcA-PDC-EcAdhE.

[0306]FIG. 42O is a schematic representation of the gene organization of the construct pSK10-PisiA-PDC-ThAdhE.

[0307]FIG. 42P is a schematic representation of the gene organization of the construct pSK10-PnblA-PDC-ThAdhE.

[0308]FIG. 42Q is a schematic representation of the gene organization of the construct pSK10-PntcA-PDC-ThAdhE.

[0309]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).

[0310]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)

[0311]FIG. 42T presents the gene organization of plasmid pRL1049-PpetE-PDC-ADHII.

[0312]FIG. 42U presents the nucleotide sequence of plasmid pRL1049-PpetE-PDC-ADHII (SEQ ID NO:106).

[0313]FIG. 42V depicts the gene organization of plasmid pRL593-PisiA-PDC-ADHII.

[0314]FIG. 42W presents the nucleotide sequence of plasmid pRL593-PisiA-PDC-ADHII (SEQ ID NO:107).

[0315]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.

[0316]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.

[0317]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.

[0318]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.

[0319]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.

[0320]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.

[0321]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.

[0322]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.

[0323]FIG. 45 is a graphic presentation of ethanol production in Synechocystis expressing different 3 variants of E. coli AdhE compared to wild-type.

[0324]FIG. 46A is a graphic representation of growth over time for the captioned mutant strains.

[0325]FIG. 46B is a graphic representation of ethanol production over time(% v/v) for the captioned mutant strains.

[0326]FIG. 46C is a graphic representation of ethanol production per growth for the captioned mutant strains.

[0327]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.

[0328]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.

[0329]FIG. 46F is a graphic depiction of the pH-dependency of acetaldehyde reduction by crude extracts containing the Synechocystis Adh.

[0330]FIG. 46G is a graphic depiction summarizing the acetaldehyde reduction rates at different cosubstrate concentrations. Measurements were performed at pH 6.1

[0331]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_m and v_max values are discussed in the text.

[0332]46-I is a photographic depiction of SDS/PAGE analysis of recombinantly expressed SynAdh showing that SynAdh was enriched, but not purified to homogeneity.

[0333]FIG. 47A presents a phylogenetic analysis examining different zinc binding ADH proteins.

[0334]FIG. 47B presents in tabular form all genes identified by the Zn-binding, SynAdh Glade.

[0335]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)

[0336]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)

[0337]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)

[0338]FIG. 47F presents the amino acid sequence of an alcohol dehydrogenase GroES-like protein of Psychrobacter cryohalolentis K5, identified by Genbank Accession No. YP_--581659.1. (SEQ ID NO:111)

[0339]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)

[0340]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)

[0341]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)

[0342]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)

[0343]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)

[0344]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)

[0345]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)

[0346]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)

[0347]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)

[0348]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)

[0349]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. (SEQ ID NO:122)

[0350]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)

[0351]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)

[0352]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)

[0353]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)

[0354]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)

[0355]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)

[0356]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)

[0357]FIG. 48A is a graphic depiction of the OD₇₅₀ growth properties of Synechocystis wild type and mutants that express Pdc/Adh enzyme and Pdc enzyme alone.

[0358]FIG. 48B is a graphic depiction of ethanol production for Synechocystis wild type and mutants that express Pdc/Adh enzyme and Pdc enzyme alone.

[0359]FIG. 48C is a graphical presentation of data for an ethanol concentration time course under limiting CO2 conditions; these data are presented in tabular form in FIG. 48C.

[0360]FIG. 48D is a graphical presentation of data for an ethanol concentration time course under limiting CO2 conditions; these data are presented in tabular form in FIG. 48E.

[0361]FIG. 48E is a graphical presentation of data for an ethanol concentration time course under limiting CO₂ conditions; these data are presented in tabular form in FIG. 48G.

[0362]FIG. 48F is a graphical presentation of data for an ethanol concentration time course under limiting CO₂ conditions; these data are presented in tabular form in FIG. 48I.

[0363]FIG. 49A is a tabular presentation of cyanobacterial promoters used to express ethanologenic enzymes in Synechocystis 6803.

[0364]FIG. 49B is a graphic depiction of growth properties of 6803 transformed with pVZ321b-PisiA-PDC/ADH as monitored by determining the OD₇₅₀.

[0365]FIG. 49C is a graphic depiction of iron-induced ethanol production of 6803 transformed with pVZ321b-PisiA-PDC/ADH.

[0366]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.

[0367]FIG. 49E is a graphic depiction of the growth properties of cells with PnirA-PDC when nitrogen is provided by ammonia or nitrate.

[0368]FIG. 49F is a graphic depiction of ethanol production of cells with PnirA-PDC when nitrogen is provided by ammonia or nitrate.

[0369]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.

[0370]FIG. 49H is a graphic depiction of growth of Synechocystis 6803 pVZ321b-PpetJ-PDC/ADH.

[0371]FIG. 49I is a graphic depiction of ethanol production of Synechocystis 6803 pVZ321b-PpetJ-PDC/ADH.

[0372]FIG. 49J is a graphic depiction ethanol productivity per growth of Synechocystis 6803 pVZ321b-PpetJ-PDC/ADH.

[0373]FIG. 49K is a graphic depiction of the growth of Synechocystis 6803 pVZ321b-PpetE-PDC/ADH.

[0374]FIG. 49L is a graphic depiction ethanol production of Synechocystis 6803 pVZ321b-PpetE-PDC/ADH.

[0375]FIG. 49M is a graphic depiction of ethanol production of Synechocystis 6803 pVZ321b-PcrhC-PDC/ADH.

[0376]FIG. 49N is a graphic depiction of growth properties of Synechocystis 6803 pVZ321b-PhspA-PDC, pVZ321b-PhtpG-PDC, pVZ321b-PhliB-PDC and pVZ321b-PclpB1-PDC.

[0377]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.

[0378]FIG. 49P is a graphic presentation of growth properties under different conditions of cells containing pVZ321b-PpetJ-PDC/SynADH.

[0379]FIG. 49Q is a graphic presentation of ethanol production under different growth conditions of cells containing pVZ321b-PpetJ-PDC/SynADH.

[0380]FIG. 49R is a graphic presentation of ethanol production per OD under different growth conditions of cells containing pVZ321b-PpetJ-PDC/SynADH.

[0381]FIG. 50A presents the nucleotide sequence of ScPDC1. (SEQ ID NO:130)

[0382]FIG. 50B presents the amino acid sequence of ScPDC1. (SEQ ID NO:131)

[0383]FIG. 50C presents the nucleotide sequence of ScADH1. (SEQ ID NO:132)

[0384]FIG. 50D presents the amino acid sequence of ScADH1. (SEQ ID NO:133)

[0385]FIG. 50E presents the nucleotide sequence of Chlamydomonas Pcyc6. (SEQ ID NO:134)

[0386]FIG. 50F presents the nucleotide sequence of Chlamydomonas FEA1. (SEQ ID NO:135)

[0387]FIG. 50G presents the nucleotide sequence of a synthetic ble marker gene. (SEQ ID NO:136)

[0388]FIG. 50H presents the nucleotide sequence of ARG7 gene of Chlamydomonas. (SEQ ID NO:137)

[0389]FIG. 50I is a schematic presentation of gene organization of pSP124S.

[0390]FIG. 50J presents the nucleotide sequence of pSP124S. (SEQ ID NO:138)

[0391]FIG. 50K is a schematic presentation of gene organization of pXX311.

[0392]FIG. 50L presents the nucleotide sequence of pXX311. (SEQ ID NO:139)

[0393]FIG. 50M is a schematic presentation of gene organization of ARG7_pKS.

[0394]FIG. 50N is a schematic presentation illustrating the construction of ScPDC1 3'UTR pKS.

[0395]FIG. 50O is a schematic presentation illustrating the construction of Pcyc6 ScPDC1 3'UTR pKS.

[0396]FIG. 50P is a schematic presentation illustrating the construction of Pcyc6ScPDC1 3'UTR Pcyc6 ScADH1 3'UTR pKS.

[0397]FIG. 50Q is a schematic presentation of gene organization for pCYC6-PDC1-ADH1 pSP124S.

[0398]FIG. 50R is a schematic presentation of gene organization for pFEA1-PDC1-ADH1 pSP124S.

[0399]FIG. 50S is a schematic presentation of gene organization for pCYC6-PDC1-ADH1 ARG7.

[0400]FIG. 50T is a schematic presentation of gene organization for pFEA1-PDC1-ADH1 ARG7.

[0401]FIG. 50U is a schematic presentation of gene organization for ScPDC1-pXX311.

[0402]FIG. 50V is a graphic presentation of Chlamydomonas ethanol production.

[0403]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).

[0404]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.

[0405]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.

[0406]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.

[0407]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.

[0408]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.

[0409]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

[0410]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.

[0411]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.

[0412]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).

[0413]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

[0414]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.

[0415]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.).

[0416]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%.

[0417]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).

[0418]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 acids, 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.

[0419]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).

[0420]Database entry numbers given in the following are for the CyanoBase, the genome database for cyanobacteria (http://bacteria.kazusa.or.jp/cyanobase/index.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

[0421]It is one object of embodiments of the invention to provide a genetically modified host cell, which can be used for production of ethanol.

[0422]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.

[0423]Embodiment of genetic knockout and/or overexpression of metabolic pathway enzymes

[0424]In a first aspect the invention provides a genetically modified photoautotrophic, ethanol producing host cell comprising: [0425]at least one first genetic modification changing the enzymatic activity or affinity of an endogenous host cell enzyme, [0426]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, [0427]at least one second genetic modification different from the first genetic modification comprising an overexpressed enzyme for the formation of ethanol.

[0428]Acetaldehyde, pyruvate and acetyl-coA or their precursors are important metabolic intermediates for energy production in cells. In photoautotrophic cells, which use light, CO₂, 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 CO₂ fixation in the Calvin-cycle, for example 3-phosphoglycerate. Pyruvate, acetyl-CoA and their precursors are important metabolic intermediates obtained e.g. by photosynthetic CO₂ fixation in photoautotrophic cells. Acetaldehyde is a metabolic intermediate of the anoxygenic fermentation pathway in many photoautotrophic cells.

[0429]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.

[0430]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.

[0431]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.

[0432]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.

[0433]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.

[0434]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_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_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_max has to be increased, whereas for increasing the affinity K_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).

[0435]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.

[0436]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.

[0437]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.

[0438]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.

[0439]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.

[0440]In yet another embodiment of the genetically modified host cell [0441]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. [0442]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.

[0443]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.

[0444]The Calvin-cycle is an important part of photosynthesis and includes the light-independent reactions, where CO₂ is captured from the environment of the cell and converted into organic compounds, for example three-carbon compounds such as 3-phosphoglycerate. CO₂ 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.

[0445]Photosynthetic CO₂ fixation can lead to the production of carbon storage compounds such as reserve carbohydrates like glycogen, starch or sucrose.

[0446]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.

[0447]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 carbo-hydrates, 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.

[0448]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.

[0449]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.

[0450]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⁺→malate+NAD⁺

Malic enzyme catalyzes the conversion of malate into pyruvate using NADP⁺:

malate+NADP⁺→pyruvate+CO₂+NADPH

[0451]In C4-plants the released CO₂ can be fixed by ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) and NADPH can be used for CO₂-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 CO₂-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 CO₂-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 CO₂ and HCO₃^- and to raise the CO₂-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 CO₂-concentrations.

[0452]In order to enhance the carbon dioxide fixating activity of RubisCO random or side directed mutagenesis can be performed to achieve higher CO₂ 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).

[0453]Another way of increasing the enzymatic activity of RubisCO according to the invention involves overexpressing heterologous RubisCO in order to increase the CO₂ 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).

[0454]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.

[0455]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.

[0456]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-phosphoglycerate 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.

[0457]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.

[0458]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.

[0459]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 CO₂ to phosphoenolpyruvate (PEP) to form the four-carbon compound oxaloacetate (OAA). This PEP-carboxylase catalyzed reaction is used for CO₂ fixation and can enhance the photosynthetic activity leading to higher CO₂ fixation, which can be used for ethanol formation.

[0460]In particular the enzymatic activity or affinity of PEP-carboxylase, malate dehydrogenase and malic enzyme can be enhanced concomitantly. This leads to a higher CO₂ 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 CO₂ partial pressure leading to an increased efficiency of the Calvin cycle. PEP-carboxylase is used for CO₂ fixation in C4-plants and can also be found in cyanobacteria.

[0461]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 slr 2072, which is annotated as ilvA (threonine dehydratase), EC 4.3.1.19, can catalyze the deamination of serine to pyruvate.

[0462]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.

[0463]According to another embodiment of the invention in combination with enhancing the enzymatic activity or affinity of phosphoketolase enzyme, the polyhydroxybutyrate (PHB) pathway is knocked out in order to avoid PHB accumulation due to an increased level of acetyl-CoA biosynthesis (Control of Poly-β-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.

[0464]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.

[0465]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.

[0466]For example, alcohol dehydrogenase enzymes are often NAD⁺/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)⁺ transhydrogenases, which transfer reduction equivalents between NADP(H) to NAD(H). These NAD(P)⁺ transhydrogenases are oxidoreductases.

[0467]Furthermore the host cell can comprise a host NADH dehydrogenase converting NADH to NAD⁺ wherein the activity of the NADH dehydrogenase is reduced compared to the wild type host cell.

[0468]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.

[0469]Alternatively, in order to enhance the enzymatic activity of an enzyme, which is NADP⁺/NADPH cofactor dependent as, for example the malic enzyme, the level of NADP⁺/NADPH in the host cell also can be increased.

[0470]In many of photoautotrophic cells the level of NAD⁺ plus NADH to NADP⁺ plus NADPH is around 1:10. Due to this high imbalance of NADH to NADPH, the conversion of an NAD⁺/NADH cofactor specific enzyme via site directed mutagenesis or random mutagenesis of the enzyme into an NADP⁺/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).

[0471]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, polyhydroxy-octanoate, 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 1.1.1.36) 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 be 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° 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° 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, whereinthe 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 Fe²+ 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, acetyl-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 have 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° C. to 40° C. In contrast to that, a cold shock can be induced by reducing the growth temperature of the cell culture from 30° C. to 20° 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 vivo 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× sodium chloride/sodium citrate (SSC) buffer at about 45 degrees Celsius, followed by one or more washes in 0.2×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 acid 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 Zn²+-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 Zn²+ 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-Glade B of the GroES-like domain alcohol dehydrogenases Glade (see FIG. 47A). The Zn²+ dependent alcohol dehydrogenase enzyme can furthermore be selected from a cyanobacterial Zn²+ dependent alcohol dehydrogenase enzyme. In yet another embodiment of the invention the Zn²+ 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 OD₇₅₀ nm 1, 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 OD₇₅₀ nm 1, 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 OD₇₅₀ 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 OD₇₅₀ 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 OD₇₅₀ 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 self-replicating construct pVZ-PhspA-Pdc-SynAdh in a for example a cyanobacterial host cell. If normalized to OD₇₅₀ 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 OD₇₅₀ 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 Zn2+ 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 dehydrogenase converting acetylphosphate into acetaldehyde.

[0645]Another embodiment of the invention provides a genetically modified photoautotrophic, ethanol producing host cell comprising an overexpressed NAD⁺/NADH cofactor specific alcohol dehydrogenase, wherein the host cell comprises an enhanced level of NAD⁺/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⁺/NADH biosynthesis.

[0647]For example, such a genetically modified host cell can comprise a host NADH dehydrogenase converting NADH to NAD⁺ 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)⁺ transhydrogenase converting NADPH to NADH (electrons are transferred from NADPH to NAD+, so NADP+ and NADH are generated) wherein the activity of this NAD(P)⁺ 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)⁺ 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 NH₄⁺ or nitrate NO₃^-. The nirA promoter for example can be induced by a switch from NH₄⁺ to NO₃^- 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, lrtA, 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° C. to 40° C.), cold shock (reducing the growth temperature of the cell culture from 30° C. to 20° 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 CO₂, 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 (N₂) fixing so called diazotrophic host strains. Upon reaching the condition of nitrogen starvation, these strains can switch to nitrogen fixation. The reduction of N₂ to ammonia is catalyzed by the nitrogenase, an enzyme complex, which is irreversibly inactivated by O₂. Therefore photosynthetic N₂-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 N₂-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]This 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 (HCO₃^-, 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 CO₂ by the enzyme RubisCO most of the carbon that is taken up by the cell can be HCO₃^-, which is than converted into CO₂ by the enzyme carbonic anhydrase before its fixation as CO₂ 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 CO₂-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:

HCO₃^-+H⁺→H₂CO₃→CO₂+H₂O

[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 can 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 ethanol 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° C. or even at 45° 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 CO₂ 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 μE/m²s per day and then it can be increased to 120 μE/m²s and further to 220 μE/m²s once the stationary phase is reached. In general the light intensity can vary between 40 μE/m²s and 100 μE/m²s. 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 μE/m²s) 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 above-mentioned 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 μMol O₂/hmg chlorophyll (180 μMol O₂/hmg chlorophyll 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 CO₂ 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 transformablefrom 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 μMol O₂/hmg chlorophyll, more preferred 200 μMol, most preferred at least 250 μMol O₂/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 ethanol 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° 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. 50-13. 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 under http://www.pasteur.fr/recherche/banques/PCC or the SAG, the so-called "Algensammlung aus Gottingen" algal collection of the university of Gottingen found under http://www.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 CO₂ 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 tricornutum". 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 tricornutum (Bacillariophyceae) with a variety of selectable marker and reporter genes". 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 microalgal species, for example Chlamydomonas. An overview of different methods of genetic transformation of microalgae is presented in the review article of Banares et 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 intercellularily 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 thereafterA2. 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 lrtA 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° C. to, for example, 40° C. or by reducing the temperature of a growing culture from 30° C. to 20° 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 μM and 200 μM.

[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 dehydrogenase 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 (http://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 by fragment containing the major part of the coding sequence from glycogen synthase 1 (sll0945) was amplified by PCR using the following primers:

TABLE-US-00001 (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 KpnI restriction site for cloning (marked in bold letters).

[0851]The PCR fragment was digested with HindIII and KpnI 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 by fragment containing the entire coding sequence from glycogen synthase 2 (sll1393) was amplified by PCR using the following primers:

TABLE-US-00002 (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 clone 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 ΔglgA1 was selected. In a second step, gene sll1393 (glgA2) was knocked out in the ΔglgA1 mutant and the double mutant ΔglgA1/ΔglgA2 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]NaNO₃: 1.5 g [0861]K₂HPO₄: 0.04 g [0862]MgSO₄.7H₂O: 0.075 g [0863]CaCl₂.2H₂O: 0.036 g [0864]Citric acid: 0.006 g [0865]Ferric ammonium citrate: 0.006 g [0866]EDTA (disodium salt): 0.001 g [0867]NaCO₃: 0.02 g [0868]Trace metal mix A5_--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]H₃BO₃: 2.86 g [0874]MnCl₂.4H₂O: 1.81 g [0875]ZnSO₄.7H₂O: 0.222 g [0876]NaMoO₄.2H₂O: 0.39 g [0877]CuSO₄.5H₂O: 0.079 g [0878]Co(NO₃)₂.6H₂O: 49.4 mg [0879]Distilled water: 1.0 L) [0880]Add 1-10 μg 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 μl 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 μl of the corresponding antibiotic in a suitable concentration under the agar for the selection of mutant clones (initial concentration for chloramphenicol: 1 μg/ml BG11 agar; initial concentration for kanamycin: 5 μg/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 μg/ml BG11, for chloramphenicol from initially 1 to 15 μg/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 μg/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° C. under continuous illumination with white light (intensity: 40 μm^-2 s^-1) and aeration with air. For cultivation of mutants, the appropriate antibiotics were added to the medium (kanamycin 75 mg/l; chloramphenicol 15 mg/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:a) alanine dehydrogenase (ald)b) ADP-glucose pyrophosphorylase (glgC)c) pyruvate water dikinase (ppsA)d) lactate dehydrogenase (ldh)e) acetate kinase (ack)f) phosphoacetyltransacetylase (pta)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 29413Protocols for Generation of Knock-Down Mutants of Synechocystis sp. PCC 6803 and Other Cyanobacteria Affecting the Following Gene:a) pyruvate dehydrogenase (pdhB) [0890]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

[0891]In general:

[0892]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.

[0893]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 under: http://www.ncbi.nlm.nih.gov/sites/entrez?term=X06404&cmd=Search&db=nuccor- e&QueryKey=1 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.

[0894]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 (Aald)

[0895]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.

[0896]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 ald ORF) using the following primers:

TABLE-US-00003 (SEQ ID NO: 144) #Ald50.fw: 5'-GGCTGACCCCCAGTAGTGTA-3 (SEQ ID NO: 1454) #Ald1042.rv: 5'-ATTTTCCGGCTTGAACATTG-3'

[0897]A 993 by ald PCR fragment was amplified by a BIOTAQ® DNA Polymerase (BIOLINE), cloned into the pGEM-T vector (Promega) and restricted with SmaI (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.

[0898]A construct designated as pGEM-T/Δald-antisense has the structure presented schematically in FIG. 5B.

[0899]The sequence of the insert for this construct (pGEM-T/Δald-antisense) is presented in FIG. 5C.

[0900]In the other construct, designated as pGEM-T/Δald-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 (ΔglgC)

[0901]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.

[0902]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.

[0903]The following primers were used for PCR

TABLE-US-00004 EcoRI: G↓AATTC (SEQ ID NO: 146) #GlgC5.fw: 5'-GTTGTTGGCAATCGAGAGGT-3' (SEQ ID NO: 147) #GlgCiR.rv: 5'-GTCTGCCGGTTTGAAACAAT-3' (SEQ ID NO: 148) BsaBI: GATNN↓NNATC (SEQ ID NO: 149) #GlgCiR.fw: 5'-ACCCCATCATCATACGAAGC-3' (SEQ ID NO: 150) #GlgC1233.rv: 5'-AGCCTCCTGGACATTTTCCT-3'

[0904]The first 1579 by glgC PCR fragment was amplified by a BIOTAQ® 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).

[0905]Plasmids were analyzed by restriction digest in order to select constructs with both orientations of the inserted kanamycin cartridge, respectively.

[0906]The construct pGEM-T/ΔglgC-KMantisense has the structure shown in FIG. 6B, and its insert the nucleotide sequence presented in FIG. 6C.

[0907]In the other construct, designated as pGEM-T/ΔglgC-KMsense the kanamycin resistance cartridge is inserted in the other direction.

[0908]The second 1453 by glgC PCR fragment was amplified by a BIOTAQ® 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).

[0909]Plasmids were analyzed by restriction digest in order to select constructs with both orientations of the inserted chloramphenicol resistance (Cm) cartridge, respectively.

[0910]A construct designated as pDrive/ΔglgC-CMantisense was selected; its structure is presented schematically in FIG. 6D and the nucleotide sequence of the insert is presented in FIG. 6E.

[0911]In the other construct, designated as pDrive/ΔglgC-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 (ΔppsA)

[0912]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.

[0913]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-00005 (SEQ ID NO: 151) #PpsA547.fw: 5'-TTCACTGACCGGGCTATTTC-3' (SEQ ID NO: 152) #PpsA2329.rv: 5'-CTTGGCCACAGATACCGATT-3'

[0914]A 1783 by ppsA PCR fragment was amplified by a BIOTAQ® DNA Polymerase (BIOLINE), cloned into the pGEM-T vector (Promega) and restricted with SmaI (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.

[0915]The construct used, designated as pGEM-T/ΔppsA-antisense, has the structure presented in FIG. 7B. The nucleotide sequence of it insert is presented in FIG. 7C.

[0916]In the other construct, designated as pGEM-T/ΔppsA-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 (Δldh)

[0917]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.

[0918]A 1931 by fragment containing the entire coding sequence from lactate dehydrogenase (slr1556) was amplified by PCR using the following primer:

TABLE-US-00006 (SEQ ID NO: 153) #ldh-1fw: 5'-GCGAACTACCCAACGCTGACCGG-3' (SEQ ID NO: 154) #ldh-2rv: 5'-GCATCAAGTGTTGGGGGATATCCCTG-3', primer contains a EcoRV restriction site (GATATC) for cloning (marked in bold letters).

[0919]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.

[0920]The knock-out-construct used, named pBlue ldh-Kan-a, has the structure presented in FIG. 8C, and the nucleotide sequence for its insert is presented in FIG. 8D.

e) Construction of a DNA-Vector for Generation of an Acetate Kinase Knock-Out Mutant (Rack)

[0921]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.

[0922]A 2316 by fragment containing the entire coding sequence from acetate kinase (sll1299) was amplified by PCR using the following primer:

TABLE-US-00007 (SEQ ID NO: 155) #ack-1 fw: 5'-CCGGGACGTGACAGAACGGGTGG-3' (SEQ ID NO: 156) #ack-2 rv: 5'-GCGTTGGCGATCGCCGTCACTAG-3'

[0923]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 pUC4K and ligated into the HpaI restriction sites of slr1299. A restriction enzyme map of this region is presented in FIG. 9B.

[0924]The orientation of the kanamycin resistance cassette was either in the same direction as sll1299 (designed "a") or in the opposite direction (designed "b").

[0925]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. 9D.

f) Construction of a DNA-Vector for Generation of a Phosphoacetyltransacetylase (Phosphoacyltransferase) Knock-Out Mutant (Δpta)

[0926]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.

[0927]A 2869 by fragment containing the entire coding sequence from phosphoacetyl-transacetylase (slr2132) was amplified by PCR using the following primer:

TABLE-US-00008 (SEQ ID NO: 157) #pta-1fw: 5'-GCCATTGTGGGGGTGGGTCAG-3' (SEQ ID NO: 158) #pta-2rv: 5'-CAGTTTATGCCCCGCTACCGGG-3',

[0928]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.

[0929]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 (ΔphaC)

[0930]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.

[0931]One construct was generated for knock out of poly(3-hydroxyalkanoate) synthase by deletion/insertion (resistance cartridge: kanamycin) mutagenesis.

TABLE-US-00009 (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'

[0932]Two PCR products (pos. 24 to pos. 404; pos. 711 to pos. 1133) of the phaC fragment were amplified by a BIOTAQ® 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 (http://seq.yeastgenome.org/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.

[0933]The construct selected is pIC2OH/ΔphaC-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 in the Filamentous, Diazotrophic Cyanobacteria Nostoc/Anabaena spec. PCC7120 and Anabaena Variabilis ATCC 29413

[0934]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.

[0935]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.

[0936]Constructs for conjugation into Anabaena PCC7120 were cloned as followed:

[0937]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-00010 (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)

[0938]Inserted restriction sites in primer sequences are marked in bold letters

[0939]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, 68, 119-138.]

[0940]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.

[0941]The following constructs have been generated:

pRL271 agp (a114645)::C.K3pRL271 agp (a114645)::C.K3-PpetE-pdc-AdhIIpRL271 agp (a114645)::C.K3-PpetE-pdc

[0942]The structures of the constructs are depicted in FIG. 11-2.

[0943]The sequence of the insert of pRL271 agp (all4645)::C.K3-PpetE-pdc-AdhII is shown in FIG. 11F.

[0944]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.

[0945]Glucose-1-phosphate adenylyltransferase (ADP-glucose-pyrophosphorylase, agp, glgC), EC 2.7.7.27, of Anabaena variabilis ATCC29314 is encoded by ORF Ava_--2020, Ac. No. Q3 MBJ4, and has the amino acid sequence as shown in FIG. 11G.

[0946]For PCR amplification of the genomic fragments of Anabaena variabilis the following primers were used:

TABLE-US-00011 (SEQ ID NO: 167) #agp1.2 5'-GAGGCAATGAGCTCCACTGGACG-3' (SacI site inserted) (SEQ ID NO: 168) #agp2.2 5'-CTGGCGTTCCACTCGAGCTTGG-3' (XhoI site inserted) (SEQ ID NO: 169) #agp3.1 5'-CGCATTGGTTTCTAGATGGCGC-3' (XbaI site inserted) (SEQ ID NO: 170) #agp4.2 5'-CGATAACTCTAGACGAGTCATCG-3' (XbaI site inserted)

[0947]Inserted restriction sites in primer sequences are marked in bold letters.

[0948]Generation of the constructs was exactly as described for the constructs of Anabaena PCC7120.

[0949]The following constructs have been generated:

pRL271 agp::C.K3pRL271 agp::C.K3-PpetE-pdc-AdhIIpRL271 agp::C.K3-PpetE-pdc

[0950]All described plasmids were conjugated into Anabaena strains according the following method:

Conjugation of Nostoc spec. PCC7120/Anabaena variabilis

Cargoplasmids

[0951]Cargoplasmids (pRL593, pRL1049 or pRL271) were transformed into competent E. coli HB101 (pRL528_{helperplasmid})

In Preparation for Conjugation

[0952]E. coli Cultures: [0953]inoculation of overnight cultures in LB with the appropriate antibiotics from [0954]Cargoplasmid in E. coli HB101 (pRL528_{helperplasmid}) [0955]Helperstrain E. coli J53 (RP4) [0956]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° C. [0957]spin down the well grown E. coli cultures in "Falcons" 10 min 4800 rpm. [0958](for J53 culture: take 2 Falcons). [0959]"wash"/resuspend cells in equal volume of LB without antibiotics. [0960]for each conjugation spin 10 ml of resuspended HB101 (culture carrying pRL528+cargo plasmid) in 15 ml Falcon tube, remover supernatant [0961]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 μl LB and incubate for 2 h at 30° C.

Cyanos

[0961] [0962]determine the chlorophyll concentration of well grown Anabaena cultures [0963]for each conjugation, culture corresponding to about 10 μg Chlorophyll is needed. [0964]spin down the equivalent volume of Anabaena culture and resuspend to a volume corresponding to 10 μg Chlorphyll/100 μl BG11 medium.

Conjugation

[0964] [0965]for each conjugation place one HATF filter on a plate (BG11) [0966]mix 100 μl E. coli suspension=100 μl Anabaena culture and plate on filter [0967]incubate plates at 30° C. overnight wrapped in paper [0968]next day remove paper [0969]after one day transfer filter on plates containing antibiotics.

Construction of DNA-Vectors for Generation of Knock-Down Mutants

[0970]a) Construction of a DNA-Vector for Generation of a Pyruvate Dehydrogenase (pdhB) Knock-Down Mutant

[0971]The open reading frame (ORF) sll1721 encodes the β-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.

[0972]Two strategies were considered for knock-down of the pyruvate dehydrogenase. 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.

[0973]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®; Finnzymes), adenylated (BIOTAQ® DNA Polymerase; BIOLINE), cloned into the pDrive vector (Qiagen) and restricted with ClaI/BglII (i) or NdeI/BglII¹ (ii) (Fermentas). These fragments were cloned into the pSK9 vector, digested with ClaI/BglII (i) or NdeI/BglII (ii). 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. ¹ BglII 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 and do not affect the translation termination loop.

TABLE-US-00012 (SEQ ID NO: 171) #PdhBantiClaI.fw: 5'-ATCGATATAATTTCCGGGTCGTAGCC- 3', this primer contains a ClaI restriction site for cloning (marked in bold letters) (SEQ ID NO: 172) #PdhBantioopBglII.rv: 5'GATCTGGAATAAAAAACGCCCGGCGGCAACCGAGCGGCAGCC ATTCGGGATAATAA-3', this primer contains a BglII restriction site for cloning (marked in bold letters) and the oop terminator region of the lambda phage (underlined) (SEQ ID NO: 173) #PdhBNdeI.fw: 5'-CATATGGCTGAGACCCTACTGTTT-3', this primer contains a NdeI restriction site for cloning (marked in bold letters) (SEQ ID NO: 174) #PdhB1061ClaI.rv: 5'-ATCGATCTTACAAGCTCCCGGACAAA- 3', this primer contains a ClaI restriction site for cloning (marked in bold letters)

[0974]The 1142 by pdhB PCR fragment for the knock-out of the original wild type gene was amplified by a BIOTAQ® 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 pUC4K vector with EcoRI (5' overhangs; Fermentas) and a following "fill in reaction" via the T4 DNA Polymerase (Promega) and ligated into the Eco147I 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-00013 (SEQ ID NO: 175) # PdhB.fw: 5'-AATCGACATCCACCCTTGTC-3' (SEQ ID NO: 176) # PdhB.rv: 5'-GCCTTAACTGCGTCCACAAT-3'

[0975](i) Knock-Down by Regulation of the Expression of the Adequate Antisense RNA

[0976]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

[0977]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.

[0978]The knock-out construct used, designated as pGEM-T/ΔpdhB-KMantisense, has the structure presented in FIG. 12F. The sequence for the insert in this clone is presented in FIG. 12G.

[0979]In the other construct, designated as pGEM-T/ΔpdhB-KMsense the kanamycin resistance cartridge is inserted in the other direction.

[0980]In the following the cloning vectors, which were used are described.

a) Cloning Vector pGEM®-T Structure and Sequence

[0981]PCR cloning vector pGEM®-T was from Promega corp., Madison Wis., USA. The structure of the plasmid is presented in FIG. 13A, and its nucleotide sequence is presented in FIG. 13B.

b) Cloning Vector pDrive Structure and Sequence

[0982]Cloning vector pDrive was from Qiagen, Hilden, Germany. The structure of this plasmid is presented in FIG. 14A and its nucleotide sequence in FIG. 14B.

c) Cloning Vector pBlueSK+ Structure and Sequence

[0983]Cloning vector pBluescript II® SK+ (Ac. No X52328) was from Stratagene, La Jolla, Calif., USA.

[0984]The structure of this plasmid is presented in FIG. 15A and, its nucleotide sequence is presented in FIG. 15B.

d) Cloning Vector pUC19Structure and Sequence

[0985]Cloning vector pUC19 (Ac. No M7779) is presented schematically in FIG. 16A, and its nucleotide sequence is presented in FIG. 16B.

e) Plasmid pSK9 Structure and Sequence

[0986]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 enzymeb) malate dehydrogenasec) malic enzyme and malate dehydrogenased) pyruvate kinase 1e) pyruvate kinase 2f) pyruvate kinase, enolase and phosphoglycerate mutaseg) enolaseh) phosphoglycerate mutasei) pyruvate kinase (1 or 2)/enolase/phosphoglycerate mutasej) phosphoketolasek) phosphoacetyltransacetylasel) phosphoketolase/phosphoacetyltransacetylasem) acetaldehyde dehydrogenasen) PEP carboxylaseo) ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO)

Construction of DNA-Vectors for Overexpression

[0987]In general:

[0988]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.).

[0989]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.

[0990]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

[0991]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.

[0992]For overexpression of malic enzyme, the encoding me gene together with its gene-specific terminator region was PCR-amplified using the following primer:

TABLE-US-00014 (SEQ ID NO: 177) Mae-NdeI.fw: 5'-CATATGGTTAGCCTCACCCCCAAT-3', primer contains a NdeI restriction site for cloning (marked in bold letters) (SEQ ID NO: 178) MeLongClaI.rv: 5'-ATCGATCGGGATGGCCTATTTATGG-3', primer contains a ClaI restriction site for cloning (marked in bold letters)

[0993]The PCR fragment was amplified by a High-Fidelity DNA Polymerase (Phusion®; Finnzymes), adenylated (BIOTAQ® 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.

[0994]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 Dehydrogenase

[0995]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.

[0996]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:

TABLE-US-00015 (SEQ ID NO: 179) Mdh-NdeI.fw: 5'-CATATGAATATTTTGGAGTATGCTCC-3', primer contains a NdeI restriction site for cloning (marked in bold letters) (SEQ ID NO: 180) Mdh-ClaI.rv: 5'-ATCGATAAGCCCTAACCTCGGTG-3', primer contains a ClaI restriction site for cloning (marked in bold letters)

[0997]The PCR fragment was amplified by a High-Fidelity DNA Polymerase (Phusion®; Finnzymes), adenylated (BIOTAQ® 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.

[0998]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 Malic Enzyme and Malate Dehydrogenase

[0999]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.

[1000]The following primers were used for amplification [1001]PCR fragment I:

TABLE-US-00016 [1001](SEQ ID NO: 181) Mae-NdeI.fw: 5'-CATATGGTTAGCCTCACCCCCAAT-3', primer contains a 'NdeI restriction site for cloning (marked in bold letters) (SEQ ID NO: 182) MeShortClaI.rv: 5'-ATCGATACAATTCCCGATTAACTATTGACC- 3', primer contains a ClaI restriction site for cloning (marked in bold letters)

[1002]PCR fragment II:

TABLE-US-00017 [1002](SEQ ID NO: 183) MdhRBSClaI.fw: 5'-ATCGATTTTTCTCCACCATCAACACC-3', primer contains a ClaI restriction site for cloning (marked in bold letters) (SEQ ID NO: 184) MdhBglII.rv: 5'-AGATCTAAGCCCTAACCTCGGTG-3', primer contains a BglII restriction site for cloning (marked in bold letters)

[1003]The PCR fragments were amplified by a High-Fidelity DNA Polymerase (Phusion®; Finnzymes), adenylated (BIOTAQ® 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.

[1004]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

[1005]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.

[1006]For the construct with the genomic 5'-region of the pyruvate kinase gene itself serving as promoter, a 2376 by fragment containing the entire coding sequence from pyruvate kinase 1 (sll 0587) plus 770 by upstream of the gene (promoter region) and 320 by downstream of the gene (terminator region) was amplified by PCR using the following primer:

TABLE-US-00018 (SEQ ID NO: 185) #pykA-5fw: 5'-CCTGTTATTGGCCACGGGCAGTA-3' (SEQ ID NO: 186) #pykA-2rv: 5'-GGTTTACCCTGGGCTCGAGAATTTAGG-3', primer contains a XhoI restriction site (CTCGAG) for cloning (marked in bold letters).

[1007]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).

[1008]The construct used, named pVZ321-pyk1, has the structure presented in FIG. 20B, and its insert nucleotide sequence is presented in FIG. 20C.

[1009]For the construct on which pyruvate kinase 1 is under control of the inducible promoter PpetJ, a 1763 by fragment containing the entire coding sequence from pyruvate kinase 1 (sll 0587) plus 320 by downstream of the gene (terminator region) was amplified by PCR using the following primer:

TABLE-US-00019 (SEQ ID NO: 187) #pykA-3fw: 5'-CCCGGTGAAGCATATGAGACCCCT-3', 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. (SEQ ID NO: 188) #pykA-2rv: 5'-GGTTTACCCTGGGCTCGAGAATTTAGG-3', primer contains a XhoI restriction site (CTCGAG) for cloning (marked in bold letters).

[1010]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).

[1011]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

[1012]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.

[1013]For the construct with the genomic 5' region of the pyk2 gene itself serving as promoter, a 2647 by fragment containing the entire coding sequence from pyk 2 (sll 1275) plus 600 by upstream of the gene (promoter region) and 280 by downstream of the gene (terminator region) was amplified by PCR using the following primer:

TABLE-US-00020 (SEQ ID NO: 189) #pykB-1fw: 5'-CCTAAATTCAGGTCGACCGGCAAAC-3', primer contains a SalI restriction site (GTCGAC) for cloning (marked in bold letters). (SEQ ID NO: 190) #pykB-2rv: 5'-CACCAACCAGGCTCGAGTGGG-3', primer contains a XhoI restriction site (CTCGAG) for cloning (marked in bold letters).

[1014]The PCR fragment was digested with SalI/XhoI and ligated into the E. coli-Synechocystis shuttle vector pVZ321 (self replicating plasmid).

[1015]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.

[1016]For the construct on which pyruvate kinase 2 is under control of the inducible promoter PpetJ, a 2057 by fragment containing the entire coding sequence from pyruvate kinase 2 (sll 1275) plus 280 by downstream of the gene (terminator region) was amplified by PCR using the following primer:

TABLE-US-00021 (SEQ ID NO: 191) #pykB-3fw: 5'-CCTAATTTCAGCCCCATATGCAAACG-3', 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. (SEQ ID NO: 192) #pykB-2rv: 5'-CACCAACCAGGCTCGAGTGGG-3', primer contains a XhoI restriction site (CTCGAG) for cloning (marked in bold letters).

[1017]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).

[1018]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

[1019]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.

[1020]The insert of plasmid #67 has the structure presented in FIG. 22A.

[1021]The insert of plasmid #67 contains a 357 bases long cyanobacterial 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_--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_--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.

[1022]The amino acid sequences of the enzymes encoded by the described insert are presented in FIG. 22B for pyruvate kinase I (E. coli K₁₂); in FIG. 22C for enolase (Zymomonas mobilis); and in FIG. 22D for phosphoglycerate mutase (Zymomonas mobilis).

[1023]The nucleotide sequence of the described insert of plasmid #67 is presented in FIG. 22E.

[1024]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.

[1025]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

[1026]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.

[1027]A construct was generated for overexpression of enolase under control of the inducible promoter PpetJ.

[1028]The construct includes the petJ promoter, the 1299 by coding sequence for enolase (slr0752) and 214 by downstream of the gene (terminator region). The enolase gene was amplified by PCR using the following primer:

TABLE-US-00022 (SEQ ID NO: 193) #Eno-SacI-ATG 5'-TAGAGCTCTTAAGTAAAGTCCCCGCCAC CAT-3', (SEQ ID NO: 194) #Eno-XhoI-rev 5'-TACTCGAGGTCATTGCTTCCTTGGCTTA GAAC-3',

[1029]Primers contain a Sad or XhoI restriction site, respectively, for cloning (marked in bold letters).

[1030]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 pVZ321 (self replicating plasmid).

[1031]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

[1032]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.

[1033]A construct was generated for overexpression of phosphoglycerate mutase under control of the inducible promoter PpetJ.

[1034]The construct includes the petJ promoter, the 1047 by coding sequence for phosphoglycerate mutase (slr1124) and 143 by downstream of the gene (terminator region). The phosphoglycerate mutase gene was amplified by PCR using the following primer:

TABLE-US-00023 (SEQ ID NO: 195) #Pgm-SacI-ATG 5'-TAGAGCTCACCAAAGACGATGTGGCCC ACCAA-3' (SEQ ID NO: 196) #Pgm-XhoI-rev 5'-TACTCGAGTATGACCCCGCTGTTGCAG TTC-3'

[1035]Primers contain a SacI or XhoI restriction site, respectively, for cloning (marked in bold letters).

[1036]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).

[1037]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

[1038]Further plasmids were generated in order to overexpress the three glycolytic enzymes pyruvate kinase 1 or 2, enolase and phosphoglycerate mutase from one transcript.

[1039]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.

[1040]In both constructs the overexpression of the three genes is under control of the inducible promoter PpetJ.

[1041]The glycolytic genes were amplified by PCR using the following primers:

TABLE-US-00024 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'

[1042]All primers contain restriction sites for cloning (marked in bold letters).

[1043]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 pIC-PpetJ as followed:

TABLE-US-00025 5'-XhoI-pIC-PpetJ-NdeI-3' 5'-NdeI-pyk1-SacI-3' 5'-SacI-eno-BglII-3' 5'-BglII-pgm-XhoI-3'

[1044]The same construct was generated using fragment 5'-NdeI-pyk2-SacI-3' instead of 5'-NdeI-pyk1-SacI-3'.

[1045]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).

[1046]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. 24G.

j) Construction of DNA-Vector for Overexpression of Phosphoketolase

[1047]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.

[1048]A construct was generated for overexpression of phosphoketolase under control of the inducible promoter PpetJ.

[1049]The construct includes the petJ promoter, the 2418 by coding sequence for phosphoketolase (slr0453) and 307 by downstream of the gene (terminator region). The phosphoketolase gene was amplified by PCR using the following primer:

TABLE-US-00026 (SEQ ID NO: 205) #phk1-NdeI 5'-GTGTCTCATATGGTTACATCCCCCTTTTCCCTT-3' (SEQ ID NO: 206) #phk2-XhoI 5'-CGAGCCCTGCTCGAGCAGGC-3'

[1050]Primers contain a NdeI or XhoI restriction site, respectively, for cloning (marked in bold letters).

[1051]The PCR fragment was digested with NdeI/XhoI and ligated downstream of the PpetJ promoter into pIC-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).

[1052]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

[1053]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.

[1054]A construct was generated for overexpression of phosphoacetyltransacetylase under control of the inducible promoter PpetJ.

[1055]The construct includes the petJ promoter, the 2094 by coding sequence from ORF slr2132 and 258 by downstream of the gene (terminator region). The phosphoacetyltransacetylase gene was amplified by PCR using the following primer:

TABLE-US-00027 (SEQ ID NO: 207) #pta_pPETJ1-NdeI 5'-GTGCCTCATATGACGAGTTCCCTTTATTTA AGCAC-3' (SEQ ID NO: 208) #pta_pPETJ2-XhoI 5'-CGGTTGCTCGAGCATCTGGAACGGTTGGGT AAAT-3'

[1056]Primers contain a NdeI or XhoI restriction site, respectively, for cloning (marked in bold letters).

[1057]The PCR fragment was digested with NdeI/XhoI and ligated downstream of the PpetJ promoter into pIC-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).

[1058]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

[1059]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-00028 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'

[1060]All primers contain restriction sites for cloning (marked in bold letters).

[1061]PCR fragments were cut with the appropriate restriction enzymes and ligated downstream of the PpetJ promoter into pIC-PpetJ as followed:

TABLE-US-00029 5'-XhoI-pIC-PpetJ-NdeI-3' 5'-NdeI-phk-BglII-3' 5'-BglII-pta-XhoI-3'

[1062]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).

[1063]The construct named pVZ322-PpetJ-phk-pta has the structure presented in FIG. 26D, and the nucleotide sequence of the insert of pVZ322-PpetJ-phk-pta is presented in FIG. 26E.

m) Construction of DNA-Vector for Overexpression of Aldehyde Dehydrogenase

[1064]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.

[1065]A construct was generated for overexpression of aldehyde dehydrogenase under control of the inducible promoter PpetJ. The construct includes the petJ promoter, the 1369 by aldehyde dehydrogenase fragment containing the entire coding sequence from ORF slr0091 and 205 by downstream of the gene (terminator region). The aldehyde dehydrogenase (aldh) gene was amplified by PCR using the following primer:

TABLE-US-00030 (SEQ ID NO: 213) #aldh1-NdeI-fw 5'-GTGCCTCATATGAATACTGCTAAAACTGTTGT TGC-3' (SEQ ID NO: 214) #aldh2-XhoI-rev 5'-GATCTCCTCGAGGTAAAGAATCAGCATAGGT CTGG-3'

[1066]Primers contain a NdeI or XhoI restriction site, respectively, for cloning (marked in bold letters).

[1067]The PCR fragment was digested with NdeI/XhoI and ligated downstream of the PpetJ promoter into pIC-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).

[1068]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

[1069]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.

[1070]One construct was generated for overexpression of phosphoenolpyruvate carboxylase under control of the inducible promoter PpetJ.

[1071]The construct includes the petJ promoter, the 3105 by ppc-fragment containing the entire coding sequence from phosphoenolpyruvate carboxylase (sll 0920) and 59 by downstream of the gene (terminator region) was amplified by PCR using the following primer:

TABLE-US-00031 (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).

[1072]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.

[1073]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)

[1074]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.

[1075]The entire rbc operon from Synechocystis sp. PCC6803 was amplified by PCR using the primer pairs: [1076]SynRbc-BglII-fw and SynRbc-PstI-rev for the over-expression from the rbcL-promoter, which are shown in FIGS. 28D and 28E, respectively. [1077]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.

[1078]The database entry numbers for the CyanoBase, the genome database for cyanobacteria (http://bacteria.kazusa.or.jp/cyanobase/index.html) for the Synechocystis rbcL-rbcX-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.

[1079]Mutants were selected on streptomycin plates and grown in BG11 medium containing the appropriate antibiotics (kanamycin 100 mg/l; streptomycin 10 mg/l).

[1080]In Synechocystis sp.PCC6803 mutants were generated by transforming the cells with the plasmid pVZ321b-Prbc-SynRbcLXS (FIG. 28K).

[1081]In the following the vectors, which were used are described.

a) Plasmid pSK9 Structure and Sequence

[1082]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

[1083]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

[1084]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 pIC20H

[1085]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 pIC 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 pIC-PeptJ.

[1086]The plasmid pIC-PpetJ has the structure presented schematically in FIG. 32A, and the nucleotide sequence of pIC PpetJ is presented in FIG. 32B.

Generation of Additional Knock-Out/Knock-Down Mutants of Synechocystis Sp. PCC 6803: Methods and Results

[1087]The following Knock-Out construct sequences have been conveniently described and provided herein: (a) alanine dehydrogenase (ald), (b) ADP-glucose pyrophosphorylase (glgC), (c) pyruvate water dikinase (ppsA), (d) lactate dehyrogenase (ldh), (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

[1088]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 μg/ml BG11, for chloramphenicol from initially 1 to 15 μg/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 μg/ml agar is increased gradually over the course of 4 weeks.

Molecular Analysis of Mutant Host Cells

[1089]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.

[1090]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.

[1091]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

[1092]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° C. under continuous illumination with white light (intensity: 40 μE m^-2 s^-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.

[1093]All other knock-out or knock-down mutants will be grown under standard culture conditions known in the art.

[1094]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.

[1095]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.

[1096]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

[1097]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

[1098]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

[1099]Wild type and mutant strains of Synechocystis PCC 6803 were grown as batch cultures in BG11 medium at 28-29° C. For cultivation of mutants the appropriate antibiotics were added to the medium (kanamycin 75 mg/l; chloramphenicol 10 mg/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×Cu for PpetJ; 3×Fe for PisiA).

[1100]Prior to characterization experiments, pre-cultures were grown in BG11 medium (no excess of Cu or Fe) and aeration with 0.5% CO₂ in air.

[1101]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).

[1102]The total culture volume in characterization experiments was 300 mL in a 500 mL Schott-Flask; the initial OD750 was 1. Cultures were aerated with 0.5% CO₂ in air.

[1103]All mutants were characterized under constant light conditions (75-100 μE m^-2 s^-1). In fast growing cultures, the light intensity was increased during the growth experiment (75-100 μE m^-2 s^-1 up to OD5; then light intensity was increased to 200 μE m^-2 s^-1).

[1104]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 μE m^-2 s^-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:

[1105]Ethanol is oxidized by nicotinamide-adenine dinucleotide (NAD⁺) 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 (Al-DH) (reaction 2).

##STR00001##

[1106]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:

[1107]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 (A₁). 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 (A₂). The absorption measurements can be performed using a photometer or a microplate reader. For plate reader measurements all volumes are downscaled.

[1108]From the measured absorbance difference ΔA=(A₂-A₁) the ethanol concentration in the sample is calculated with the equation:

c = V × MG × d × v × 2 × 1000 × Δ 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×mmol^-1×cm^-1 at 340 nm); d, light path [cm]; v, sample volume [mL]

Literature:

[1109]Protocol of the kit Ethanol, UV method for the determination of ethanol in in foodstuff and other materials, Cat. No. 10176290035, R-Biopharm AG, Darmstadt, Germany.

[1110]H.-O. Beutler (1984) in: Methods in Enzymatic Analysis (Bergmeyer, H. U. ed.) 3^rd ed. Vol. VI, pp. 598-606, Verlag Chemie, Weinheim, Germany.

Growth Properties

[1111]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 O₂ production was determined several times during exponential growth phase using a Clark electrode as followed:

Measurement of Photosynthetic Oxygen Evolution

[1112]Cell are washed 2× with fresh growth medium by centrifugation (3000×g, 10 min, room temperature) and resuspension. The cells are finally resuspended in growth medium to a chlorophyll concentration of 10 to 15 μg chlorophyll/ml. 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.

[1113]The excitation light for photosynthesis experiments is provided by a slide projector with a 150-watt lamp (Osram, Xenophot HLX Germany).

[1114]The oxygen concentration in the chamber is recorded continuously with chart recorder (REC 112, Amersham Pharmacia Biotech) connected to the electrode. The chamber of the oxygen electrode is maintained at 25° 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° 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

[1115]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.

[1116]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 μmol 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)

[1117]For the comparison of ethanol production and photosynthesis, ethanol production rates and rates of photosynthetic oxygen evolution are measured simultaneous in a single assay.

[1118]Cells are washed 2× with fresh growth medium by centrifugation (3000×g, 10 min, room temperature) and resuspension. Cells are resuspended in growth medium to a chlorophyll concentration of 10 to 15 μg 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° 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 μm^-2 s^-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 μm^-2 s^-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×g, 10 min, 4° 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 μmol ethanol*h^-1*mg chlorophyll^-1, the chlorophyll content measured at the beginning of the experiment is then used.

Determination of Intra- and Extracellular Metabolites

[1119]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 mill". 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

[1120]use 5 ml culture. [1121]Centrifuge for 10 min, 4500 rpm. [1122]Resuspend the pellet in 1 ml dd water. [1123]Centrifuge 5 min with 14000 rpm. Discard the supernatant. [1124]Resuspend the pellet in 1 ml double distilled water. [1125]Centrifuge 5 min, 14000 rpm, 4° C. Discard the complete supernatant.

[1126]Continue or store the pellet by -20° C. under Argon atmosphere. [1127]Add 600 ml of extraction buffer. [1128]Extraction buffer: 10:3:1-methanol:chloroform:water [1129]Vortex briefly. [1130]Shake at 4 degrees for 10 min. [1131]Centrifuge 5 min with 14000 rpm. [1132]Transfer 500 μl to a new tube. [1133]Add 200 μl chloroform and 200 μl water. [1134]Centrifuge 5 min with 14000 rpm. [1135]Transfer 500 μl of the upper phase to a new tube and speed vac to dry. [1136]Resuspend the pellet in 100 μl double distilled water. [1137]Shake at 4 degrees for 20 min. Centrifuge 5 min with 14000 rpm. [1138]Transfer 95 μl to a vial for IC.

Extraction of Metabolites Using a Retsch Mill:

[1139]The protocol for extraction of intracellular metabolites was designed by Dr. M. Grundel.

Protocol:

[1140]Cells (150 ml cell culture) are harvested by centrifugation and resuspended in 400 μl buffer (100 mM Tris/HCl, pH 7.5) to which 200 μl 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° C.). After removal of glass beads, remaining intact cells and cell debris was removed by centrifugation (10 minutes, 4° 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 K₂CO₃ 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 μl are used in the optical tests.

Extraction of Metabolites Using Ice Cold Methanol (Snap Shot Extraction):

Literature Describing the Method:

[1141]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.

[1142]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:

[1143]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×g, temperature as low as possible). The pellet is extracted a second time with cold 50% methanol (-20° C.). Supernatants are combined. Methanol is removed by evaporation at 35° C. under vacuum using a rotavapor apparatus. The remaining solution is lyophilized, the residue is resuspended in a minimal volume of water.

[1144]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.

[1145]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.

[1146]Pyruvate and phosphoenolpyruvate are quantified using an optic enzymatic test of Hausler et al. (2000), Anal. Biochem, 281:1-8. This method allows for the quantification of pyruvate and phosphoenolpyruvate in one test.

Protocol:

[1147]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.

[1148]To 450 μl master mix (9 μl 20 mM NADH, 12 μl 1 M MgCl2, 46 μl 1 M KCl, 12 μl 100 mM ADP, 360 μl 100 mM HEPES, 10 μl H₂O) 520 μl sample (if necessary diluted with H₂O) are added. Add 2 μl 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; E340-380=4.83 l×cm×mmol-1) or the absorbance at 340 nm is measured against water (ε340=6.28 l×cm×mmol-1). After complete reaction of pyruvate, 2 μl 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:

[1149]1. Lactate dehydrogenase suspension from bovine heart (L-LDH, Sigma L2625-2.5KU, suspension with 5629.5 U/ml), diluted 1:102. Pyruvate Kinase from rabbit muscle (P K, Serva 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 H₂O

7. 1 M KCl

[1150]Photometric Quantification of Pyruvate (and/or Lactate) in an Enzymatic Cycling System

Method:

[1151]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

[1152]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:

[1153]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 μl) consisting of 50 mM TRIS-buffer, pH 7.5, 256 μM NADH, 1.8 μg lactate dehydrogenase and 60 μg 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 H2O

[1154]3. 0.25 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:

[1155]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:

[1156]Cuvettes contained a final volume of 1000 μl: 100-500 μl sample; 10 μl ammonium sulfate; 10 μl NADH; 10 μl ADP; 10 μl glutamate dehydrogenase solution; TRIS buffer added to a final volume of 1000 μl. The reaction is started by the addition of glutamate dehydrogenase.

Chemicals and Solutions:

[1157]1. 1 M ammonium sulfate

2. 20 mM NADH

3. 0.1 M ADP

[1158]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/HCl pH 8.0

[1160]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.

[1161]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.

[1162]Short description of the UV-method for the determination of acetic acid in foodstuff and other materials from Boehringer Mannheim/R-Biopharm, Darmstadt, Germany

[1163]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).

Acetate+ATP+CoA ACS acetyl-CoA+AMP+PP (1)

[1164]Acetyl-CoA reacts with oxaloacetate to citrate in the presence of citrate synthase (CS) (2).

Acetyl-CoA+oxaloacetate+H2O CS citrate+CoA (2)

[1165]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.

L-malate+NAD+L-MDH oxaloacetate+NADH+H+ (3)

[1166]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).

[1167]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.

[1168]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-Integrating 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 ¹³C-NMR and gas chromatography. Mass spectroscopy (GCMS) measurements.

[1169]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-00032 Metabolite Metabolite Intracellular Extracellular level in mmol level in mmol per liter per liter OD₇₅₀ wt mutant wt mutant 1.0 +N A A + Δ F F + Δ -N, B B + Δ G G + Δ 0 h -N, C C + Δ H H + Δ 3.5 h -N, D D + Δ I I + Δ 6 h -N, E E + Δ J J + Δ 24 h Data will be verified by repetitions. A-J represent wild type values for the indicated conditions Δ represents an increment relative to the wt measurement

[1170]The table shows an example for such an experiment. In other experiments the optical density (OD₇₅₀) at the beginning of the experiment and the time points can be different

TABLE-US-00033 Metabolite Intracellular Metabolite level in mmol Extracellular per liter level in mmol (calculated per per liter Time packed cell culture of volume¹) volume cultivation wt mutant wt Mutant T1 A A + Δ E E + Δ T2 B B + Δ F F + Δ T3 C C + Δ G G + Δ T4 D D + Δ H H + Δ Data will be verified by repetitions. A-H represent wild type values Δ represents an increment relative to the wt measurement

[1171]Parameters such as OD₇₅₀nm, Chlorophyll content, protein content and cell number will also be measured in standardizing and evaluating metabolite values at different time points.

[1172]In addition, measurements can be obtained for variations in culture conditions such as light intensity, growth in darkness and in day/night cycles respectively, CO₂ 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.

[1173]The data will be analyzed and presented graphically as previously described herein.

Analysis of Ethanol Production

[1174]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.

[1175]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

[1176]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

[1177]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 μg/ml Bg11, for chloramphenicol from initially 1 to 15 μg/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 μg/ml agar is increased gradually over the course of 4 weeks.

Molecular Analysis of Mutant Host Cell

[1178]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.

[1179]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

[1180]wild type (WT) and mutant strains will be grown under standard culture conditions.

[1181]Nitrogen step-down conditions will be as previously described herein.

[1182]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.

[1183]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.

[1184]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.

[1185]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.

[1186]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-00034 Metabolite Metabolite Intracellular Extracellular level in mmol level in mmol per liter per liter OD₇₅₀ wt mutant wt mutant 1.0 +N A A + Δ F F + Δ -N, B B + Δ G G + Δ 0 h -N, C C + Δ H H + Δ 3.5 h -N, D D + Δ I I + Δ 6 h -N, E E + Δ J J + Δ 24 h Data will be verified by repetitions. A-J represent wild type values for the indicated conditions Δ represents an increment relative to the wt measurement

[1187]The table shows an example for such an experiment. In other experiments the optical density (OD₇₅₀) at the beginning of the experiment and the time points can be different

TABLE-US-00035 Metabolite Intracellular Metabolite level in mmol Extracellular per liter level in (calculated per mmol per liter Time packed cell culture of volume¹) volume cultivation wt mutant wt mutant T1 A A + Δ E E + Δ T2 B B + Δ F F + Δ T3 C C + Δ G G + Δ T4 D D + Δ H H + Δ Data will be verified by repetitions. A-H represent wild type values Δ represents an increment relative to the wt measurement

[1188]Parameters such as OD₇₅₀nm, Chlorophyll content, protein content and cell number will also be measured in standardizing and evaluating metabolite values at different time points.

[1189]In addition, measurements can be obtained for variations in culture conditions such as light intensity, growth in darkness and in day/night cycles respectively, CO₂ 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.

[1190]The data will be analyzed and presented graphically as previously described herein.

Analysis of Ethanol Production

[1191]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.

[1192]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.

X. Experimental Data for Characterization of Metabolic Mutants Containing at Least One First or One First and One Second Genetic Modification

[1193]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

[1194]Characterization of the glycogen deficient glycogen synthase double knock out mutants of Synechocystis PCC 6803:

Nomenclature:

TABLE-US-00036 [1195] Enzyme: Glycogen Glycogen (starch) synthase 1 synthase 2 EC no.: EC 2.4.1.21 EC 2.4.1.21 Gene name: glgA1 glgA2 Gene in sll0945 sll1393 Synechocystis PCC 6803:

[1196]Theoretical background: Diverting the production of storage reserves into an enhanced production of pyruvate/ethanol [1197]Genetic Manipulation: double knockout by insertion of a chloramphenicol cassette (ΔglgA1) and kanamycin cassette (ΔglgA2) 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).

[1198]Determination of intracellular Glycogen before and after a N step down

[1199]The procedure is an adaptation of the method described by Ernst et al. (A. Ernst, H. Kirschenloher, J. Diez, P. Boger. 1984. Arch. Microbiol. 140:120-125). 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.

[1200]Protocol: [1201]Spin down 1-4 ml of Synechocystis culture before and after N step down resp. at RT and remove the supernatant [1202]Add 200 μl KOH (30% w/v) to the pellet and incubate 90 minutes at 95° C. in a heating block [1203]Add 600 μl cold ethanol (96%) and incubate 90 min on ice [1204]Spin down and discard the supernatant [1205]Wash once with ethanol (70%) and once with ethanol (96%) [1206]Dry the pellet in a vacuum centrifuge [1207]Dissolve the pellet in 45-90 μl acetate buffer [1208]Add 5-10 μl enzyme mix (amyloglucosidase+alpha-amylase from Bacillus amyloliquefaciens, purchased from Roche) and incubate 90 min at 45° C. [1209]Use 10-40 μl of the resulting sample for the determination of glucose after manufacturer's instruction (Infinity glucose hexokinase liquid stable reagent for optical test at 340 nm; Cat No. TR15421Thermo Electron Corporation)

Reaction:

##STR00002##

[1210]Chemicals and solutions:1. aqueous solution of KOH (30% w/v)2. ethanol 96% v/v3. 100 mM acetate buffer, adjusted to pH 5.0 with NaOH4. 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)

[1211]Quantification of intracellular and extracellular pyruvate and oxoglutarate levels before and after nitrogen deprivation ("N step down")

Explanation for "N Step Down":

[1212]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 μE m^-2 s^-1), BG11, 29° C.: [1213]Growth properties: no difference between wild type (wt) and mutant (M8) (the growth of M8 is impaired under High Light conditions [130 μE m^-2 s^-1] and low inoculi [initial OD₇₅₀<0.1]) [1214]Pigmentation: no difference between wt and mutant [1215]Storage substances: no glycogen production by the mutants in contrast to the wtContinuous Light (40 μEm^-2s^-1), BG11 without nitrogen (24 h, 48 h), 29° C.:(N starvation) [1216]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. [1217]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 M8Pyruvate level:

TABLE-US-00037 [1217] Intracellular level in mmol per Extracellular liter level in mmol (calculated per liter per packed culture cell volume¹) volume OD₇₅₀ 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

[1218]Growth properties and extracellular pyruvate levels of the ΔglgA1/ΔglgA2 double mutant (M8) under nitrogen replete and nitrogen starved conditions are presented in FIG. 32C.

[1219]The glycogen deficient mutant M8 was grown up to an OD₇₅₀ 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-00038 [1220] Intracellular level in mmol Hours per liter Extracellular after (calculated per level in mmol nitrogen packed cell per liter step volume¹) culture volume 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 ¹The packed cell volume is less than 1% of the culture volume nd, not detectable

Light/Dark Cycle (16 h/8 h), BG11, 29° C.:

[1221]Growth properties: no difference between wt and mutants M1 and M8

[1222]Further mutant characterization of the glycogen deficient mutant M8 in comparison with the wild type strain of Synechocystis sp. PCC6803

Culture Conditions:

[1223]Continuous light (150 μE m^-2 s^-1), 28° C.: [1224]Aeration with air (no additional CO₂ supplementation) [1225]Culturing in glass flasks with 5 cm diameter, 400 ml culture volume [1226]Media: BG11 buffered with TES buffer (Sigma-Aldrich Inc.) at pH 8

Storage Substances:

[1226] [1227]No glycogen production by the mutants in contrast to the wild type. [1228]Pyruvate concentrations in the media determined by using an optical enzymatic test:

TABLE-US-00039 [1228] Pyr- Pyr- Pyr- uvate uvate Pyruvate uvate 0 h 3.5 h 6 h 24 h after after after after N step N step N step N step OD₇₅₀ Chlorophyll down down down down WT 1.2 6.18 μg/ml 0 μM 5.1 μM 4.0 μM 2.5 μM M8 1.1 3.60 μg/ml 0 μM 37 μM 79 μM 473 μM mutant

[1229]Pyruvate concentrations in the media determined by ion chromatography:

TABLE-US-00040 [1229] Pyruvate Pyruvate 0 h after 24 h after N step N step OD₇₅₀ Chlorophyll down down WT 1.2 6.18 μg/ml 0 μM 13.4 μM M8 mutant 1.1 3.60 μg/ml 0 μM 511 μM

Pyruvate Concentrations in the Media Plus Cells (Snap Shot Extraction) Determined by Ion Chromatography:

TABLE-US-00041 [1230] Pyruvate Pyruvate 0 h 24 h after after N step N step OD₇₅₀ Chlorophyll down down WT 1.2 6.18 μg/ml 0 μM 6.12 μM M8 1.1 3.60 μg/ml 0 μM 523 μM mutant

[1231]Wildtype and mutant were transferred into a medium without combined nitrogen and grown 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

[1232]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.

[1233]Data results are presented graphically in FIGS. 32D and 32E.

Summary Pertaining to Ethanol Production:

[1234]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 (OD₇₅₀ 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.

[1235]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:

[1236]Glycogen synthesis requires energy (ATP): [1237]Photosynthesis→glucose phosphate [1238]glucose phosphate+ATP→ADP-glucose+pyrophosphate [1239]n ADP-glucose→glycogen+n ADP

[1240]During the night, glycogen will be degraded: [1241]glycogen+n phosphate→n glucose phosphate [1242]glucose phosphate→→pentose phosphate+CO₂↑ [1243]pentose phosphate→→pyruvate pyruvate ethanol+CO₂↑

Conclusions:

[1243] [1244]Ethanol production via glycogen requires more energy and releases 50% more CO₂ than direct production. [1245]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.

[1246]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-pdc/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).

[1247]Both graphs depict on the Y-axis the concentrations of pyruvate and ethanol in μM normalized to the cell density measured at 750 nm (OD₇₅₀nm). The x-axes denote the course of the experiments in hours.

[1248]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.

[1249]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 M8 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.

[1250]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 OD₇₅₀nm were determined over the course of 15 days. Results are depicted graphically in FIG. 32H.

[1251]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.

TABLE-US-00042 ΔglgA1/ΔglgA2 mutant μmol μmol % of μmol O₂/ EtOH/ EtOH/ theoretical mg Chl * h mg Chl * h μmol O₂ fixed CO2 S. PCC6803 98.3 5.0 0.051 15.4 pVZ321b-PisiA- PDC-ADHII ΔglgA1/A2 34.8 5.4 0.154 46.2 pVZ321b-PisiA- PDC-ADHII

[1252]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 CO₂ 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 (ΔglgA1/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 (ΔGLGC) as a First Genetic Modification

[1253]Construction of the DNA-vector pGEM-T/ΔglgC-KM, which was used for generation of ΔglgC mutant, was already described herein. The obtained ΔglgC 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.

[1254]The partially segregated mutant ΔglgC was examined in comparison to Synechocystis wild-type strain under constant light conditions as described herein.

Growth Characteristics Under Constant Light Conditions

[1255]The ΔglgC mutant is generally more sensitive to light 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 ΔglgC 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., 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).

[1256]Like in the ΔglgA mutant described above, in the ΔglgC mutant the extracellular pyruvate level is strongly increased. Data from one representative experiment are shown in the following table:

TABLE-US-00043 7 days 4 days pyr- 9 days pyruvate uvate pyruvate OD₇₅₀ [mM] OD₇₅₀ [mM] OD₇₅₀ [mM] PCC6803 Wt 1.7 0.009 2.0 0.001 2.4 0.003 ΔglgC 1.1 0.087 2.0 0.093 2.2 0.199

[1257]In wild type cells glycogen synthesis is increased during nitrogen starvation. Therefore, in the ΔglgC mutant, that is not able to produce glycogen, an additional increase of the pyruvate level was achieved by a nitrogen step down.

[1258]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 ΔglgA 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-00044 +N -N OD₇₅₀ pyruvate [mM] OD₇₅₀ pyruvate [mM] PCC 6803 Wt 1.7 0.012 1.2 0.010 ΔglgC 1.3 0.295 1.2 0.361

ADP Glucose Pyrophosphorylase (GlgC) Knock-Out Mutant Expressing PDC and ADH

[1259]The DNA-vector pGEM-T/ΔglgC-KM was transformed into the PDC-ADHII expressing mutant Synechocystis PCC6803 pSK10-PpetJ-PDC-ADHII. The obtained mutant ΔglgC pSK10-PpetJ-PDC-ADHII was fully segregated and was grown in BG11 medium containing 100 mg/l kanamycin and 10 mg/l streptomycin.

[1260]Ethanol production was induced by copper starvation and compared to that of Synechocystis wild-type pSK10-PpetJ-PDC-ADHII.

[1261]In short term experiments under optimal conditions (light, CO₂) the overall as well as the relative (to photosynthetic activity) ethanol production rate of the ΔglgC 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 ΔglgC pSK-PpetJ-PDC-ADHII mutant. (Data are the mean of 2 measurements)

TABLE-US-00045 ΔglgC mutant μmol μmol % of μmol O₂/ EtOH/ EtOH/ theoretical mg Chl * h mg Chl * h μmol O₂ fixed CO2 S. PCC6803 pSK- 250 4 0.016 4.8 PpetJ-PDC-ADHII ΔglgC pSK-PpetJ- 125 9 0.072 21.6 PDC-ADHII

[1262]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 (ΔPPSA) as a First Genetic Modification

[1263]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/ΔppsA, 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.

[1264]The mutant ΔppsA was characterized in comparison to the Synechocystis wild-type strain under constant light conditions as described herein.

[1265]No significant differences could be detected in cell growth, chlorophyll content and photosynthetic oxygen production between Synechocystis PCC6803 wild-type and the ΔppsA mutant. However, in several independent growth experiments the extracellular pyruvate level of the ΔppsA mutant was increased at the end of the log-phase. Data from one representative experiment are shown in the following table:

TABLE-US-00046 10 days 4 days pyr- 14 days pyruvate uvate pyruvate OD₇₅₀ [mM] OD₇₅₀ [mM] OD₇₅₀ [mM] PCC6803 Wt 2.0 0 12.8 0.009 13.3 0.010 ΔppsA 1.8 0.014 8 0.010 10.8 0.073

X.4 Metabolic Mutant Harbouring a Knock Out of Either Acetatekinase (ΔACK) or a Double Knock Out of Acetatekinase and Phosphoacetyltransacetylase (RACK/PTA) as a First Genetic Modification

[1266]The following knock-out mutants were generated: the single-mutants Δack and Δpta and the double mutant Δack/Δpta. 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 Δack 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 Δpta mutant is described above. The double knock-out mutant Δack/Δpta was generated by transformation of pBlue-ack-Kan into the Δpta mutant.

[1267]All mutants were fully segregated. Mutants were grown in BG11 medium containing the appropriate antibiotics (kanamycin 75 mg/l; chloramphenicol 10 mg/l).

[1268]Mutants Δack, Δpta, Δack/pta and Synechocystis wild-type strains were examined under constant light conditions as described.

Results:

[1269]No significant differences could be detected in cell growth, chlorophyll content and photosynthetic oxygen production between the Synechocystis PCC6803 wild type and mutants Δack, Δpta and double mutant Δack/Δpta.

[1270]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 (OD₇₅₀nm) and the concentration of pyruvate in the medium are given at two time points at the end of the log phase.

TABLE-US-00047 10 days 14 days OD₇₅₀ nm pyruvate [mM] OD₇₅₀ nm pyruvate [mM] PCC6803 wt 4.6 0.006 6.2 0.012 Δack 6.6 0.009 7.0 0.025 Δpta 6.7 0.010 6.3 0.019

TABLE-US-00048 10 days 12 days OD₇₅₀ nm pyruvate [mM] OD₇₅₀ nm pyruvate [mM] PCC6803 wt 8 0.003 8 0.011 Δack/Δpta 6 0.004 7 0.026

Acetatekinase (ack) and Acetatekinase (ack)/Phosphoacetyltransacetylase (pta) Knock-Out Mutants Expressing PDC and ADH

[1271]The self-replicating plasmid pVZ321b-PpetJ-PDC-ADHII was conjugated into each of the mutants: Δack, and double mutant Δack/pta, resulting in mutants Δack pVZ321b-PpetJ-PDC-ADHII, and Δack/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:

[1272]In several independent growth experiments, the double mutant Δack/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 Δack, 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.

[1273]Data from one representative experiment are shown in the following table. FIGS. 32I and 32J depict a graphical presentation of these data.

TABLE-US-00049 time [days] 0 6 d 11 d 13 d PCC6803 pVZ321b- OD₇₅₀ nm 1.2 2.5 3.2 3.9 PpetJ-PDC-ADHII EtOH [%] 0.000 0.030 0.060 0.072 Δack/pta pVZ321b- OD₇₅₀ nm 1.2 2.3 2.6 2.7 PpetJ-PDC-ADHII EtOH [%] 0.000 0.044 0.098 0.121 Δack pVZ321b-PpetJ- OD₇₅₀ nm 1.3 2.8 3.9 4.8 PDC-ADHII EtOH [%] 0.000 0.034 0.082 0.094

[1274]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 OD₇₅₀nm and day.

TABLE-US-00050 EtOH [%] after 13 EtOH production days of growth rate [%/OD₇₅₀nm * d] PCC6803 pVZ321b- 0.072 0.001 PpetJ-PDC-ADHII Δack/pta pVZ321b- 0.121 0.0039 PpetJ-PDC-ADHII Δack pVZ321b- 0.094 0.001 PpetJ-PDC-ADHII

[1275]When mutants Δack pVZ321b-PpetJ-PDC-ADHII, and Δack/pta pVZ321b-PpetJ-PDC-ADHII and the reference strain S. PCC6803 pVZ321b-PpetJ-PDC-ADHII were grown under day/night cycle conditions, similar results were obtained. After induction of PDC and ADHII by copper starvation, strains Δack/pta pVZ321b-PpetJ-PDC-ADHII and Δack pVZ321b-PpetJ-PDC-ADHII showed higher ethanol production rates compared to the reference strain S. PCC6803 pVZ321b-PpetJ-PDC.

[1276]At three consecutive days during the logarithmic growth phase, photosynthetic capacity and ethanol production was measured in the oxygen electrode as described.

[1277]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.

[1278]Following the reaction equation of photosynthesis 6 CO₂+12H₂O→C₆H₁₂O₆+6 O₂+6H₂O, the photosynthetic capacity [μmol O₂/mg Chl*h] is equivalent to the maximal carbon fixation [μmol CO₂/mg Chl*h]. Therefore the factor (μmol EtOH per/μmol O₂) given in the following table puts EtOH production into perspective of carbon fixation/photosynthesis.

[1279]Values are the mean of three consecutive measurements.

TABLE-US-00051 EtOH PS capacity production [μmol O₂/ [μmol EtOH/ μmol EtOH/ mg Chl * h] mg Chl * h] μmol O₂ PCC6803 pVZ321b- 221 3.6 0.016 PpetJ-PDC-ADHII Δack/pta pVZ321b- 241 6.1 0.025 PpetJ-PDC-ADHII Δack pVZ321b- 301 7.2 0.024 PpetJ-PDC-ADHII

Conclusions:

[1280]Ethanol production in the double mutant Δack/pta, harboring pVZ321b-PpetJ-PDC-ADHII, was significantly enhanced compared to the reference strain (wt) and also in comparison to the single mutant Δack pVZ321b-PpetJ-PDC-ADHII. For the single mutant Δack 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

[1281]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_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_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:

[1282]No significant differences could be detected in cell growth, chlorophyll content and photosynthetic oxygen production between Synechocystis PCC6803 wild type and pdhB_anti mutant. After induction of the petJ promoter, the level of extracellular pyruvate was slightly increased in the pdhB_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-00052 7 days 9 days OD₇₅₀ pyruvate [mM] OD₇₅₀ pyruvate [mM] PCC6803 wt 3.6 0 5.3 0.004 pdhB_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

[1283]Mutant and Synechocystis wild-type strains were grown at 28° C., under constant light (70 μE m^-2 s^-1) and aerated with CO₂-enriched air (0.5% CO₂). The initial OD₂₅₀ 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:

[1284]The rate of oxygen evolution was measured with a Clark-type oxygen electrode (Rank Brothers, UK). Prior to the measurement cells were washed 2× and resuspended in BG-11 medium supplemented with 25 mM NaHCO₃. Light intensity was saturating with approx. 500 μE/s*m².

[1285]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 (2×10 min). The supernatant of a centrifugation (15 min, 14000 rpm, 4° C., Micro 200R, Hettich) was used for the experiments. The protein content of cell extracts was measured with the method of Lowry.

[1286]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:

[1287]5 μl to 15 μl of cell extracts were mixed with 750 μl of 50 mM HEPES/KOH, pH 7.5, 20 mM MgCl₂, 50 mM KHCO₃, 0.15 mM NADH, 5 mM ATP, 2.5 mM Phosphocreatine, 1.5 μl carbonic anhydrase (10 U/μl in 50 mM HEPES, pH 7.5), 7.5 μl creatine kinase (0.5 U/μl) 3.75 μl of glyceraldehyde-3-phosphate dehydrogenase (12.5 mg/ml), phosphoglycerate kinase (suspension with 10 mg/ml). The assay was incubated at 30° C. for 10 min. Then the reaction was started by the addition of 7.5 μl of 250 mM ribulose-1,5-bisphosphate and the absorption of 340 nm was monitored.

Results and Conclusions:

[1288]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).

[1289]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-00053 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 Dryweight Chl a Dryweight [d] OD₇₅₀ nm [mg/l] [g/l] OD₇₅₀ 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

[1290]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-00054 TABLE 2 RuBisCO activity and photosynthetic oxygen evolution of Synechocystis wild type and a mutant overexpressing the endogenous RuBisCO operon. oxygen RuBisCO activity evolution [μmol RBP/min * mg [μmol O₂/h * protein] mg chl] PCC6803 wild type 0.23 (100%) 107.8 (100%) pVZ321b-Prbc- 0.48 (209%) 124.6 (115%) SynRbcLXS

[1291]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).

[1292]FIGS. 32L, 32M and 32N, respectively show the OD₇₅₀, the ethanol production and the ethanol production normalized to the OD₇₅₀ for the mutant Synechocystis PCC6803 harboring the pSK10-PisiA-PDC/ADHII plasmid and the mutant additionally containing the vector pVZ321b-Prbc-SynRbc.

[1293]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

[1294]Construction of the DNA-vector pVZ321-PpetJ-pyk2, which was used for the generation of a pyk2 overexpression mutant, was already described herein.

[1295]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 light conditions as described herein. Expression of pyruvate kinase gene was induced by copper starvation.

Results:

[1296]No significant differences could be detected in cell growth, chlorophyll content and photosynthetic oxygen production between Synechocystis PCC6803 wild type and mutant PCC6803 PpetJ-pyk2.

[1297]After induction of the petJ promoter, the level of extracellular pyruvate was slightly increased in the PCC6803 PpetJ-pyk2 mutant compared to the wild-type.

TABLE-US-00055 9 days 6 days pyr- 14 days pyruvate uvate pyruvate OD₇₅₀ [mM] OD₇₅₀ [mM] OD₇₅₀ [mM] PCC6803 Wt 1.3 0.018 1.9 0.005 2.5 0.009 PpetJ-pyk2 0.8 0.016 1.3 0.051 1.9 0.064

Pyruvatekinase 2 Overexpression Mutant Expressing PDC and ADH

[1298]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.

[1299]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.

TABLE-US-00056 (data are mean of two measurements) μmol μmol μmol O₂/ EtOH/mg EtOH/ mg Chl * h Chl* h μmol O₂ S. PCC6803 pSK- 164.5 9.5 0.058 PisiA-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

[1300]Two mutants have been created for overexpression of the three glycolytic genes pyruvate kinase (pyk), enolase (eno) and phosphoglycerate mutase (pgm).

[1301]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.

[1302]In the other mutant the expression of additional copies of the endogenous genes pyruvate kinase 2, enolase and phosphoglycerate mutase from Synechocystis PCC6803 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.

[1303]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:

[1304]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.

[1305]Excretion of pyruvate was increased in mutant PCC6803 Prbc-pyk-eno-pgm compared to wild-type, as shown in the following table:

TABLE-US-00057 10 days 14 days pyruvate pyruvate OD₇₅₀ [mM] OD₇₅₀ [mM] PCC6803 (BG11) 4.6 0.006 6.2 0.012 PCC6803 Prbc-pyk- 3.0 0.017 6.1 0.032 eno-pgm

[1306]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-00058 7 days 14 days pyruvate pyruvate OD₇₅₀ [mM] OD₇₅₀ [mM] PCC6803 (BG11-Cu) 1.6 0 3 0.006 PCC6803 PpetJ- 3.1 0.013 3.7 0.024 pyk2-eno-pgm

Expression of Pyruvate Kinase, Enolase and Phospho-Glycerate Mutase in Synechocystis Strains Expressing Pdc Enzyme Alone as a Second Genetic Modification.

[1307]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 Sad and PstI.) The resulting mutants were cultured in BG11 containing 10 mg/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).

[1308]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-00059 μmol O₂/ μmol EtOH/ μmol EtOH/ mg Chl * h mg Chl * h μmol O₂ PCC6803 pSK-PpetJ-PDC 130 1.8 0.014 PCC6803 pSK-PpetJ-PDC 148 3.2 0.022 pVZ-Prbc-pyk-eno-pgm PCC6803 pSK-PpetJ-PDC 197 2.5 0.012 PCC6803 pSK-PpetJ-PDC 104 2.8 0.028 pVZ-PpetJ-pyk2-eno-pgm

Conclusions

[1309]These data suggest that overexpression of the glycolytic enzymes pyruvate kinase, enolase and phosphoglycerate mutase leads to a higher flux from CO₂ 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

[1310]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 mg/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:

[1311]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.

[1312]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-00060 Not induced (BG11) Induced (BG11-Cu) 10 days 10 days OD₇₅₀ pyruvate [mM] OD₇₅₀ pyruvate [mM] PCC6803 Wt 8.3 0.010 9.3 0.011 PpetJ-me 10.1 0.005 8.3 0.032 PpetJ-me-mdh 7.8 0.005 8.5 0.024

[1313]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 29413 Harbouring a Knockout of the ADP-Glucose-Pyrophosphorylase as a First Genetic Modification

[1314]In the following the EtOH production in Anabaena PCC7120 transformed with the integrative PpetE-PDC-ADHII and PpetE-PDC constructs will be discussed.

[1315]In a first test experiment EtOH production in Anabaena PCC7120 with PpetE-pdc-adhll or PpetE-pdc inserted in ADP-glucose-pyrophosphorylase gene, agp, was measured of the following mutants: A.7120 Δagp (a114645)::C.K3-PpetE-pdc-adhII, named "PpetE-pdc-adhII" and A.7120 Δagp (a114645)::C.K3-PpetE-pdc, named "PpetE-pdc". Mutant A.7120 Δagp (a114645)::C.K3, named Δagp, served as control.

[1316]Cultures of all mutants were grown at 28° C., under continuous light conditions (40 μE/m² s¹) 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 μg/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 Anabaena PCC7120 [Buikema, W. J., 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.

[1317]Expression of the ethanologenic genes was induced by addition of 1× copper (0.32 μM CuSO₄). This corresponds to the copper concentration present in BG11 medium.

[1318]As a measure of growth, chlorophyll was determined at several time points and ethanol was measured using the already described enzymatic method.

TABLE-US-00061 TABLE 1 Growth and ethanol production of Anabaena mutants expressing ethanologenic genes under control of petE promoter. Chl, chlorophyll in [μg/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" Δagp 8 0 16 0.0001 20 0.0001 25 0.0001 (con- trol)

[1319]Ethanol was produced by both integrative mutants, while in the control strain (mutant Δagp) 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 (http://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 Zn²+ Dependent Alcohol Dehydrogenase and PDC and/or ADH Enzymes Under the Control of Various Inducible Promoters

[1320]In the following further embodiments of the invention related to for example an overexpressed Zn²+ dependent alcohol dehydrogenase, an overexpressed alcohol dehydrogenase, directly converting acetyl-CoA to ethanol, or promoters 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.

[1321]Construction of selfreplicating (extrachromosomal) and chromosome-integrative vectors for the inducible overexpression of ethanologenic enzymes in cyanobacteria [1322]Construction of extrachromosomal pVZ-vectors for inducible overexpression of pyruvate decarboxylase (ZmPdc) and alcohol dehydrogenase (ZmAdhII) from Zymomonas mobilis

[1323]The construction of the certain vectors including the different promoters were done by using the following general protocol: [1324]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. [1325]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. [1326]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 by and include the ribosome binding site of the corresponding genes):

TABLE-US-00062 [1326](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'

[1327]All forward primer included the SalI restriction site, all reverse primer included a EcoRI restriction site for cloning (marked bold). [1328]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-pdc/adh [1329]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(http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nuccore&id=4323382- ) [1330]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/.

[1331]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.

[1332]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.

[1333]Remark: In all following nucleotide sequences of promoters restriction sites for clonings are marked (colored).

[1334]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.

[1335]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.

[1336]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.

[1337]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.

[1338]Introduction of further well suited inducible promoters into the existing pVZ-expression constructs (point 1).

[1339]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.

[1340]Representative new promoters include, but are not limited to, the following:

[1341](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

[1342]Biol. Chem. 2004 Feb. 20; 279(8):7229-33. Epub 2003 December. The efficient functioning of photosynthesis and respiration in Synechocystis sp. PCC 6803 strictly requires the presence of either cytochrome c6 or plastocyanin. [1343]Duran R V, Hervas M, De La Rosa M A, Navarro J A.

[1344]A plasmid created with this promoter element is presented schematically in FIG. 36B.

[1345](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

[1346]Arch Microbial, 2006 October; 186(4):273-86. Epub 2006 Jul. 26. The heat shock response in the cyanobacterium Synechocystis sp. Strain PCC 6803 and regulation of gene expression by HrcA and SigB. [1347]Singh A K, Summerfield I C, Li H, Sherman L A FEBS, Lett. 2003 Nov. 20; 554(3):357-62. Antagonistic dark/light-induced SigB/SigD, group 2 sigma factors, expression through redox potential and their roles in cyanobacteria. [1348]Imamura S, Asayama M, Takahashi H, Tanaka K, Takahashi H, Shirai M J Biol Chem. 2006 Feb. 3; 281(5):2668-75. Epub 2005 Nov. 21. Growth phase-dependent activation of nitrogen-related genes by a control network of group 1 and group 2 sigma factors in a cyanobacterium. [1349]Imamura S, Tanaka K, Shira M, Asayama M. A plasmid created with this promoter element is presented schematically in FIG. 36D.

[1350](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

[1351]Plant Physiol. 1998 May; 117(1):225-34. Transcriptional and posttranscriptional control of mRNA from lrtA, a light-repressed transcript in Synechococcus sp. PCC 7002. [1352]Samartzidou H, Widger W R

[1353]A plasmid created with this promoter element is presented schematically in FIG. 36F.

[1354](4) FIG. 36 G shows the nucleotide sequence of the lrtA promoter (Synechocystis sp. PCC 6803) lrtA gene:sll0947 (encoding the light repressed protein A homolog induced expression after light to dark transition)

REFERENCES

[1355]Plant Physiol. 1998 May; 117(1):225-34. Transcriptional and posttranscriptional control of mRNA from lrtA, a light-repressed transcript in Synechococcus sp. PCC 7002. [1356]Samartzidou H, Widger W R

[1357]A plasmid created with this promoter element is presented schematically in FIG. 36H.

[1358](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

[1359]Biochem Biophys Res Commun. 1999 Feb. 5; 255(1):47-53. Light-dependent and rhythmic psbA transcripts in homologous/heterologous cyanobacterial cells. [1360]Agrawal G K, Asayama M, Shirai M.

[1361]A plasmid created with this promoter element is presented schematically in FIG. 36J.

[1362](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

[1363]Plant Mol. Biol. 1989 December; 13(6):693-700 Influence of light on accumulation of photosynthesis-specific transcripts in the cyanobacterium Synechocystis 6803. [1364]Mohamed A, Jansson c.

[1365]A plasmid created with this promoter element is presented schematically in FIG. 36L.

[1366](7) FIG. 36M depicts the nucleotide sequence of the psaA promoter (Synechocystis sp. PCC6803); PsaA gene: slr134 (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

[1367]Plant Cell Physiol. 2005 September; 46(9):1484-93. Epub 2005 Jun. 24. Regulation of photosystem I reaction center genes in Synechocystis sp. strain PCC 6803 during Light acclimation. [1368]Herranen M, Tyystjarvi T, Aro E M. [1369]Plant Cell Phys. 2006 July; 47(7):878-90. Epub 2006 May 16. Characterization of high-light-responsive promoters of the psaAB genes in Synechocystis sp. PCC 6803. [1370]Muramatsu M, Hihara Y.

[1371]A plasmid created with this promoter element is presented schematically in FIG. 36N.

[1372](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

[1373]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. [1374]Marin K, Kanesaki Y, Los D A, Murata N, Suzuki I, Hagemann M. [1375]J. Bacteriol, 2002 June; 184(11):2870-7. Salt-dependent expression of glucosylglycerol-phosphate synthase, involved in osmolyte synthesis in the cyanobacterium Synechocystis sp. strain PCC 6803. [1376]Marin K, Huckauf J, Fulda S, Hagemann M.

[1377]A plasmid created with this promoter element is presented schematically in FIG. 36P.

[1378](9) FIG. 36Q depicts the nucleotide sequence of the nirA promoter (Synechocystis sp. PCC6803); nirA gene: sir0898 (encoding ferredoxin-nitrite reductase) induced expression after transition from ammonia to nitrate

REFERENCES

[1379]Appl Environ Microbiol. 2005 October; 71(10):5678-84. Application of the Synechococcus nirA promoter to establish an inducible expression system for engineering the Synechocystis tocopherol pathway. [1380]Qi Q, Hao M, Ng W O, Slater S C, Baszis S R, Weiss J D, Valentin H E. [1381]J. Bacterial. 1998 August; 180(16):4080-8 cis-acting sequences required for NtcB-dependent, nitrite-responsive positive regulation of the nitrate assimilation operon in the cyanobacterium Synechococcus sp. strain PCC 7942. [1382]Maeda S, Kawaguchi Y, Ohe T A, Omata T.

[1383]A plasmid created with this promoter element is presented schematically in FIG. 36R.

[1384](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

[1385]Microbiology, 1994 May; 140 (Pt 5):1151-9. Cloning, sequencing and transcriptional studies of the genes for cytochrome c-553 and plastocyanin from Anabaena sp. PCC 7120. [1386]Ghassemian M, Wong B, Ferreira F, Markley J L, Straus N A. [1387]Proc Natl Acad Sci USA. 2001 Feb. 27; 98(5):2729-34. Epub 2001 Feb. 20. Expression of the Anabaena hetR gene from a copper-regulated promoter leads to heterocyst differentiation under repressing conditions. [1388]Buikema W J, Haselkorn R.

[1389]A plasmid created with this promoter element is presented schematically in FIG. 36T

[1390](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

[1391]Curr Microbial, 2004 September; 49(3):192-8. Expression of the heat shock gene hsp16.6 and promoter analysis in the cyanobacterium, Synechocystis sp. PCC 6803. [1392]Fang F, Barnum S R. [1393]J. Exp Bot, 2006; 57(7):1573-8. Epub 2006 Mar. 30. The heat shock response of Synechocystis sp. PCC 6803 analyzed by transcriptomics and proteomics. [1394]Suzuki I, Simon W J, Slabas A R.

[1395]A plasmid created with this promoter element is presented schematically in FIG. 36V.

[1396](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

[1397]J Biol Chem. 2001 Jan. 5; 276(1):306-14. The high light-inducible polypeptides in Synechocystis PCC6803. Expression and function in high light. [1398]He Q, Dolganov N, Bjorkman O, Grossman A R. [1399]Arch. Microbiol, 2007 April; 187(4):337-42. Epub 2007 Feb. 10. 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. [1400]Kappell A D, van Waasbergen L G.

[1401]A plasmid created with this promoter element is presented schematically in FIG. 36X

[1402](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 ClpB multi-stress responsible promoter (heat, cold, salt and oxidative stress)

REFERENCES

[1403]Microbiology, 2004 May; 150(Pt 5):1271-81. Effects of high light on transcripts of stress-associated genes for the cyanobacteria Synechocystis sp. PCC 6803 and Prochlorococcus MED4 and MIT9313. [1404]Mary I, Tu C J, Grossman A, Vaulot D. [1405]J Exp Bot. 2006; 57(7):1573-8. Epub 2006 Mar. 30. The heat shock response of Synechocystis sp. PCC 6803 analysed by transcriptomics and proteomics. [1406]Suzuki I, Simon W J, Slabas A R.

[1407]A plasmid created with this promoter element is presented schematically in FIG. 36Z.

Introduction of Alternative Ethanologenic Genes to ZmPdc and ZmAdhII into the Existing pVZ-Expression Constructs (Point 1)

[1408]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-pdc/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.

[1409]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.

[1410]In this context, new alcohol dehydrogenases include the following:

[1411](1) FIG. 37A presents the nucleotide sequence for ZmADHI (adhA gene from Zymomonas mobilis ZM4) and FIG. 37B presents the amino acid sequence for ZmAdhI AAV8960 FIG. 37C presents a schematic representation of the plasmid pVZ321b-PisiA-PDC-ZmADH1. FIG. 37D presents a schematic representation of the plasmid pVZ321b-PntcA-PDC-ZmAH1. FIG. 37E presents a schematic representation of the plasmid pVZ321b-PnblA-PDC-ZmADH1.

[1412](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.

[1413]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.

[1414]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 elongates 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.

[1415](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_--415757) is presented in FIG. 39B.

[1416]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.

[1417](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.

[1418]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.

[1419]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.

[1420]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 AAM4956) is presented in FIG. 41B.

Construction of Chromosome Integrative pSK-Vectors

[1421]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.

[1422]FIG. 42A presents the nucleotide sequence of the pSK10 cloning vector (derivate of pSK9 [V. V. Zinchenko, Moscow, Russia; unpublished]). FIG. 42B presents a schematic representation of this plasmid.

[1423]Several pSK10 constructs with ZmPdc/ZmAdhII were obtained.

[1424]FIG. 42C presents a schematic diagram of pSK10-PisiA-PDC-ADHII.

[1425]FIG. 42D presents a schematic diagram of pSK10-PnblA-PDC-ADHII.

[1426]FIG. 42E presents a schematic diagram of pSK10-PntcA-PDC-ADHII.

[1427]Several pSK10 constructs with ZmPdc/ZmAdhI were obtained.

[1428]FIG. 42F presents a schematic diagram of pSK10-PisiA-PDC-ADHI.

[1429]FIG. 42G presents a schematic diagram of pSK10-PnblA-PDC-ADHI.

[1430]FIG. 42H presents a schematic diagram of pSK10-PntcA-PDC-ADHI.

[1431]Several pSK10 constructs with ZmPdc/SynAdh were obtained.

[1432]FIG. 42I presents a schematic diagram of pSK10-PisiA-PDC-SynADH.

[1433]FIG. 42J presents a schematic diagram of pSK10-PnblA-PDC-SynADH.

[1434]FIG. 42K presents a schematic diagram of pSK10-PntcA-PDC-SynADH.

[1435]Several pSK10 constructs with EcAdhE were obtained.

[1436]FIG. 42L presents a schematic diagram of pSK10-PisiA-PDC-EcAdhE.

[1437]FIG. 42M presents a schematic diagram of pSK10-PnblA-PDC-EcAdhE.

[1438]FIG. 42N presents a schematic diagram of pSK10-PntcA-PDC-EcAdhE.

[1439]Several pSK10 constructs with ThAdhE were obtained.

[1440]FIG. 42O presents a schematic diagram of pSK10-PisiA-PDC-ThAdhE.

[1441]FIG. 42P presents a schematic diagram of pSK10-PnblA-PDC-ThAdhE.

[1442]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

[1443]In order to generate ethanol producing Anabaena strains, different constructs were created for conjugation into Anabaena PCC7120 and Anabaena variabilis ATCC29413.

[1444]Nostoc/Anabaena spec. PCC7120 and Anabaena variabilis ATCC 29413 were transformed using Self-replicating plasmids.

[1445]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

[1446]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.

[1447]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.

[1448]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):

[1449]FIG. 42R depicts the crhC promoter (Anabaena sp. PCC7120) (crhC gene: a1r4718, RNA helicase crhC cold shock inducible)

[1450]FIG. 42S shows the petE promoter (Anabaena sp. PCC7120) petE gene: a110258, plastocyanin precursor (petE) induced by addition of Cu

[1451]The structure of plasmid pRL1049-PpetE-PDC-ADHII is shown in FIG. 42T.

[1452]The sequence of the plasmid pRL1049-PpetE-PDC-ADHII is shown in FIG. 42U.

pRL593 Construct

[1453]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

[1454]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.

[1455]Ethanol production in medium +N appeared higher than under condition lacking combined nitrogen (-N); but this effect was not very pronounced when calculated per OD750 nm. 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 PisiA, 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:

[1456]In order to quantify the induction rate of the used promoters, Pdc/AdhII protein levels in cultures with and without nutrient starvation were estimated by Western blot analysis.

[1457]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:

[1458]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.

[1459]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).

[1460]Results are presented in FIGS. 43A and 43B.

Ethanol Generation Rates in Cyanobacterial Mutants:

[1461]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.

[1462]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:

[1463]Mutants and Synechocystis wild-type strains were grown at 28° C., under constant light (50 μE m-2 s-1) either on a shaker (100 rpm) or in aerated culture vessels, bubbled with CO₂-enriched air (0.5% CO₂). The initial OD₇₅₀ was between 2 and 3 in a total culture volume of 50 ml in Erlenmeyer flasks or 100 ml in the aerated culture vessels.

[1464]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:

[1465]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.

[1466]Furthermore, for repression of the nirA-promoter ammonia (0.265 g/l corresponds to 5 mM NH₄Cl) 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.

BG11 Media Recipe:

NaNO₃: 1.5 g

K₂HPO₄: 0.04 g

MgSO₄.7H₂O: 0.075 g

CaCl₂.2H₂O: 0.036 g

[1467]Citric acid: 0.006 gFerric ammonium citrate: 0.006 gEDTA (disodium salt): 0.001 g

NaCO₃: 0.02 g

[1468]Trace metal mix A5 1.0 ml (see below)Distilled water: 1.0 L

Trace Metal Mix A5:

H₃BO₃: 2.86 g

MnCl₂.4H₂O: 1.81 g

ZnSO₄.7H₂O: 0.222 g

NaMoO₄.2H₂O: 0.39 g

CuSO₄.5H₂O: 0.079 g

Co(NO₃)₂.6H₂O: 49.4 mg

[1469]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

[1470]Ethanol production rates and OD₇₅₀nm values were determined as described above and are shown in FIGS. 44A, 44B and 44C.

[1471]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+→acetaldehyde+NADH+H⁺acetaldehyde+NAD⁺+H₂O→acetic acid+NADH+H⁺

P.3 Ethanol Production Rates of Genetically Modified Photoautotrophic Host Cells Containing Zymomonas Mobilis PDC And Synechocystis ADH as a Second Genetic Modification

[1472]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. 44D.

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:

[1473]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 acetylCoA 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.

[1474]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:

[1475]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 K₁₂ wild-type strain, which lead to specific amino acid exchanges.

[1476]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).

[1477]The following pVZ321b mutants were generated:

6803 pVZ321b-PpetJ-EcAdhE (wt)6803 pVZ321b-PpetJ-EcAdhE (E568K, exchange from glutamic acid at position 568 to lysine)6803 pVZ321b-PpetJ-EcAdhE (A267T/E568K, exchange of alanine at position 267 to threonine and in addition E568K)6803 pVZ321b-PpetJ-ThAdhE (AdhE from Thermosynechococcus)

Growth Conditions:

[1478]Mutants and Synechocystis wild-type strains were grown at 28° C., under constant light (50 μE m-2 s-1) on a shaker (100 rpm). The initial OD₇₅₀ was about 3 in a total culture volume of 50 ml in a 100 ml Erlenmeyer flask. The ethanol concentration was determined as described.

[1479]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:

[1480]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 over-expression 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.

[1481]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:

[1482]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 CO₂. 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.

[1483]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:

[1484]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).

[1485]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 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).

[1486]The following pVZ321b mutants were generated:

6803 pVZ321b-PpetJ-ZmPdc/ZmAdhI6803 pVZ321b-PpetJ-ZmPdc/ZmAdhII6803 pVZ321b-PpetJ-ZmPdc/SynAdh

Growth Conditions:

[1487]Mutants were grown in BG11 medium without copper at 28° C. and constant light conditions (100 μE m-2 s-1). The initial OD₂₅₀ was about 1.5 in a total culture volume of about 150 ml in a culture vessel aerated with CO₂-enriched air (0.5% CO₂). 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.

[1488]FIGS. 46A, 46B and 46C present results of growth, ethanol accumulation and ethanol production per growth of Synechocystis mutants that express ZmPdc/ZmAdhI (sqares), ZmPdc/ZmAdhII(diamonds) and ZmPdc/SynAdh (triangles), respectively.

Results and Conclusions:

[1489]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.

[1490]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/ZmAdhII (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.

[1491]Due to the fact that the ZmPdc/SynAdh expressing mutants do not convert the produced ethanol back into acetaldehyde, there is no loss 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.

[1492]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.

[1493]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.

[1494]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, 30m×0.53 mm; carrier gas: helium; temperature: 40° C. constant. An acetaldehyde standard eluted under this conditions at 3.2 min. For the standard, acetaldehyde (Carl Roth) was diluted to 1 mg/ml in water, 25 μl were injected into a 250 ml gas sampling tube, the acetaldehyde was vaporized (30 min, 60° 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.

[1495]The gas phase over the cultures was sampled with a gas tight syringe pierced into the tubing at the outlet and 250 μl were injected into the GC.

[1496]For measurement of the acetaldehyde production from ethanol Synechocystis cells were pelleted, repeatedly washed with BG-11 and dissolved to 10 μg 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 μE/s*m2). 250 μl 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-00063 TABLE 1 Ethanol and acetaldehyde in the gas phase above ethanol producing strains. acetaldehyde ethanol ethanol gas phase gas phase medium [μmol/L] [μmol/L] [μmol/L] PCC6803 wild n.d. n.d. n.d. type 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 9767 The gas phase above transgenic strains of Synechocystis PCC6803 expressing different Pdcs and Adhs using the plasmid pVZ323 PpetJ was analyzed for ethanol and acetaldehyde content. As a control the ethanol was also quantified in the culture medium. ZmPdc, Pdc of Zymomonas mobilis; ZpPdc, Pdc of Zymobacter palmae; ZmAdh I, Adh I of Zymomonas mobilis; ZmAdh II, Adh II of Zymomonas mobilis; native Adh, no expression of an heterologous Adh, the native Adh of Synechocystis is present; SynAdh, Adh of Synechocystis is overexpressed; n.d. not detectable; --, not measured

[1497]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 μmol/l) and the x-axis shows the ethanol concentration in % (v/v).

[1498]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.

[1499]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 II were prepared as well.

[1500]For preparation of crude extracts, cells were pelleted, dissolved in buffer supplemented with 1 mM DTT and broken (beadbeater, 2×10 min, glass beads with 100 μm diameter). The supernatant of a centrifugation (15 min, 14000 rpm, 4° C., Micro 200R, Hettich) was used for the experiments.

[1501]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 μl 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-00064 TABLE 2 ADH activities measured as ethanol oxidation. with Adh II with Adh Wild type Z. mobilis Synechocystis 1 mM NAD⁺ 0.4 85.2 1.4 1 mM NADP⁺ 1.6 3.3 6.8 0.1 mM NADP⁺ 2.4 3.4 8.9 1 mM NAD⁺ + 0.1 mM 2.2 65.7 8.7 NADP⁺ 1 mM NAD⁺ + 1 mM 1.3 25.5 6.4 NADP⁺ 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⁺ in the given concentrations. Shown are specific activities in nMol min^-1 mg^-1 of total protein.

[1502]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⁺ or mixtures of NAD⁺ and NADP⁺ are used as a cosubstrates.

TABLE-US-00065 TABLE 3 ADH activities measured in the direction of ethanol production. 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 2.9 3.7 2.8 NADH + 1 mM NADP⁺ The assays contained NADH or NADPH or a combination of NADH and NADP⁺. Shown are the specific activities in nMol min^-1 mg^-1 of total protein.

[1503]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.

[1504]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.

[1505]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_m and v_max for NADH were calculated with 1 mM and 1.6 μMol min^-1 ml^-1 crude extract. For NADPH K_m and v_max were 15 μM and 0.4 μMol min^-1 ml^-1 for the crude extract. The K_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

[1506]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

[1507]SynADH was overexpressed as fusion protein with glutathione S-transferase (GST) in E. coli. The fusion protein was purified by affinity chromatography (Glutathione Sepharose® 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

[1508]ORF slr1192 from Synechocystis was amplified by PCR using the primers:

TABLE-US-00066 (SEQ ID NO: 223) 5' CTCTAGGATCCATGATTAAAGCCTACG 3' and (SEQ ID NO: 224) 5' CACGGACCCAGCGGCCGCCTTTGCAGAG 3'.

[1509]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® PCR Cloning Kit, Fermentas) and E. coli cells (α-Select Chemical Competent Cells, Bioline) were transformed with the ligation assay. Plasmidic DNA was isolated (GeneJET® 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.

[1510]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 μg/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 μg/ml) and IPTG (isopropyl thiogalactoside, 0.5 mM) and cultured under shaking at 20° 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° C., Micro 200R Hettich). 4 ml column material Glutathione Sepharose® 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 μl PreScission Protease (2 units/μl). After incubation at 4° C. over night, the column was eluted with buffer A. Fractions of 1.5 ml or 1 ml were collected.

[1511]SDS Polyacrylamide gel electrophoresis was performed with standard methods using 15% polyacrylamide gels. Page Ruler® unstained protein ladder (Fermentas) was the molecular weight standard.

[1512]Alcohol dehydrogenase activity was measured in the direction of acetaldehyde reduction. The assay contained in a total volume of 1000 μl 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 and Discussion

[1513]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.

[1514]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.

[1515]Fraction 2 of the purification was used for the measurement of the kinetic parameters of the SynAdh as described in the following.

TABLE-US-00067 activity/ activity/ protein vol. protein total volume conc. [μmol/min * [μmol/min * Purification activity yield sample [ml] [mg/ml] ml] mg] [-fold] [μmol/min] [%] cell 15 14.3 7.86 0.55 1 117.9 100 lysate flow 15 11.7 3.86 0.33 57.9 49 through wash 20 1.5 0.37 0.25 7.3 6 solution 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 41.6 35 1-5 computed value

[1516]Adh enzyme activity was measured either as ethanol oxidation (back reaction) or as acetaldehyde reduction (in 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:

[1517]The assays for the determination of the K_m values for NAD⁺ and NADP⁺ contained in a total volume of 1000 μl 30 mM HEPES/KOH (pH 7.5), 150 mM KCl, 1 mM DTT, 1.5 M ethanol, purified enzyme and NAD⁺ or NADP⁺ in different concentrations. For measurements at pH 8.5 HEPES/KOH was substituted by 30 mM Tris/HCl (pH 8.5), KCl was omitted. The assays for the determination of the K_m value for ethanol contained in a total volume of 1000 μl 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 8.5), KCl was omitted.

Acetaldehyde Reduction:

[1518]The assays for the determination of the K_m values for NADH and NADPH contained in a total volume of 1000 μl 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 K_m value for acetaldehyde contained in a total volume of 1000 μl 30 mM HEPES/KOH (pH 7.5), 150 mM KCl, 1 mM DTT, 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

[1519]The K_m and v_max values of SynAdh for the different substrates were determined with Lineweaver-Burk plots. The K_m values are summarized in table 1 and table 2.

TABLE-US-00068 TABLE 1 K_m values of SynAdh for the different substrates of the acetaldehyde reduction. pH 7.5, 150 mM KCl pH 6.0 NADH 1000 μM -- NADPH 15 μM 20 μM acetaldehyde 180 μM 200 μM Shown are the K_m values for NADH, NADPH and acetaldehyde at two different conditions (see Methods); --, not measured.

TABLE-US-00069 TABLE 2 K_m values of SynAdh for the different substrates of the ethanol oxidation. pH 7.5, 150 mM KCl pH 8.5 NAD⁺ 10 mM 10 mM NADP⁺ 15 μM 15 μM ethanol 23 mM 59 mM Shown are the K_m values for NAD⁺, NADP⁺ and ethanol at two different conditions (see Methods).

Discussion

[1520]The K_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_m value, the higher the "affinity" of the enzyme to the substrate.

[1521]The K_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_m approx. 15 μM), but the affinity for NADH is very low (K_m for NADH approx. 1000 μM). 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_total (NADP⁺+NADPH) is approx. 10 fold higher than the pool of NAD_total (NAD⁺+NADH) as described in Cooley & Vermaas, J. Bacteriol. 183(14) (2001) 4251-42589. The K_m value of SynAdh for acetaldehyde was determined with approx. 200 μM. As a comparison the K_m value of ADH I and ADH II of Zymomonas mobilis given in the literature are between 8 and 21 μM for acetaldehyde and 12 to 27 μM for NADH as described in Hoppner & Doelle, Eur. J. Appl. Microbiol. Biotechnol. 17, (1983), 152-157 and Kinoshita et al., Appl. Microbiol. Biotechnol. 22, (1985), 249-254, respectively.

[1522]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_m value of ADH I and ADH II of Z. mobilis for ethanol are given in the literature with 24 μM (ADH I) and 140 μM (ADH II), the K_m for NAD⁺ with 73 μM (ADH I) and 110 μM (ADH II) [6]. The affinity of SynAdh to ethanol is by far lower, the K_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_m for NAD⁺ was determined with 10 mM, the K_m for NADP⁺ with 15 μM.

[1523]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

[1524]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.

[1525]The FIG. 47A shows a in-depth phylogenetic analysis of different alcohol dehydrogenase families. Within the Glade 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-Glade to Synechocystis Adh enzyme NP 443028. It can clearly be seen that for example the members of the sub-Glade 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 (www.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 Glade of Zinc-binding GroES-like domain alcohol dehydrogenases shown in FIG. 47A.

[1526]Genes encoding the alcohol dehydrogenase (Adh) from Synechocystis sp. PCC 6803 were compared to all proteins from the NCBI non-redundant database (http://www.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: [1527]Method: Neighbor-Joining [1528]Phylogeny Test and options: Bootstrap (100 replicates; seed=64238)

Include Sites: ==============================

[1528] [1529]Gaps/Missing Data: Pairwise Deletion [1530]Substitution Model: ============================== [1531]Model: Amino: Poisson correction [1532]Substitutions to Include: All [1533]Pattern among Lineages: Same (Homogeneous) [1534]Rates among sites: Uniform rates

No. of Sites: 315

No Of Bootstrap Reps=100

[1535]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' 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' terminal and a Rossmann-fold NAD(P)+-binding (NADB_Rossmann) domain at C' 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.

[1536]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.

[1537]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

[1538]1. S. F. Altschul et al., Nucleic Acids Res. 25, 3389 (1997). [1539]2. J. Thompson, D. Higgins, T. Gibson, Nucleic Acids Res. 22, 4673 (1994). [1540]3. S. Kumar, K. Tamura, M. Nei, Briefings in Bioinformatics 5, 150 (2004). [1541]4. H. Jornvall, B. Persson, J. Jeffery, Eur. J. Biochem. 167, 195 (1987). [1542]5. H. W. Sun, B. V. Plapp, J. Mol. Evol. 34, 522 (1992). [1543]6. B. Persson, J. Hallborn, M. Walfridsson, B. Hahn-Hagerdal, S. Keranen, M. Penttila, H. Jornvall, FEBS Lett. 324, 9 (1993). [1544]7. H. Jornvall, B. Persson, M. Krook, S. Atrian, R. Gonzalez-Duarte, J. Jeffery, D. Ghosh, Biochemistry 34, 6003 (1995).

[1545]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.

[1546]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.

[1547]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.

[1548]FIG. 47F presents the amino acid sequence of an alcohol dehydrogenase GroES-like protein of Psychrobacter cryohalolentis K₅, identified by Genbank Accession No. YP_--581659.1.

[1549]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.

[1550]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.

[1551]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.

[1552]The FIGS. 47J to 47S represent the Adh protein sequences of sub-clade A of the above phylogenetic analysis.

[1553]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_--001519107.1.

[1554]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.

[1555]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.

[1556]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.

[1557]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.

[1558]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.

[1559]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.

[1560]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.

[1561]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.

[1562]FIG. 47S presents the amino acid sequence of an unnamed protein product of Microcystis aeruginosa PCC 7806, identified by Genbank Accession No. CA090817.1.

[1563]The FIG. 47T to 47X show the amino acid sequences of the Adh enzymes of the sub-clade C of the above phylogenetic analysis.

[1564]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_--01085101.1.

[1565]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.

[1566]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.

[1567]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.

[1568]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.

P.6 Ethanol Production Rates of Genetically Modified Photoautotrophic Host Cells Containing Zymomonas Mobilis PDC as the Only Second Genetic Modification

[1569]Almost all 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.

[1570]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:

[1571]From a preexisting pVZ plasmid (pVZ321b-PisiA-Pdc/AdhII) containing respective Pdc/Adh genes from Zymomonas mobilis the coding region of AdhII 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:

[1572]Mutant and Synechocystis wild-type strains were grown in BG11 without iron, at 28° C., under constant light (100 μE m^-2 s^-1), aerated with CO₂-enriched air (0.5% CO₂). The initial OD₇₅₀ was 1.3 in a total culture volume of 300 ml in a 500 ml Schott-flask.

[1573]The FIGS. 48A and 48B show the growth as determined by measurement of the OD₂₅₀ 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:

[1574]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.

[1575]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

[1576]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 CO₂ limitation or with sufficient CO₂ supply.

[1577]The condition of CO₂ limitation was created by shaking 50 ml cyanobacterial cultures in 100 ml Erlenmeyer flasks at 28° C. at a rate of 100 rpm. The light intensity was set to 40 μE m^-2 s^-1.

[1578]The condition of sufficient CO₂ 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 CO₂ with a rate of 10 ml/min. The temperature was at 28° C. and the light intensity was set at 100 μm^-2 s^-1.

[1579]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 CO₂ limitation.

[1580]These data show that under conditions of CO₂ 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.

[1581]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 CO₂ supply.

[1582]These data suggest that at conditions of sufficient CO₂ 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

[1583]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.

[1584]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:

[1585]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 mg/l; streptomycin 10 mg/l).

Growth Conditions:

[1586]Cultures were grown in BG11 in continuous light (50-100pE) either on a shaker in 100 ml Erlenmeyer flasks (100 rpm) or in bubbling flasks (200 ml) aerated with CO₂-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 NH₄Cl. 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 nm. The ethanol production was determined in the culture supernatant by an optical enzymatic test (Boehringer Mannheim).

Results and Conclusions:

[1587]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 iron (3×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 OD₇₅₀. Thus, ethanol production in Synechocystis is excellent adjustable by using the iron depending isiA-promoter.

[1588]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.

[1589]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.

[1590]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 OD750 nm=2), one with and the other without nitrate.

[1591]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.

[1592]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.

[1593]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.

[1594]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 OD₇₅₀nm=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.

[1595]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.

[1596]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.

[1597]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 μM) already activates the petJ-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 FIG. 49H to 49J). If copper is absent the ethanol production is significant higher compared to the control cultures with 0.3 μM (1×) or 1.5 μM copper (5×) but at the same time the culture without copper grows more slowly.

[1598]Between 1× and 5× 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 OD₇₅₀nm) 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 petJ-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 light (for about 4 weeks) and about 0.007% (v/v) ethanol in day/night cycles (for about 3 weeks).

[1599]FIG. 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× copper) was divided into 3 sub-cultures (start OD₇₅₀nm=3) and different concentrations of copper were added.

[1600]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 (5× copper corresponds 1.5 μM) 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.

[1601]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× copper) was divided into 2 sub-cultures (start OD₇₅₀nm=3) with different concentrations of copper (without and 5×Cu).

[1602]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° C. (compared to reference culture at 28° 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.

[1603]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° C. and 28° C.

[1604]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.

[1605]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.

[1606]The growth experiment revealed that the mutant with the hspA-promoter was most productive till the 10^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.

[1607]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 CO₂ enriched air (0.5%).

[1608]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.

[1609]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.

[1610]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:

[1611]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:

[1612]Synechocystis mutant was grown either at 28° C., in continuous light (150 μE m^-2 s^-1) and aerated with CO₂-enriched air (0.5% CO₂) or in day/night cycles (12 h/12 h) with a temperature cycle (25° C. night/35° C. day) and aerated with 5% CO₂. The initial OD₇₅₀ 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.

Recipe for 11 Artificial Seawater (28 ppm):

TABLE-US-00070 NaCl 28.05 g MgSO₄ 6.90 g MgCl₂ 5.49 g KCl 0.67 g CaCl₂ 1.47 g

Results and Conclusions:

[1613]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.

[1614]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.

[1615]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-00071 TABLE 1 Ethanol production rates of Synechocystis sp. PCC6803 pVZ321b-PpetJ-PDC/SynADH at different growth conditions. pVZ321b-PpetJ- EtOH EtOH/ PDC/SynADH % EtOH/day OD₇₅₀ nm EtOH/OD₇₅₀ 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, 0.41 0.0108 0.0413 0.00109 contin. 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

[1616]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 (www.yeastgenome.org).

[1617]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

[1618]Wild type Chlamydomonas strains (CC-1960, CC-620 and CC-621) were obtained from the Chlamydomonas Culture Collection at Duke University (www.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° 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 μE m^-2 s^-1). Arginine was added at 200 mg l^-1 (TAP+R) when required. For solid media, 1.5% agar was used.

Pdc and Adh Genes

[1619]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) J. 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 Adh1 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.

[1620]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)

[1621]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

[1622]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

[1623]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 6:1841]. The iron-responsive element, Pfea1 was obtained from the plasmid p5'FEA1-ARS2 [Allen et al. (2007) Eukaryot Cell 6:1841], purchased from the Chlamydomonas Center www.chlamy.org). The nucleotide sequence of FEA1 is shown in FIG. 50F.

Selectable Markers (ble and ARG7)

[1624]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

[1625]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 μg zeocin ml^-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: www.chlamy.org). Capital letters in FIG. 50G represent the coding sequence.

Nucleotide Sequence of the ARG7 Marker

[1626]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 (pSP124S, pXX311 and ARG7_pKS)pSP124S

[1627]The plasmid pSP124S was purchased from the Chlamydomonas Center (www.chlamy.org). It contains the Amp^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).

[1628]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. 50-5B.

pXX311

[1629]The plasmid pXX311 was a gift from Prof. Peter Hegemann (Humboldt University Berlin). It contains an Amp^R (bla) and a Km^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.

[1630]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.

ARG7pKS

[1631]The commercial cloning vector pBluescript II contains the Amp^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

[1632]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-00072 (SEQ ID NO: 225) ScPDC1-XhoI-F catg ATGTCTGAAATTACTTTGGGTAA (SEQ ID NO: 226) ScPDC1-BamHI-R catg TTATTGCTTAGCGTTGGTAG (SEQ ID NO: 227) ScADH1-XhoI-F catg ATGTCTATCCCAGAAACTCAAA (SEQ ID NO: 228) ScADH1-BamHI-R catg TTATTTAGAAGTGTCAACAACGT

[1633]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-00073 (SEQ ID NO: 229) Pcyc6-NotI-F CACTGAAGACTGGGATGAGC (SEQ ID NO: 230) Pcyc6-NotI-SpeI-F CACTGAAGACTGGGATG AGC (SEQ ID NO: 231) Pcyc6-XhoI-R CATGTTTATGGAGTAGG (SEQ ID NO: 232) Pfea1-NotI-F AGGACAGAGTGCGTGTGG (SEQ ID NO: 233) Pfea1-NotI-SpeI-F AGGACAGAGTGCGTGTGG (SEQ ID NO: 234) Pfea1-XhoI-R CATGGTTAACTGTG

[1634]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 KpnI.

TABLE-US-00074 (SEQ ID NO: 235) 3'UTR-BamHI-F catg CCGCTCCGTGTA (SEQ ID NO: 236) 3'UTR-XbaI-KpnI-R catg CGCTTCAAATACGCCCAG

[1635]For intermediate cloning, the 3'UTR sequence was cloned into pBluescript II SK+ (pSK; BamHI/KpnI). All other PCR products were cloned into pJET1.2/blunt (Fermentas).

[1636]After cloning of ScPDC1 and ScADH1, they were subcloned in front of the 3'UTR sequence in pSK via NotI/BamHI (NotI 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.

[1637]Afterwards, the respective promoter (with NotI/XhoI sites) was connected to the ScPDC1_--3'UTR construct via NotI/XhoI restriction as shown in FIG. 50O. Similarly, the respective promoter (with NotI, SpeI/XhoI sites) was connected to the ScADH1_--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.]

[1638]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.

[1639]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.

[1640]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.

[1641]The resulting expression plasmids are depicted in FIGS. 50Q, 50R, 50S, 50T and 50U respectively.

Transformation of Chlamydomonas

[1642]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 cw¹⁵ mutants) or by treatment of wild type cells with autolysin. Prior to gene transfer, expression plasmids were linearized (XmnI).

Protocol for Transformation

[1643]1. Transformation of cw15 arg-cells with ARG7 [1644]1) Inoculate 25 ml TAP+R with a loopful of cells and grow for 3 days [1645]2) Transfer 2 ml of the preculture to 150 ml fresh TAP+R, and grow the cells for 2 days (OD₇₅₀=0.3 to 0.5) [1646]3) Collect cells by centrifugation [1647]4) Wash and resuspend cells in TAP, incubate for 2 h with gentle shaking. [1648]5) Collect the cells by centrifugation [1649]6) Resuspend the cells in 3 ml of TAP [1650]7) Glass beads transformation: [1651]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 [1652]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 [1654]8) Spread the cell suspension on 2 plates of TAP agar (1.5% sigma agar)

[1655]2. Transformation of cw15 arg-cells with ble

[1656]Steps 1)-3): same as 1 [1657]4) Wash and resuspend cells in 3 ml of TAP+R [1658]5) Glass beads transformation [1659]6) Transfer the cells in a flask, add 10 ml of TAP+R, and shake for 1 day under the growth conditions [1660]7) Collect the cells by centrifugation [1661]8) Resuspend the cells in 1 ml of TAP+R [1662]9) Spread the cells on 4 plates of TAP+R agar (1.5% Bacto-Agar) that contain 5-20 μg/ml zeocin

[1663]3. Transformation of CC-1960 cells with ble [1664]1) Inoculate 25 ml TAP with a loopful of cells and grow for 3 days [1665]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 [1667]4) Wash and resuspend the cells in 25 ml of autolysin preparation (see below). Incubate for 1 h with gentle shaking. [1668]5) Wash and resuspend the cells in 3 ml TAP [1669]6) Glass beads transformation [1670]7) Transfer the cells in a flask, add 10 ml of TAP, and shake for 1 day under the growth conditions [1671]8) Centrifuge and resuspend the cells in 1 ml of TAP [1672]9) Spread the cells on 4 plates of TAP agar (1.5% Bacto-Agar) that contain 5-20 μg/ml zeocin

[1673]4. Preparation of Autolysin [1674]1) Cultivate the two different mating types of Chlamydomonas (CC-620 & CC-621) into early exponential phase (3×10⁶ cells ml^-1) (use 250 ml TAP medium in a 1 L flask) [1675]2) Collect cells and resuspend in TAP-N(NH₄Cl was replaced with the same concentration of KCl) (use 1 L TAP-N in a 2 L flask) [1676]3) Shake gently under light for 24 h (induction of gamete formation) [1677]4) Harvest cells and resuspend each culture in 200 ml TAP-N [1678]5) Mix both cultures in a 2 L flask [1679]6) Keep the flask in the light without shaking for 1-2 h (mating) [1680]7) Remove the cells by centrifugation [1681]8) Freeze the supernatant (clued extract of autolysin) and store at -80° C.

[1682]Transformation of the Chlamydomonas wild type (strain CC-1960) and mutant strain cw¹⁵ arg^- (defective in cell wall and arginine biosynthesis) was carried out with the expression constructs listed below.

TABLE-US-00075 Expressed Selectable C. reinhardtii gene(s) Promoter marker Plasmid strain(s) ScPDC1 CYC6 ARG pKS cw¹⁵ arg.sup.ScADH1 (pXX311) ScPDC1 FEA1 ARG pKS cw¹⁵ arg.sup.ScADH1 (pXX311) ScPDC1 CYC6 BLE pSP124S CC-1960 & ScADH1 cw¹⁵ arg.sup.ScPDC1 FEA1 BLE pSP124S CC-1960 & ScADH1 cw¹⁵ arg.sup.ScPDC1 CYC6 ARG pXX311 cw¹⁵ arg.sup.

[1683]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

[1684]EtOH production was assayed by an optic enzymatic test (as described herein). Cells were grown in TAP medium at 25° C. on a rotary shaker in continuous light. For transformants carrying the synthetic ble gene as a marker, zeocine (3 μg ml^-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.

[1685]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-00076 EtOH content [μM] 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-transformed background strain 0 20 45 45 43 (cw¹⁵ arg^-) [mean of 4 independent cultures] Non-induced transformant (cw¹⁵ arg^-) 0 20 30 45 90 [pKS ARG Pcyc6 PDC ADH] [mean of 5 independent cultures] Time(d) after induction Induced transformant (cw¹⁵ arg^-) 0 40 80 150 225 [pKS ARG Pcyc6 PDC ADH] [mean of 6 independent cultures] Induced transformant (cw¹⁵ arg^-) 0 20 110 170 240 [pKS ARG Pfeal PDC ADH] [mean of 4 independent cultures]

[1686]The Chlamydomonas transformants pKS_ARG_Pcyc6_PDC_ADH and pKS_ARG_Pfea1_PDC_ADH, both generated in the cw¹⁵ arg^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 μM ethanol were reached in the medium. The non-transformed control strains (wild type strain CC1960 as well as background strain cw¹⁵ arg^-) 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.

[1687]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.

Detailed Description of Various Embodiments for Testing a Photoautotrophic Strain for a Desired Growth Property

[1688]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

[1689]testing of all strains for tolerance against ethanol by stepwise increasing of ethanol concentration in 5% steps up to an concentration of 20% [1690]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. [1691]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:

[1691] [1692]20 ml of culture are transferred into 100 ml Erlenmeyer flask [1693]taking 1 ml culture for measuring start --OD (photometer by 750 nm) [1694]adding 1 ml ethanol up to an end concentration of 5% ethanol in the culture [1695]after 10 minutes at 5% ethanol end concentration taking 1 ml culture for measuring of OD [1696]macroscopic observation of the culture by eyes as well as microscopic analysis (as described above) [1697]adding 1 ml ethanol up to an end concentration of 10% ethanol in the culture.

2nd Day:

[1697] [1698]after 24 hours at 10% ethanol taking 1 ml culture for measuring of OD and microscopic analysis [1699]adding 1 ml ethanol up to an end concentration of 15% ethanol in the culture

3rd Day:

[1699] [1700]after another 24 hours at 15% ethanol taking 1 ml culture for measuring of OD and microscopic analysis [1701]adding 1 ml ethanol up to an end concentration of 20% ethanol in the culture [1702]after 2 hours at 20% ethanol taking 1 ml culture for measuring of OD and microscopic analysis

[1703]If the OD₇₅₀ 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:

[1704]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) [1705]if strains are self-sedimenting, ethanol containing media is removed after self-sedimentation of cells [1706]cell pellets are resuspended in 20 ml fresh media and transferred into 100 ml flasks [1707]1 ml is taken for measuring OD [1708]cultures are cultivated for 72 hours and OD was measured again after 24, 48 and 72 hours respectively.

[1709]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

[1710]testing of all strains for tolerance against ethanol by continuous and fast increasing of ethanol concentration up to a concentration of 20% (v/v) [1711]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) [1712]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 daysMic=abbreviation for microscopic analysis

1st Day:

[1712] [1713]650 ml of culture were transferred into a 2 l Erlenmeyer flask [1714]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 [1715]start of adding ethanol with MS-pumps to an end concentration of 10% ethanol in the culture after 18 h

2nd Day:

[1715] [1716]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 [1717]after 20 h recultivation of the 50 ml sample taken at 10% EtOH concentration [1718]after 20.5 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 [1719]after 22.5 h recultivation of the 50 ml sample taken at 15% EtOH concentration [1720]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 [1721]after 26 h recultivation of the 50 ml sample taken at 20% EtOH concentration

Long Term Ethanol Tolerance Test

Method

[1721] [1722]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% [1723]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

[1723] [1724]20 ml of culture are transferred into two 100 ml Erlenmeyer flasks, [1725]taking 1 ml culture for measuring start --OD (Spectrophotometer at 750 nm) [1726]adding ethanol up to an end concentration of e.g. 1% and 5% ethanol in the culture [1727]daily taking 1 ml culture for measuring of OD and analysis of the cells under fluorescence microscope [1728]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 [1729]the evaporation of water is compensated by adding corresponding amounts of sterile water whenever necessary [1730]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. [1731]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.

[1732]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.

[1733]Thermo Tolerance and Mechanical Stress Tolerance Test

Method

[1734]testing of all strains against higher temperature and mechanical stress tolerance

Protocol

[1734] [1735]40 ml of culture are transferred into 100 ml Erlenmeyer flasks [1736]for every culture 3 parallels at the same light conditions e.g. of 40 μE/m2*s are observed [1737]Blind culture: 28° C. on shaker [1738]Thermo stress: 45° C. on shaker [1739]Mechanical stress: magnetic stirrer in culture flask under highest rotations (5.000 rpm; max. speed) [1740]1 ml sample is taken after 48 and 96 hours for measuring OD [1741]these samples are also microscopically checked and observations are noticed [1742]at the end of experiment macroscopic photos were taken [1743]in case of non-unicellular cultures also microscopic photos were takentake samples after 48 and 96 h for OD₇₅₀ nm and LM-microscopic analysis [1744]compare growth of control culture to that of stressed cultures and evaluate the results as follows: [1745]positive=same or faster growth of the stressed culture than growth of the control culture; [1746]positive/negative results or indefinite results=slower growth of the stressed culture than control [1747]negative result=death of the stressed culture (within these 4 days).

Test for Growth in Salty Medium

[1748]Freshwater strains are investigated for their ability to grow in marine medium. [1749]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 [1750]grow cells in 0.5× salty medium for one week [1751]wash and cultivate cells in 1× salty medium (start--OD750 nm e.g. Synechocystis 1, 5-2) [1752]parallel growth of the same culture in freshwater medium (same start--OD750 nm) for control [1753]cultivate cells for 4 weeks; sampling two times a week: OD750 nm and chlorophyll content of cells [1754]comparison of both growth curves of the stressed culture in salty medium and the control culture for analysiscompare growth of control culture to that of stressed cultures and evaluate the results as follows: [1755]positive=same or faster growth of the stressed culture than growth of the control culture; [1756]positive/negative results or indefinite results=slower growth of the stressed culture than control [1757]negative result=death of the stressed culture.

[1758]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).

[1759]Recipe for 1 l of artificial seawater:

[1760]Recipe for 1 l artificial seawater (28 ppm):

TABLE-US-00077 NaCl 28.05 g MgSO4 6.90 g MgCl2 5.49 g KCl 0.67 g CaCl2 1.47 g

HPLC Analysis for Natural Product Content

Protocol for Natural Product Extraction and Sample Preparation for HPLC/MS Analysis

[1761]50 ml of cell culture (optical density around 1) is centrifuged, supernatant is discarded [1762]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) [1763]extract is centrifuged (6000 rpm=about 6.000 g, 10 min, 4° C.), supernatant is transferred into a new tube [1764]cell pellet is resuspended in 2 ml 50% methanol and cells are broken by ultrasonic bar as before [1765]extract is centrifuged (6000 rpm, 10 min, 4° C.), supernatant is united with the first one [1766]cell pellet is resuspended in 2 ml 80% methanol and cells are broken by ultrasonic bar as before [1767]extract is centrifuged (6000 rpm, 10 min, 4° C.), supernatant united with the other ones [1768]cell pellet is resuspended in 2 ml 80% methanol and cells are broken by ultrasonic bar as before [1769]extract is centrifuged (6000 rpm, 10 min, 4° C.), supernatant united with the other ones [1770]drying the supernatant in a vacuum rotator until pellet is dry [1771]the pellet is resuspend in 1.6 ml 20% methanol, centrifuged at 4° C., 13000 rpm (about 15.000 g) and filtered (0.45 μm CA membrane) [1772]HPLC/MS analysis (Detector: ELSD, PDA, MS)

Exact Growth Test

Method

[1772] [1773]determination of growth speed and max. optical density [1774]stepwise increase of light intensity and CO₂ supplementation [1775]samples are taken for analysis of metabolites at certain growth phases

Protocol

[1775] [1776]500 ml of culture in 11 culture vessel:

TABLE-US-00078 [1776] height about 9 cm diameter about 11 cm volume of vessel 1 l used volume 500 ml

[1777]light conditions starts with 40pE/m²*s; culture conditions of 30° C. or 21° C. respectively. CO₂ concentration starts with 2% [1778]when growth becomes stationary light conditions are increased in 2 steps (120pE/m²*s and 220pE/m²*s) [1779]daily taking samples of 1 ml for OD and microscopic observations, the evaporation is compensated by adding sterile water whenever necessary [1780]a growth curve is drawn and the growth rate can be calculated (optical density versus time) [1781]start optical density is about 0.2. [1782]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

[1782] [1783]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) [1784]Measurement of optical density using a photometer plate reader at 750 nm. [1785]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. [1786]strains which need more than 48 hours for doubling fail, 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):

[1787]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)

[1788]Centrifugation of the culture and resuspension of the cells in fresh BG11-Medium adjusting a chlorophyll concentration of about 10 μg/ml

[1789]Addition of 25 mM NaHCO₃ as carbon source

[1790]Cultures are then filled into a 2.5 ml cuvette with an integrated Clark-electrode (oxygen electrode).

[1791]Measurement of oxygen generation using light saturated conditions (about 500 μE/m²×s) at 25° C. over the time and record of data using a chart recorder.

[1792]Calculation of oxygen generation using the following formula: oxygen rate in μmol O₂ per h and μg chlorophyll=Δunits of measurement×0.253 μmol per ml×60/chlorophyll concentration in μg per ml×Δunits of calibration×Δt of measurement in min.

[1793]The Δunits are recorded by the chart recorder. The Δunits of calibration are determined by measuring the amplitude of difference of a O₂ saturated water solution and a water solution without any O₂ after adding of Sodium dithionite(zero point). That means Δunits of calibration correspond directly to the oxygen concentration of air-saturated water at 25° C. of 0.253 μmol per ml.

[1794]A photoautotrophic strain passes this test if a photosynthetic oxygen evolution of at least 150 μmol O₂/h*mg chl can be detected.

Photometric Quantification of Chlorophyll in Cyanobacterial Cultures

Chemicals and Solutions:

[1795]100% methanol (4° C.)

Principle of the Method:

[1796]Cyanobacterial cells are extracted with methanol (90%, v/v). The chlorophyll content in the extract is measured spectrophotometrically.

Method:

[1797]Batches of cyanobacteria cultures are centrifuged. The pellets are resuspended in 90% methanol, for example by leaving 100 μl of the supernatant and addition of 900 μl of 100% methanol. After resuspension and incubation (at 4° 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:

A₆₆₅×13.9=chlorophyll [μg/ml]

[1798]For the calculation of the chlorophyll content of the cyanobacteria culture the dilution factor has to be considered.

[1799]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 Algensammlung (SAG) have been identified, which are prime candidates for genetic modification due to their positive behavior during the above screening procedures:

[1800]SAG 37.79, PCC 7715, Calothrix thermalis

[1801]PCC 8937, Lyngbya sp.

[1802]SAG 12.89, Phormidium africanum

[1803]PCC 7321, Pleurocapsa sp.

[1804]PCC 6715, Synechococcus sp.

[1805]In particular these strains performed during the screening procedures as indicated in the below table:

TABLE-US-00079 Initial Exact Mechanical ethanol ethanol- Thermo stress tolerance tolerance Recul- tolerance tolerance Strain test test tivation test test SAG 12.89 up to up to up to pos. pos. 20% 20% 15% PCC 8937 up to up to pos. pos. 20% 20% PCC 7321 up to up to up to pos./neg. pos. 20% 20% 10% (2 h) SAG 37.79 up to neg. pos. 20% PCC 6715 up to up to up to pos. pos. 20% 20% 0%

[1806]Further examples of photoautotrophic strains which passed and failed the screening test are shown in FIG. 50-13.

[1807]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

[1808]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).

[1809]Methods: Synechocystis PCC 6803 wild type and the transgenic strain "6803-pVZ-PisiA", corresponding to the above described Synechocystis pVZ-PisiA-Pdc-AdhII, were washed twice with BG11 (centrifugation 15 min, 4500 rpm, 4° 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 μl 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° C., subsequently the ethanol content was measured.

[1810]Ethanol was quantified with a described protocol. The method is based on oxidation of ethanol catalyzed by alcohol dehydrogenase (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:

[1811]Ethanol is oxidized by nicotinamide-adenine dinucleotide (NAD⁺) 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 (Al-DH) (reaction 2).

##STR00003##

[1812]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:

[1813]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 (A₁). 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 (A₂). The absorption measurements can be performed using a photometer or a microplate reader. For plate reader measurements all volumes are downscaled.

[1814]From the measured absorbance difference ΔA=(A₂-A₁) the ethanol concentration in the sample is calculated with the equation:

c = V × MG × d × v × 2 × 1000 × Δ 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×mmol^-1×cm^-1 at 340 nm); d, light path [cm]; v, sample volume [mL]

Literature:

[1815]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.

[1816]H.-O. Beutler (1984) in: Methods in Enzymatic Analysis (Bergmeyer, H. U. ed.) 3^rd ed. Vol. VI, pp. 598-606, Verlag Chemie, Weinheim, Germany.

[1817]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.

[1818]For preparation of crude extracts, cells were harvested, washed with 40 mM MES/Tris (pH 6.5), 1 mM DTT and broken (beadbeater, 2×10 min). The supernatant of a centrifugation (15 min, 14000 rpm, 4° C., Micro 200R, Hettich) was used for the determination of Adh activity in cells.

[1819]Assays for measurement of the Adh activity in the direction of ethanol formation contained in a total volume of 800 μl 40 mM MES adjusted with Tris base to pH 6.5, 1 mM DTT, different concentrations of acetaldehyde, 50 μl crude extract and 0.3 M NADH. The initial velocity was calculated from the dE/min at 340 nm.

[1820]Results: Addition of acetaldehyde to final concentrations in the range of 6.6 μM to 200 μM 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).

[1821]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.

[1822]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_m and V_max were calculated from a "Lineweaver-Burk" plot (1/v versus 1/[S]; FIG. 6) with K_m for acetaldehyde=18 μM and V_max=3.2 μMol L^-1 min^-1. OD₇₅₀ of the culture was 0.56.

[1823]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.

[1824]FIG. 51C presents a Lineweaver-Burk-Plot. Reciprocal of the initial velocity versus the reciprocal of the acetaldehyde concentration. Intact cells were used.

[1825]This experiment was repeated with a different culture of strain 6803-pVZ-PisiA-PDC/ADHII of OD₇₅₀ of 1.353 and a chlorophyll concentration of 4.6 μg/ml. Similar results were obtained. The K_m for acetaldehyde was calculated with 25 μM (FIG. 51D). V_max was 4.35 μMol L^-1 min^-1, or 0.95 μMol L^-1 mg^-1 using chlorophyll as reference.

[1826]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 FIG. 51A to 51C.

[1827]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_m for acetaldehyde was calculated with 45 μM and V_max was 7.2 μMol L^-1 mg^-1 chlorophyll.

[1828]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_max was calculated with 0.89 μMol L^-1 mg^-1 chlorophyll, the K_m for acetaldehyde was determined in this experiment with 100 μM (FIG. 9).

[1829]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.

[1830]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.

[1831]Summary: Acetaldehyde added to the medium is absorbed and converted into ethanol by intact cells. The K_m for acetaldehyde of the entire process of uptake and ethanol formation was determined with approx. 20 to 25 μM. This value is similar to the K_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 μM 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.

[1832]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

2361477PRTSynechocystis sp. PCC 6803 1Met Lys Ile Leu Phe Val Ala Ala Glu Val Ser Pro Leu Ala Lys Val1 5 10 15Gly Gly Met Gly Asp Val Val Gly Ser Leu Pro Lys Val Leu His Gln 20 25 30Leu Gly His Asp Val Arg Val Phe Met Pro Tyr Tyr Gly Phe Ile Gly 35 40 45Asp Lys Ile Asp Val Pro Lys Glu Pro Val Trp Lys Gly Glu Ala Met 50 55 60Phe Gln Gln Phe Ala Val Tyr Gln Ser Tyr Leu Pro Asp Thr Lys Ile65 70 75 80Pro Leu Tyr Leu Phe Gly His Pro Ala Phe Asp Ser Arg Arg Ile Tyr 85 90 95Gly Gly Asp Asp Glu Ala Trp Arg Phe Thr Phe Phe Ser Asn Gly Ala 100 105 110Ala Glu Phe Ala Trp Asn His Trp Lys Pro Glu Ile Ile His Cys His 115 120 125Asp Trp His Thr Gly Met Ile Pro Val Trp Met His Gln Ser Pro Asp 130 135 140Ile Ala Thr Val Phe Thr Ile His Asn Leu Ala Tyr Gln Gly Pro Trp145 150 155 160Arg Gly Leu Leu Glu Thr Met Thr Trp Cys Pro Trp Tyr Met Gln Gly 165 170 175Asp Asn Val Met Ala Ala Ala Ile Gln Phe Ala Asn Arg Val Thr Thr 180 185 190Val Ser Pro Thr Tyr Ala Gln Gln Ile Gln Thr Pro Ala Tyr Gly Glu 195 200 205Lys Leu Glu Gly Leu Leu Ser Tyr Leu Ser Gly Asn Leu Val Gly Ile 210 215 220Leu Asn Gly Ile Asp Thr Glu Ile Tyr Asn Pro Ala Glu Asp Arg Phe225 230 235 240Ile Ser Asn Val Phe Asp Ala Asp Ser Leu Asp Lys Arg Val Lys Asn 245 250 255Lys Ile Ala Ile Gln Glu Glu Thr Gly Leu Glu Ile Asn Arg Asn Ala 260 265 270Met Val Val Gly Ile Val Ala Arg Leu Val Glu Gln Lys Gly Ile Asp 275 280 285Leu Val Ile Gln Ile Leu Asp Arg Phe Met Ser Tyr Thr Asp Ser Gln 290 295 300Leu Ile Ile Leu Gly Thr Gly Asp Arg His Tyr Glu Thr Gln Leu Trp305 310 315 320Gln Met Ala Ser Arg Phe Pro Gly Arg Met Ala Val Gln Leu Leu His 325 330 335Asn Asp Ala Leu Ser Arg Arg Val Tyr Ala Gly Ala Asp Val Phe Leu 340 345 350Met Pro Ser Arg Phe Glu Pro Cys Gly Leu Ser Gln Leu Met Ala Met 355 360 365Arg Tyr Gly Cys Ile Pro Ile Val Arg Arg Thr Gly Gly Leu Val Asp 370 375 380Thr Val Ser Phe Tyr Asp Pro Ile Asn Glu Ala Gly Thr Gly Tyr Cys385 390 395 400Phe Asp Arg Tyr Glu Pro Leu Asp Cys Phe Thr Ala Met Val Arg Ala 405 410 415Trp Glu Gly Phe Arg Phe Lys Ala Asp Trp Gln Lys Leu Gln Gln Arg 420 425 430Ala Met Arg Ala Asp Phe Ser Trp Tyr Arg Ser Ala Gly Glu Tyr Ile 435 440 445Lys Val Tyr Lys Gly Val Val Gly Lys Pro Glu Glu Leu Ser Pro Met 450 455 460Glu Glu Glu Lys Ile Ala Glu Leu Thr Ala Ser Tyr Arg465 470 4752491PRTSynechocystis sp. PCC 6803 2Met Tyr Ile Val Gln Ile Ala Ser Glu Cys Ala Pro Val Ile Lys Ala1 5 10 15Gly Gly Leu Gly Asp Val Ile Tyr Gly Leu Ser Arg Glu Leu Glu Leu 20 25 30Arg Gly His Cys Val Glu Leu Ile Leu Pro Met Tyr Asp Cys Met Arg 35 40 45Tyr Asp His Ile Trp Gly Leu His Asp Ala Tyr Arg Asn Leu Glu Val 50 55 60Pro Trp Tyr Gly Ser Ser Ile Phe Cys Asp Val Phe Cys Gly Trp Val65 70 75 80His Gly Arg Leu Cys Phe Phe Ile Gln Pro Lys Ser Ser Asp Asn Phe 85 90 95Phe Asn Arg Gly His Tyr Tyr Gly Ala Leu Asp Asp His Met Arg Phe 100 105 110Ala Phe Phe Ser Lys Ala Ala Met Glu Phe Leu Leu Arg Ser Asn Lys 115 120 125Arg Pro Asp Ile Ile His Cys His Asp Trp Gln Thr Gly Leu Val Pro 130 135 140Val Leu Leu Tyr Glu Ile Tyr Arg Phe His Gly Met Asp His Gln Arg145 150 155 160Val Cys Tyr Thr Ile His Asn Phe Lys His Gln Gly Ile Ala Gly Ala 165 170 175Asn Ile Leu His Ala Thr Gly Leu Asn Asn Asp Ser Tyr Tyr Phe Ser 180 185 190Tyr Asp Arg Leu Gln Asp Asn Phe Asn Pro Asn Ala Ile Asn Phe Met 195 200 205Lys Gly Gly Ile Val Tyr Ser Asn Tyr Val Asn Thr Val Ser Pro His 210 215 220His Ala Trp Glu Ala Arg Phe Ser Asp Ile Ser Cys Gly Leu Gly His225 230 235 240Thr Leu Glu Ile His Gln Gln Lys Phe Gly Gly Ile Leu Asn Gly Leu 245 250 255Asp Tyr Glu Val Trp Asn Pro Glu Ile Asp Pro Leu Leu Ala Ser Asn 260 265 270Phe Ser Val Lys Thr Phe Gly Asp Lys Ala Lys Asn Lys Gln Ala Leu 275 280 285Arg Glu Arg Leu Leu Leu Glu Thr Asp Asp Lys Lys Pro Met Leu Cys 290 295 300Phe Ile Gly Arg Leu Asp Gly Gln Lys Gly Val His Leu Val His His305 310 315 320Ser Ile Tyr Tyr Ala Leu Ser Gln Gly Ala Gln Phe Val Leu Leu Gly 325 330 335Ser Ala Thr Glu Pro Asn Leu Ser Lys Trp Phe Trp His Glu Lys Gln 340 345 350His Leu Asn Asp Asn Pro Asn Val His Leu Glu Leu Gly Phe Asp Glu 355 360 365Glu Leu Ala His Leu Ile Tyr Gly Ala Ala Asp Ile Ile Val Val Pro 370 375 380Ser Asn Tyr Glu Pro Cys Gly Leu Thr Gln Met Ile Gly Leu Arg Tyr385 390 395 400Gly Ala Val Pro Val Val Arg Gly Val Gly Gly Leu Val Asn Thr Val 405 410 415Phe Asp Arg Asp Tyr Asp Gln Asn His Pro Pro Glu Lys Arg Asn Gly 420 425 430Phe Val Phe Tyr Gln Pro Asp Glu Tyr Ala Leu Glu Thr Ala Leu Ser 435 440 445Arg Ala Ile Ala Leu Tyr Lys Asp Asp Pro Val Ala Phe Lys Thr Leu 450 455 460Ala Leu Gln Gly Met Ala Tyr Asp Tyr Ser Trp Asn Lys Pro Gly Leu465 470 475 480Gln Tyr Val Glu Ala Tyr Glu Tyr Ile Arg Ala 485 49035069DNAartificialconstruct 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 Val1 5 10 15Gly Leu Thr Pro Ser Ser Val Arg Ala Leu Leu Ser Gln Gly His Gln 20 25 30Val Phe Val Glu Glu Gly Ala Gly Val Gly Ser Gly Phe Pro Asp Gly 35 40 45Ala Tyr Ala Lys Ala Gly Ala Glu Leu Val Ala Thr Ala Lys Glu Ala 50 55 60Trp Asn Arg Glu Leu Val Val Lys Val Lys Glu Pro Leu Pro Glu Glu65 70 75 80Tyr Glu Tyr Leu Thr Leu Pro Lys Leu Leu Phe Thr Tyr Leu His Leu 85 90 95Ala Ala Glu Arg Thr Leu Thr Glu Ala Leu Ile Lys Ser Gly Ile Thr 100 105 110Ala Ile Ala Tyr Glu Thr Val Glu Leu Ala Asp Gly Gln Leu Pro Leu 115 120 125Leu Ala Pro Met Ser Arg Ile Ala Gly Arg Leu Ala Val Gln Met Gly 130 135 140Ala His Tyr Leu Glu Lys Gln Gln Gly Gly Arg Gly Val Leu Leu Gly145 150 155 160Gly Val Pro Gly Val Lys Ala Gly Gln Val Thr Ile Leu Gly Gly Gly 165 170 175Val Val Gly Thr Glu Ala Ala Lys Met Ala Ile Gly Leu Gly Ala Met 180 185 190Val Thr Ile Leu Asp Ile Asn Val Asp Arg Leu Asn Gln Leu Gly Glu 195 200 205Leu Phe Gly Ser Arg Val Asp Leu Arg Tyr Ser Asn Ala Ser Gln Ile 210 215 220Glu Asp Leu Leu Pro His Thr Asp Leu Leu Ile Gly Ala Val Leu Ile225 230 235 240Thr Gly Lys Arg Ala Pro Val Leu Val Ser Arg Gln Glu Val Glu Gln 245 250 255Met Leu Pro Gly Ala Val Ile Met Asp Val Ala Ile Asp Gln Gly Gly 260 265 270Cys Val Glu Thr Leu Arg Val Thr Ser His Ser Gln Pro Ser Tyr Ile 275 280 285Glu Ala Glu Val Val His Val Gly Ile Pro Asn Met Pro Gly Ala Thr 290 295 300Pro Trp Thr Ala Thr Gln Ala Leu Asn Asn Ser Thr Leu Arg Tyr Val305 310 315 320Leu Lys Leu Ala Asn Leu Gly Glu Gln Ala Trp Glu Asn Asp Leu Pro 325 330 335Leu Ala Lys Gly Val Asn Val Gln Ala Gly Lys Leu Val Gln Gly Ala 340 345 350Val Lys Thr Val Phe Pro Asp Leu 355 36062292DNAartificialconstruct 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 Ala1 5 10 15Ile Ile Leu Gly Gly Gly Ala Gly Thr Arg Leu Tyr Pro Leu Thr Lys 20 25 30Leu Arg Ala Lys Pro Ala Val Pro Leu Ala Gly Lys Tyr Arg Leu Ile 35 40 45Asp Ile Pro Val Ser Asn Cys Ile Asn Ser Glu Ile Val Lys Ile Tyr 50 55 60Val Leu Thr Gln Phe Asn Ser Ala Ser Leu Asn Arg His Ile Ser Arg65 70 75 80Ala Tyr Asn Phe Ser Gly Phe Gln Glu Gly Phe Val Glu Val Leu Ala 85 90 95Ala Gln Gln Thr Lys Asp Asn Pro Asp Trp Phe Gln Gly Thr Ala Asp 100 105 110Ala Val Arg Gln Tyr Leu Trp Leu Phe Arg Glu Trp Asp Val Asp Glu 115 120 125Tyr Leu Ile Leu Ser Gly Asp His Leu Tyr Arg Met Asp Tyr Ala Gln 130 135 140Phe Val Lys Arg His Arg Glu Thr Asn Ala Asp Ile Thr Leu Ser Val145 150 155 160Val Pro Val Asp Asp Arg Lys Ala Pro Glu Leu Gly Leu Met Lys Ile 165 170 175Asp Ala Gln Gly Arg Ile Thr Asp Phe Ser Glu Lys Pro Gln Gly Glu 180 185 190Ala Leu Arg Ala Met Gln Val Asp Thr Ser Val Leu Gly Leu Ser Ala 195 200 205Glu Lys Ala Lys Leu Asn Pro Tyr Ile Ala Ser Met Gly Ile Tyr Val 210 215 220Phe Lys Lys Glu Val Leu His Asn Leu Leu Glu Lys Tyr Glu Gly Ala225 230 235 240Thr Asp Phe Gly Lys Glu Ile Ile Pro Asp Ser Ala Ser Asp His Asn 245 250 255Leu Gln Ala Tyr Leu Phe Asp Asp Tyr Trp Glu Asp Ile Gly Thr Ile 260 265 270Glu Ala Phe Tyr Glu Ala Asn Leu Ala Leu Thr Lys Gln Pro Ser Pro 275 280 285Asp Phe Ser Phe Tyr Asn Glu Lys Ala Pro Ile Tyr Thr Arg Gly Arg 290 295 300Tyr Leu Pro Pro Thr Lys Met Leu Asn Ser Thr Val Thr Glu Ser Met305 310 315 320Ile Gly Glu Gly Cys Met Ile Lys Gln Cys Arg Ile His His Ser Val 325 330 335Leu Gly Ile Arg Ser Arg Ile Glu Ser Asp Cys Thr Ile Glu Asp Thr 340 345 350Leu Val Met Gly Asn Asp Phe Tyr Glu Ser Ser Ser Glu Arg Asp Thr 355 360 365Leu Lys Ala Arg Gly Glu Ile Ala Ala Gly Ile Gly Ser Gly Thr Thr 370 375 380Ile Arg Arg Ala Ile Ile Asp Lys Asn Ala Arg Ile Gly Lys Asn Val385 390 395 400Met Ile Val Asn Lys Glu Asn Val Gln Glu Ala Asn Arg Glu Glu Leu 405 410 415Gly Phe Tyr Ile Arg Asn Gly Ile Val Val Val Ile Lys Asn Val Thr 420 425 430Ile Ala Asp Gly Thr Val Ile 43582522DNAartificialconstruct 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 Thr1 5 10 15Ala Leu Ile Leu Trp Phe Glu Glu Val Gly Thr His Asp Val Gly Leu 20 25 30Val Gly Gly Lys Asn Ser Ser Leu Gly Glu Met Ile Gln Gln Leu Thr 35 40 45Asn Lys Gly Val Asn Val Pro Ser Gly Phe Ala Thr Thr Ala Tyr Ala 50 55 60Tyr Arg Tyr Phe Ile Gln Glu Ala Gly Leu Glu Gln Lys Leu Arg Asp65 70 75 80Leu Phe Thr Asp Leu Asp Val Asn Asp Met Ala Asn Leu Gln Glu Arg 85 90 95Gly His Leu Ala Arg Gln Leu Ile Leu Asp Thr Pro Phe Pro Gln Asn 100 105 110Leu Gln Thr Ala Ile Ala Glu Ala Tyr Gly Ala Met

Cys Glu Arg Tyr 115 120 125Gly Gln Lys Met Gly Arg Thr Gly Val Asp Val Ala Val Arg Ser Ser 130 135 140Ala Thr Ala Glu Asp Leu Pro Glu Ala Ser Phe Ala Gly Gln Gln Glu145 150 155 160Thr Tyr Leu Asn Val His Ser Leu Ser Cys Val Leu Glu Ser Cys His 165 170 175Lys Cys Phe Ala Ser Leu Phe Thr Asp Arg Ala Ile Ser Tyr Arg His 180 185 190His Asn Gly Phe Asp His Phe Ala Val Ala Leu Ser Val Gly Val Gln 195 200 205Lys Met Val Arg Ser Asp Leu Ala Thr Ser Gly Val Met Phe Ser Ile 210 215 220Asp Thr Glu Thr Gly Phe Lys Asn Ala Ala Leu Ile Thr Ala Ala Tyr225 230 235 240Gly Leu Gly Glu Asn Val Val Gln Gly Ala Val Asn Pro Asp Glu Tyr 245 250 255Phe Val Phe Lys Pro Thr Leu Lys Glu Gly Phe Lys Pro Ile Leu Glu 260 265 270Lys Arg Leu Gly Ser Lys Ala Ile Lys Met Val Tyr Asp Val Gly Gly 275 280 285Ser Lys Leu Thr Lys Asn Val Glu Val Ala Glu Pro Glu Arg Glu Lys 290 295 300Tyr Cys Ile Asn Asp Glu Glu Ile Leu Gln Leu Ala Arg Trp Ala Cys305 310 315 320Ile Ile Glu Asp His Tyr Ser Gly Val Arg Gly Val Tyr Thr Pro Met 325 330 335Asp Ile Glu Trp Ala Lys Asp Gly Gln Thr Gly Glu Leu Phe Ile Val 340 345 350Gln Ala Arg Pro Glu Thr Val Gln Ser Gln Lys Ser Ala Asn Val Ile 355 360 365Lys Thr Tyr Glu Leu Lys Asp His Ser Gln Val Leu Ala Thr Gly Arg 370 375 380Ser Val Gly Ala Ala Ile Gly Gln Gly Lys Ala Gln Val Ile Arg Asn385 390 395 400Val Ser Gln Ile Asn Gln Phe Arg Pro Gly Glu Val Leu Ile Thr Asn 405 410 415Arg Thr Asp Pro Asp Trp Glu Pro Ile Met Lys Gln Ala Ser Ala Ile 420 425 430Val Thr Asn Gln Gly Gly Lys Thr Cys His Ala Ala Ile Ile Ala Arg 435 440 445Glu Met Gly Ile Pro Ala Ile Val Gly Cys Gly Asp Ala Thr Asp Thr 450 455 460Ile Lys Thr Gly Glu Asp Val Thr Ile Cys Cys Ser Glu Gly Asp Glu465 470 475 480Gly Ser Val Tyr Ser Gly Ile Leu Asn Tyr Glu Val His Glu Thr Glu 485 490 495Leu Ser Asn Leu Pro Arg Thr Lys Thr Gln Ile Leu Met Asn Val Gly 500 505 510Asn Pro Glu Gln Ala Phe Gly Phe Ala Ser Tyr Pro Ala Asp Gly Val 515 520 525Gly Leu Ala Arg Leu Glu Phe Ile Ile Ala Asn His Ile Lys Ala His 530 535 540Pro Leu Ala Leu Met Lys Phe Asp Glu Leu Glu Asp Pro Leu Ala Lys545 550 555 560Ala Glu Ile Ala Glu Leu Thr Lys Leu Tyr Ala Gly Asp Arg Pro Arg 565 570 575Phe Phe Val Asp Lys Leu Ala His Gly Ile Ala Met Ile Ala Ala Ala 580 585 590Phe Tyr Pro Lys Pro Val Val Val Arg Met Ser Asp Phe Lys Ser Asn 595 600 605Glu Tyr Ala Asn Leu Leu Gly Gly Arg Gln Phe Glu Pro Lys Glu Glu 610 615 620Asn Pro Met Ile Gly Trp Arg Gly Ala Ser Arg Tyr Tyr Asp Pro Asn625 630 635 640Tyr Arg Glu Ala Tyr Ala Leu Glu Cys Gln Ala Leu Lys Arg Val Arg 645 650 655Asp Glu Met Gly Leu Thr Asn Val Ile Pro Met Ile Pro Phe Cys Arg 660 665 670Thr Pro Asp Glu Gly Arg Lys Val Ile Ala Glu Met Ala Lys His Gly 675 680 685Leu Lys Gln Gly Lys Asn Gly Leu Glu Ile Tyr Val Met Cys Glu Leu 690 695 700Pro Ser Asn Val Ile Leu Ala Asp Glu Phe Ser Glu Val Phe Asp Gly705 710 715 720Phe Ser Ile Gly Ser Asn Asp Leu Thr Gln Leu Thr Leu Gly Leu Asp 725 730 735Arg Asp Ser Ser Leu Val Ala His Leu Phe Asp Glu Arg Asn Leu Gly 740 745 750Val Lys Arg Met Val Lys Met Ala Ile Glu Thr Ala Lys Ala Asn Gly 755 760 765Arg Lys Ile Gly Ile Cys Gly Gln Ala Pro Ser Asp Tyr Pro Glu Phe 770 775 780Ala Glu Phe Leu Val Glu Leu Gly Ile Asp Ser Ile Ser Leu Asn Pro785 790 795 800Asp Ser Val Leu Lys Thr Val Leu Arg Ile Ala Glu Val Glu Lys Ala 805 810 815Leu Gly113082DNAartificialconstruct 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 Phe1 5 10 15Gln Gln Ala Asn His Pro His Gln Arg Glu Met Val Phe Phe Asp Ala 20 25 30Gln Leu Asn Leu Asp Thr Ala Ile Leu Ala Glu Asp Cys Pro Val Ile 35 40 45Cys Leu Phe Val Asn Asp Gln Ala Pro Ala Pro Val Leu Glu Lys Leu 50 55 60Ala Ala Gln Gly Thr Lys Leu Ile Ala Leu Arg Ser Ala Gly Tyr Asn65 70 75 80Asn Val Asp Leu Lys Thr Ala Ala Asp Leu Gly Leu Lys Val Val His 85 90 95Val Pro Ser Tyr Ser Pro His Ala Val Ala Glu His Thr Val Gly Leu 100 105 110Ile Leu Ala Leu Asn Arg Lys Leu Tyr Arg Ala Tyr Asn Arg Val Arg 115 120 125Asp Asp Asn Phe Ser Leu Glu Gly Leu Leu Gly Phe Asp Leu His Gly 130 135 140Thr Thr Val Gly Val Ile Gly Thr Gly Lys Ile Gly Leu Ala Phe Ala145 150 155 160Gln Ile Met Asn Gly Phe Gly Cys His Leu Leu Gly Tyr Asp Ala Phe 165 170 175Pro Asn Asp Lys Phe Thr Ala Ile Gly Gln Ala Leu Tyr Val Ser Leu 180 185 190Asn Glu Leu Leu Ala His Ser Asp Ile Ile Ser Leu His Cys Pro Leu 195 200 205Leu Pro Glu Thr His Tyr Leu Ile Asn Thr Asn Thr Ile Ala Gln Met 210 215 220Lys Pro Gly Val Met Leu Ile Asn Thr Ser Arg Gly His Leu Ile Asp225 230 235 240Thr Gln Ala Val Ile Gln Gly Ile Lys Ser His Lys Ile Gly Phe Leu 245 250 255Gly Ile Asp Val Tyr Glu Glu Glu Glu Glu Leu Phe Phe Thr Asp His 260 265 270Ser Asp Thr Ile Ile Gln Asp Asp Thr Phe Gln Leu Leu Gln Ser Phe 275 280 285Pro Asn Val Met Ile Thr Ala His Gln Gly Phe Phe Thr His Asn Ala 290 295 300Leu Gln Thr Ile Ala Ala Thr Thr Leu Ala Asn Ile Ala Glu Phe Glu305 310 315 320Gln Asn Lys Pro Leu Thr Tyr Gln Val Ile Cys Pro His 325 330132520DNAartificialconstruct 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 Cys1 5 10 15Leu Tyr Glu Leu Thr Gly Asp Arg Leu Pro Glu Thr Ile Pro Glu Pro 20 25 30Leu Trp Glu Ala Phe Ile Asp Trp Thr Val Leu Ala Asn Gln Gly Arg 35 40 45Leu Thr Val Glu Thr Ala Gly Gln Lys Gln Val Ile Ile Leu Glu Thr 50 55 60Gly Asp Arg Gln Gln Gly Ile Ala Arg Met Leu Asp Thr Leu Val Thr65 70 75 80Gly Asp Asp Ala Val Leu Lys Ser Leu Ala Glu Ile Asp Leu Val Gly 85 90 95His Arg Val Val His Gly Gly Thr Asp His Ala Glu Ala Thr Leu Ile 100 105 110Thr Pro Glu Val Gln Gln Ala Ile Ala Asp Leu Ile Pro Leu Ala Pro 115 120 125Ala His Asn Pro Ala His Leu Glu Gly Ile Glu Ala Ile Ser Ala Leu 130 135 140Leu Val Leu Gly Glu Val Pro Gln Ile Ala Val Phe Asp Thr Ala Phe145 150 155 160His Arg Thr Ile Pro Thr Pro Ala Ala Glu Tyr Pro Ile Pro Gln Ala 165 170 175Trp Thr Asn Leu Gly Ile Arg Arg Tyr Gly Phe His Gly Thr Ser His 180 185 190Lys Tyr Cys Ala Gln Lys Thr Ala Glu Ile Leu Gly Lys Pro Leu Ala 195 200 205Asp Leu Lys Leu Ile Thr Cys His Ile Gly Asn Gly Ala Ser Leu Thr 210 215 220Ala Ile Lys Asn Gly Val Ser Ile Asp Thr Thr Met Gly Phe Thr Pro225 230 235 240Leu Glu Gly Leu Met Met Gly Ala Arg Ser Gly Ser Ile Asp Pro Ala 245 250 255Ile Leu Leu Phe Leu Gln Glu Thr Gln Gly Leu Thr Pro Ala Glu Ile 260 265 270Asn Thr Thr Leu Asn Lys Lys Ser Gly Leu Leu Gly Val Ser Gly Leu 275 280 285Ser Ala Asp Leu Arg Thr Ile Leu Gln Ala Lys Ala Glu Gly Asn Glu 290 295 300Gln Ala Gln Leu Ala Tyr Val Met Tyr Ile His Arg Phe Arg Ser Cys305 310 315 320Leu Gly Gln Met Ile Ala Ser Leu Glu Gly Leu Asp Thr Leu Val Phe 325 330 335Thr Ala Gly Val Gly Glu Asn Ala Ala Thr Val Arg Ala Asp Val Cys 340 345 350Gln Ala Phe Glu Phe Leu Gly Leu Lys Leu Asp Pro Glu Leu Asn Asn 355 360 365Arg Ser Pro Arg Asp Thr Val Ile Ser His Ser Asp Ser Leu Val Thr 370 375 380Val Leu Ile Val His Thr Glu Glu Asp Trp Ala Ile Ala Gln Asp Cys385 390 395 400Trp His Trp Trp His Ser Gln Gly Gln Arg Lys Gln Ser 405 410152754DNAartificialconstruct 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 Lys1 5 10 15Ser Leu Val Val Leu Gly Ile Leu Asp Leu Ile Leu Lys Lys Thr Thr 20 25 30Arg Ile Ala Tyr Phe Arg Pro Ile Ile Gln Asp Pro Val Asn Gly Lys 35 40 45His Asp Asn Asn Ile Ile Leu Val Leu Glu Asn Phe Arg Leu Gln Gln 50 55 60Thr Tyr Thr Asp Ser Phe Gly Leu Tyr Phe His Glu Ala Val Ser Leu65 70 75 80Ala Ser Asp Gly Ala Ile Asp Gln Val Leu Asp Arg Ile Leu Ala Lys 85 90 95Tyr Arg His Leu Ala Asp Gln Val Asp Phe Ile Leu Cys Glu Gly Ser 100 105 110Asp Tyr Leu Gly Glu Glu Ser Ala Phe Glu Phe Asp Leu Asn Thr Thr 115 120 125Ile Ala Lys Met Leu Asn Cys Pro Ile Leu Leu Leu Gly Asn Ala Met 130 135 140Gly Asn Thr Ile Ala Asp Ser Leu Gln Pro Ile Asp Met Ala Leu Asn145 150 155 160Ser Tyr Asp Gln Glu Ser Cys Gln Val Val Gly Val Ile Ile Asn Arg 165 170 175Val Gln Pro Glu Leu Ala Thr Glu Ile Gln Ala Gln Leu Glu Gln Arg 180 185 190Tyr Gly Asp Arg Pro Met Val Leu Gly Thr Ile Pro Gln Asp Ile Met 195 200 205Leu Lys Ser Leu Arg Leu Arg Glu Ile Val Ser Gly Leu Asn Ala Gln 210 215 220Val Leu Ser Gly Ala Asp Leu Leu Asp Asn Leu Val Tyr His His Leu225 230 235 240Val Val Ala Met His Ile Ala His Ala Leu His Trp Leu His Glu Lys 245 250 255Asn Thr Leu Ile Ile Thr Pro Gly Asp Arg Gly Asp Ile Ile Leu Gly 260 265 270Val Met Gln Ala His Arg Ser Leu Asn Tyr Pro Ser Ile Ala Gly Ile 275 280 285Leu Leu Thr Ala Asp Tyr His Pro Glu Pro Ala Ile Met Lys Leu Ile 290 295 300Glu Gly Leu Pro Asp Ala Pro Pro Leu Leu Leu Thr Ser Thr His Thr305 310 315 320His Glu Thr Ser Ala Arg Leu Glu Thr Leu His Pro Ala Leu Ser Pro 325 330 335Thr Asp Asn Tyr Lys Ile Arg His Ser Ile Ala Leu Phe Gln Gln Gln 340 345 350Ile Asp Gly Glu Lys Leu Leu Asn Tyr Leu Lys Thr Ile Arg Ser Lys 355 360 365Gly Ile Thr Pro Lys Leu Phe Leu Tyr Asn Leu Val Gln Ala Ala Thr 370 375 380Ala Ala Gln Arg His Ile Val Leu Pro Glu Gly Glu Glu Ile Arg Ile385 390 395 400Leu Lys Ala Ala Ala Ser Leu Ile Asn His Gly Ile Val Arg Leu Thr 405 410 415Leu Leu Gly Asn Ile Glu Ala Ile Glu Gln Thr Val Lys Ile Asn His 420 425 430Ile Asp Leu Asp Leu Ser Lys Val Arg Leu Ile Asn Pro Lys Thr Ser 435 440 445Pro Asp Arg Glu Arg Tyr Ala Glu Thr Tyr Tyr Gln Leu Arg Lys His 450 455 460Lys Gly Val Thr Leu Ala Met Ala Arg Asp Ile Leu Thr Asp Ile Ser465 470 475 480Tyr Phe Gly Thr Met Met Val His Leu Gly Glu Ala Asp Gly Met Val 485 490 495Ser Gly Ser Val Asn Thr Thr Gln His Thr Val Arg Pro Ala Leu Gln 500 505 510Ile Ile Lys Thr Gln Pro Gly Phe Ser Leu Val Ser Ser Val Phe Phe 515 520 525Met Cys Leu Glu Asp Arg Val Leu Val Tyr Gly Asp Cys Ala Val Asn 530 535 540Pro Asp Pro Asn Ala Glu Gln Leu Ala Glu Ile Ala Leu Thr Ser Ala545 550 555 560Ala Thr Ala Lys Asn Phe Gly Ile Glu Pro Arg Val Ala Leu Leu Ser 565 570 575Tyr Ser Ser Gly Ser Ser Gly Gln Gly Ala Asp Val Glu Lys Val Arg 580 585 590Gln Ala Thr Ala Ile Ala Lys Glu Arg Glu Pro Asp Leu Ala Leu Glu 595 600 605Gly Pro Ile Gln Tyr Asp Ala Ala Val Asp Ser Thr Val Ala Ala Gln 610 615 620Lys Met Pro Gly Ser Ala Val Ala Gly Lys Ala Thr Val Phe Ile Phe625 630 635 640Pro Asp Leu Asn Thr Gly Asn Asn Thr Tyr Lys Ala Val Gln Arg Glu 645 650 655Thr Lys Ala Ile Ala Ile Gly Pro Ile Leu Gln Gly Leu Asn Lys Pro 660 665 670Val Asn Asp Leu Ser Arg Gly Cys Leu Val Glu Asp Ile Ile Asn Thr 675 680 685Val Val Ile Thr Ala Leu Gln Val Lys 690 695173654DNAartificialconstruct 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 Pro1 5 10 15Phe Phe Ala Gln Val Gly Leu Glu Glu Asn Leu His Glu Thr Leu Asp 20 25 30Phe Thr Glu Lys Phe Leu Ser Gly Leu Glu Asn Leu Gln Gly Leu Asn 35 40 45Glu Asp Asp Ile Gln Val Gly Phe Thr Pro Lys Glu Ala Val Tyr Gln 50 55 60Glu Asp Lys Val Ile Leu Tyr Arg Phe Gln Pro Val Val Glu Asn Pro65 70 75 80Leu Pro Ile Pro Val Leu Ile Val Tyr Ala Leu Val Asn Arg Pro Tyr 85 90 95Met Val Asp Leu Gln Glu Gly Arg Ser Leu Val Ala Asn Leu Leu Lys 100 105 110Leu Gly Leu Asp Val Tyr Leu Ile Asp Trp Gly Tyr Pro Ser Arg Gly 115 120 125Asp Arg Trp Leu Thr Leu Glu Asp Tyr Leu Ser Gly Tyr Leu Asn Asn 130 135 140Cys Val Asp Ile Ile Cys Gln Arg Ser Gln Gln Glu Lys Ile Thr Leu145 150 155 160Leu Gly Val Cys Gln Gly Gly Thr Phe Ser Leu Cys Tyr Ala Ser Leu 165 170 175Phe Pro Asp Lys Val Lys Asn Leu Val Val Met Val Ala Pro Val Asp 180 185 190Phe Glu Gln Pro Gly Thr Leu Leu Asn Ala Arg Gly Gly Cys Thr Leu 195 200 205Gly Ala Glu Ala Val Asp Ile Asp Leu Met Val Asp Ala Met Gly Asn 210 215 220Ile Pro Gly Asp Tyr Leu Asn Leu Glu Phe Leu Met Leu Lys Pro Leu225 230 235 240Gln Leu Gly Tyr Gln Lys Tyr Leu Asp Val Pro Asp Ile Met Gly Asp 245 250 255Glu Ala Lys Leu Leu Asn Phe Leu Arg Met Glu Lys Trp Ile Phe Asp 260 265 270Ser Pro Asp Gln Ala Gly Glu Thr Tyr Arg Gln Phe Leu Lys Asp Phe 275 280 285Tyr Gln Gln Asn Lys Leu Ile Lys Gly Glu Val Met Ile Gly Asp Arg 290 295 300Leu Val Asp Leu His Asn Leu Thr Met Pro Ile Leu Asn Leu Tyr Ala305 310 315 320Glu Lys Asp His Leu Val Ala Pro Ala Ser Ser Leu Ala Leu Gly Asp 325 330 335Tyr Leu Pro Glu Asn Cys Asp Tyr Thr Val Gln Ser Phe Pro Val Gly 340 345 350His Ile Gly Met Tyr Val Ser Gly Lys Val Gln Arg Asp Leu Pro Pro 355 360 365Ala Ile Ala His Trp Leu Ser Glu Arg Gln 370 375192121DNAartificialconstruct 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 Arg1 5 10 15Leu Tyr Pro Leu Thr Lys Leu Arg Ala Lys Pro Ala Val Pro Val Ala 20 25

30Gly Lys Tyr Arg Leu Ile Asp Ile Pro Val Ser Asn Cys Ile Asn Ser 35 40 45Glu Ile Phe Lys Ile Tyr Val Leu Thr Gln Phe Asn Ser Ala Ser Leu 50 55 60Asn Arg His Ile Ala Arg Thr Tyr Asn Phe Ser Gly Phe Ser Glu Gly65 70 75 80Phe Val Glu Val Leu Ala Ala Gln Gln Thr Pro Glu Asn Pro Asn Trp 85 90 95Phe Gln Gly Thr Ala Asp Ala Val Arg Gln Tyr Leu Trp Met Leu Gln 100 105 110Glu Trp Asp Val Asp Glu Phe Leu Ile Leu Ser Gly Asp His Leu Tyr 115 120 125Arg Met Asp Tyr Arg Leu Phe Ile Gln Arg His Arg Glu Thr Asn Ala 130 135 140Asp Ile Thr Leu Ser Val Ile Pro Ile Asp Asp Arg Arg Ala Ser Asp145 150 155 160Phe Gly Leu Met Lys Ile Asp Asn Ser Gly Arg Val Ile Asp Phe Ser 165 170 175Glu Lys Pro Lys Gly Glu Ala Leu Thr Lys Met Arg Val Asp Thr Thr 180 185 190Val Leu Gly Leu Thr Pro Glu Gln Ala Ala Ser Gln Pro Tyr Ile Ala 195 200 205Ser Met Gly Ile Tyr Val Phe Lys Lys Asp Val Leu Ile Lys Leu Leu 210 215 220Lys Glu Ala Leu Glu Arg Thr Asp Phe Gly Lys Glu Ile Ile Pro Asp225 230 235 240Ala Ala Lys Asp His Asn Val Gln Ala Tyr Leu Phe Asp Asp Tyr Trp 245 250 255Glu Asp Ile Gly Thr Ile Glu Ala Phe Tyr Asn Ala Asn Leu Ala Leu 260 265 270Thr Gln Gln Pro Met Pro Pro Phe Ser Phe Tyr Asp Glu Glu Ala Pro 275 280 285Ile Tyr Thr Arg Ala Arg Tyr Leu Pro Pro Thr Lys Leu Leu Asp Cys 290 295 300His Val Thr Glu Ser Ile Ile Gly Glu Gly Cys Ile Leu Lys Asn Cys305 310 315 320Arg Ile Gln His Ser Val Leu Gly Val Arg Ser Arg Ile Glu Thr Gly 325 330 335Cys Met Ile Glu Glu Ser Leu Leu Met Gly Ala Asp Phe Tyr Gln Ala 340 345 350Ser Val Glu Arg Gln Cys Ser Ile Asp Lys Gly Asp Ile Pro Val Gly 355 360 365Ile Gly Pro Asp Thr Ile Ile Arg Arg Ala Ile Ile Asp Lys Asn Ala 370 375 380Arg Ile Gly His Asp Val Lys Ile Ile Asn Lys Asp Asn Val Gln Glu385 390 395 400Ala Asp Arg Glu Ser Gln Gly Phe Tyr Ile Arg Ser Gly Ile Val Val 405 410 415Val Leu Lys Asn Ala Val Ile Thr Asp Gly Thr Ile Ile 420 425215916DNAartificialinsert 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 Arg1 5 10 15Leu Tyr Pro Leu Thr Lys Leu Arg Ala Lys Pro Ala Val Pro Val Ala 20 25 30Gly Lys Tyr Arg Leu Ile Asp Ile Pro Val Ser Asn Cys Ile Asn Ser 35 40 45Glu Ile Phe Lys Ile Tyr Val Leu Thr Gln Phe Asn Ser Ala Ser Leu 50 55 60Asn Arg His Ile Ala Arg Thr Tyr Asn Phe Ser Gly Phe Ser Glu Gly65 70 75 80Phe Val Glu Val Leu Ala Ala Gln Gln Thr Pro Glu Asn Pro Asn Trp 85 90 95Phe Gln Gly Thr Ala Asp Ala Val Arg Gln Tyr Leu Trp Met Leu Gln 100 105 110Glu Trp Asp Val Asp Glu Phe Leu Ile Leu Ser Gly Asp His Leu Tyr 115 120 125Arg Met Asp Tyr Arg Leu Phe Ile Gln Arg His Arg Glu Thr Asn Ala 130 135 140Asp Ile Thr Leu Ser Val Ile Pro Ile Asp Asp Arg Arg Ala Ser Asp145 150 155 160Phe Gly Leu Met Lys Ile Asp Asn Ser Gly Arg Val Ile Asp Phe Ser 165 170 175Glu Lys Pro Lys Gly Glu Ala Leu Thr Lys Met Arg Val Asp Thr Thr 180 185 190Val Leu Gly Leu Thr Pro Glu Gln Ala Ala Ser Gln Pro Tyr Ile Ala 195 200 205Ser Met Gly Ile Tyr Val Phe Lys Lys Asp Val Leu Ile Lys Leu Leu 210 215 220Lys Glu Ser Leu Glu Arg Thr Asp Phe Gly Lys Glu Ile Ile Pro Asp225 230 235 240Ala Ser Lys Asp His Asn Val Gln Ala Tyr Leu Phe Asp Asp Tyr Trp 245 250 255Glu Asp Ile Gly Thr Ile Glu Ala Phe Tyr Asn Ala Asn Leu Ala Leu 260 265 270Thr Gln Gln Pro Met Pro Pro Phe Ser Phe Tyr Asp Glu Glu Ala Pro 275 280 285Ile Tyr Thr Arg Ala Arg Tyr Leu Pro Pro Thr Lys Leu Leu Asp Cys 290 295 300His Val Thr Glu Ser Ile Ile Gly Glu Gly Cys Ile Leu Lys Asn Cys305 310 315 320Arg Ile Gln His Ser Val Leu Gly Val Arg Ser Arg Ile Glu Thr Gly 325 330 335Cys Val Ile Glu Glu Ser Leu Leu Met Gly Ala Asp Phe Tyr Gln Ala 340 345 350Ser Val Glu Arg Gln Cys Ser Ile Asp Lys Gly Asp Ile Pro Val Gly 355 360 365Ile Gly Pro Asp Thr Ile Ile Arg Arg Ala Ile Ile Asp Lys Asn Ala 370 375 380Arg Ile Gly His Asp Val Lys Ile Ile Asn Lys Asp Asn Val Gln Glu385 390 395 400Ala Asp Arg Glu Ser Gln Gly Phe Tyr Ile Arg Ser Gly Ile Val Val 405 410 415Val Leu Lys Asn Ala Val Ile Thr Asp Gly Thr Ile Ile 420 42523324PRTSynechocystis sp. strain PCC6803 23Met Ala Glu Thr Leu Leu Phe Ala Ala Leu Arg Gln Ala Leu Asp Glu1 5 10 15Glu Met Gly Arg Asp Val Asn Val Leu Val Leu Gly Glu Asp Val Gly 20 25 30Leu Tyr Gly Gly Ser Tyr Lys Val Thr Lys Asp Leu Tyr Glu Lys Tyr 35 40 45Gly Glu Met Arg Val Leu Asp Thr Pro Ile Ala Glu Asn Ser Phe Thr 50 55 60Gly Met Ala Val Gly Ala Ala Met Thr Gly Leu Arg Pro Val Ile Glu65 70 75 80Gly Met Asn Met Gly Phe Leu Leu Leu Ala Phe Asn Gln Ile Ala Asn 85 90 95Asn Ala Gly Met Leu Arg Tyr Thr Ser Gly Gly Asn Tyr Gln Ile Pro 100 105 110Met Val Ile Arg Gly Pro Gly Gly Val Gly Arg Gln Leu Gly Ala Glu 115 120 125His Ser Gln Arg Leu Glu Ala Tyr Phe His Ala Val Pro Gly Leu Lys 130 135 140Ile Val Ala Cys Ser Thr Pro Tyr Asn Ala Lys Gly Leu Leu Lys Ala145 150 155 160Ala Ile Arg Asp Asn Asn Pro Val Leu Phe Phe Glu His Val Leu Leu 165 170 175Tyr Asn Leu Lys Glu Asn Leu Pro Asp Tyr Glu Tyr Ile Val Pro Leu 180 185 190Asp Lys Ala Glu Val Val Arg Pro Gly Lys Asp Val Thr Ile Leu Thr 195 200 205Tyr Ser Arg Met Arg His His Cys Leu Gln Ala Leu Lys Thr Leu Glu 210 215 220Lys Glu Gly Tyr Asp Pro Glu Ile Ile Asp Leu Ile Ser Leu Lys Pro225 230 235 240Phe Asp Met Glu Thr Ile Ser Ala Ser Val Lys Lys Thr His Arg Val 245 250 255Ile Ile Val Glu Glu Cys Met Lys Thr Gly Gly Ile Gly Ala Glu Leu 260 265 270Ile Ala Leu Ile Asn Asp His Leu Phe Asp Glu Leu Asp Gly Pro Val 275 280 285Val Arg Leu Ser Ser Gln Asp Ile Pro Thr Pro Tyr Asn Gly Met Leu 290 295 300Glu Arg Leu Thr Ile Val Gln Pro Pro Gln Ile Val Asp Ala Val Lys305 310 315 320Ala Ile Ile Gly24257DNAartificialinsert 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 Leu1 5 10 15Glu Leu Pro Asn His Ala Gly Thr Leu Ala Ser Val Thr Gln Ala Ile 20 25 30Ala Asp Ala Gly Gly Ser Phe Gly Gln Ile Ser Leu Ile Glu Ser Asn 35 40 45Leu Lys Leu Thr Arg Arg Glu Ile Ala Val Asp Ala Ser Ser Ser Glu 50 55 60His Ala Glu Lys Ile Ile Gly Ala Val Lys Ala Leu Asp Asn Val Lys65 70 75 80Leu Leu Lys Val Ser Asp Arg Thr Phe Asp Leu His Arg Gln Gly Lys 85 90 95Ile Ser Val Val Ser Arg Ile Pro Leu Thr Ser Gln Ser Asp Leu Ala 100 105 110Met Ala Tyr Thr Pro Gly Val Gly Arg Ile Cys Arg Ala Ile Ala Glu 115 120 125Asp Pro Glu Lys Val Tyr Ser Leu Thr Ile Lys Ser Asn Thr Val Ala 130 135 140Val Val Thr Asp Gly Ser Ala Val Leu Gly Leu Gly Asn Leu Gly Pro145 150 155 160Glu Ala Ala Leu Pro Val Met Glu Gly Lys Ala Met Leu Phe Lys Glu 165 170 175Phe Ala Gln Leu Asp Ala Phe Pro Ile Cys Leu Asp Thr Gln Asp Thr 180 185 190Glu Glu Ile Ile Arg Thr Val Lys Ala Ile Ala Pro Val Phe Gly Gly 195 200 205Val Asn Leu Glu Asp Ile Ala Ala Pro Arg Cys Phe Glu Ile Glu Ala 210 215 220Arg Leu Lys Lys Glu Leu Asn Ile Pro Val Phe His Asp Asp Gln His225 230 235 240Gly Thr Ala Ile Val Thr Leu Ala Ala Leu Leu Asn Ala Leu Lys Phe 245 250 255Val Gly Lys Ala Met Ala Ala Val Arg Ile Val Ile Asn Gly Ala Gly 260 265 270Ala Ala Gly Leu Ala Ile Ala Glu Leu Leu Lys Glu Ser Gly Ala Thr 275 280 285Asp Ile Trp Ile Cys Asp Ser Lys Gly Ile Val Gly Lys His Arg Thr 290 295 300Asp Leu Asn Ser Lys Lys Gln Ser Phe Ala Val Asp Ala Glu Gly Thr305 310 315 320Leu Ala Asp Ala Met Ala Gly Ala Asp Val Phe Leu Gly Val Ser Ala 325 330 335Pro Gly Val Val Thr Lys Glu Met Val Gln Ser Met Ala Lys Asp Pro 340 345 350Ile Val Phe Ala Met Ala Asn Pro Ile Pro Glu Ile Gln Pro Glu Leu 355 360 365Ile Gln Glu Asp Ala Ala Val Ile Ala Thr Gly Arg Ser Asp Tyr Pro 370 375 380Asn Gln Ile Asn Asn Val Leu Ala Phe Pro Gly Val Phe Arg Gly Ala385 390 395 400Ile Asp Cys Arg Ala Ser Ile Ile Thr Thr Thr Met Cys Ile Glu Ala 405 410 415Ala Lys Ala Ile Ala Ser Leu Val His Ser Asn Thr Leu Asp Ser Glu 420 425 430His Ile Ile Pro Ser Val Phe Asp Asn Arg Val Ala Thr Thr Val Ala 435 440 445Ser Ala Val Gln Leu Ala Ala Arg Asn Glu Gly Val Ala Gly Gln 450 455 460331545DNAartificialinsert 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 Val1 5 10 15Thr Val Val Gly Ala Gly Asn Val Gly Arg Thr Leu Ala Gln Arg Leu 20 25 30Val Gln Gln Asn Val Ala Asn Val Val Leu Leu Asp Ile Val Pro Gly 35 40 45Leu Pro Gln Gly Ile Ala Leu Asp Leu Met Ala Ala Gln Ser Val Glu 50 55 60Glu Tyr Asp Ser Lys Ile Ile Gly Thr Asn Glu Tyr Glu Ala Thr Ala65 70 75 80Gly Ser Asp Val Val Val Ile Thr Ala Gly Leu Pro Arg Arg Pro Gly 85 90 95Met Ser Arg Asp Asp Leu Leu Gly Lys Asn Ala Asn Ile Val Ala Gln 100 105 110Gly Ala Arg Glu Ala Leu Arg Tyr Ser Pro Asn Ala Ile Leu Ile Val 115 120 125Val Thr Asn Pro Leu Asp Val Met Thr Tyr Leu Ala Trp Lys Val Thr 130 135 140Gly Leu Pro Ser Gln Arg Val Met Gly Met Ala Gly Val Leu Asp Ser145 150 155 160Ala Arg Leu Lys Ala Phe Ile Ala Met Lys Leu Gly Ala Cys Pro Ser 165 170 175Asp Ile Asn Thr Leu Val Leu Gly Gly His Gly Asp Leu Met Leu Pro 180 185 190Leu Pro Arg Tyr Cys Thr Val Ser Gly Val Pro Ile Thr Glu Leu Ile 195 200 205Pro Pro Gln Thr Ile Glu Glu Leu Val Glu Arg Thr Arg Asn Gly Gly 210 215 220Ala Glu Ile Ala Ala Leu Leu Gln Thr Gly Thr Ala Tyr Tyr Ala Pro225 230 235 240Ala Ser Ser Ala Ala Val Met Val Glu Ser Ile Leu Arg Asn Gln Ser 245 250 255Arg Ile Leu Pro Ala Ala Thr Tyr Leu Asp Gly Ala Tyr Gly Leu Lys 260 265 270Asp Ile Phe Leu Gly Val Pro Cys Arg Leu Gly Cys Arg Gly Val Glu 275 280 285Asp Ile Leu Glu Val Gln Leu Thr Pro Glu Glu Lys Ala Ala Leu His 290 295 300Leu Ser Ala Glu Ala Val Arg Leu Asn Ile Asp Val Ala Leu Ala Met305 310 315 320Val Ser Asp Gly351027DNAartificialinsert 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 Pro1 5 10 15Ala Ser Ser Ser Val Glu Val Ile Arg Gln Met Val Asp Ala Gly Met 20 25 30Asn Val Ala Arg Leu Asn Phe Ser His Gly Ser Tyr Glu Asp His Ala 35 40 45Thr Met Val Arg Leu Leu Arg Ser Val Glu Gln Glu Met Asp Thr Pro 50 55 60Ile Thr Leu Leu Gln Asp Leu Gln Gly Pro Lys Ile Arg Ile Gly Gln65 70 75 80Leu Pro Gly Gly Glu Lys Gln Leu Arg Glu Gly Glu Lys Val Ser Leu 85 90 95Val Pro Val Glu Ile Gly Asp Arg His Pro Gly Ala Val Gly Ile Asp 100 105 110Tyr Pro His Leu Ala Thr Glu Ala Lys Val Gly Glu Arg Ile Leu Leu 115 120 125Asp Asp Gly Leu Leu Glu Met Lys Val Val Ser Ile Gln Asp Pro Glu 130 135 140Val Ile Cys Glu Val Val Thr Gly Gly Ile Leu Lys Ser Arg Lys Gly145 150 155 160Val Asn Leu Pro Gly Leu Val Leu Thr Leu Pro Ser Met Thr Thr Lys 165 170 175Asp Lys Gln Asp Leu Glu Phe Gly Leu Ser Gln Gly Ile Asp Trp Val 180 185 190Ser Leu Ser Phe Val Arg Lys Gly Glu Asp Ile His Thr Leu Lys Gln 195 200 205Phe Leu Ala Glu Arg Gly His Pro Asp Leu Pro Val Ile Ala Lys Ile 210 215 220Glu Lys Pro Gln Ala Ile Asp Asn Leu Glu Glu Ile Val Ala Val Ser225 230 235 240Asn Gly Ile Met Val Ala Arg Gly Asp Leu Gly Val Glu Val Asn Pro 245 250 255Glu Lys Val Pro Arg Leu Gln Lys Glu Ile Ile Arg Arg Cys Asn Val 260 265 270Arg Ala Ile Pro Val Ile Thr Ala Thr Gln Met Leu Asp Ser Met Ile 275 280 285Gln Asn Ser Arg Pro Thr Arg Ala Glu Ala Ser Asp Val Ala Asn Ala 290 295 300Ile Leu Asp Gly Thr Asp Ala Val Met Leu Ser Gly Glu Ser Ala Val305 310 315 320Gly Gln Tyr Pro Val Lys Ser Val Gln Met Leu Arg Lys Ile Ala Glu 325 330 335Glu Thr Glu Val Gly Leu His Leu Val Asn Asn Pro Pro Ile Glu Asn 340 345 350Thr Glu Thr His Ala Leu Ser Glu Ala Leu Val Val Ile Asp Gly Ile 355 360 365Leu Asp Leu Lys Tyr Ile Val Thr Phe Thr Thr Ser Gly Phe Thr Ser 370 375 380Leu Leu Ala Ser Asn Gln Arg Pro Ser Val Pro Val Ile Ala Phe Thr385 390 395 400Pro Ser Glu Lys Val Tyr His Ser Leu Asn Leu Val Trp Gly Ile Ile 405 410 415Pro Phe Leu Ile Asn Glu Glu Phe Asp Thr Phe Glu Asp Leu Ile Gln 420 425 430Gln Ala Glu Val Leu Leu Arg Asp Arg Lys Met Val Glu Lys Gly Asp 435 440 445Gln Leu Leu Ile Met Ala Gly Ile Pro Thr Lys Ile Pro Arg Gly Thr 450 455 460Asn Phe Leu Lys Ile His Arg Ile Ser465 470382350DNAartificialinsert 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 Ile1 5 10 15Gly Pro Ala Thr Gln Ser Lys Glu Val Leu Arg Gln Leu Ile Gln Ala 20 25 30Gly Ala Thr Thr Phe Arg Leu Asn Phe Ser His Gly Asp His Ala Tyr 35 40 45His Gln Gln Ser Ile Arg Leu Ile Arg Gln Ile Ala Phe Glu Leu Asn 50 55 60Gln Pro Val Gly Ile Leu Gln Asp Leu Gln Gly Pro Lys Ile Arg Val65 70 75 80Gly Lys Phe Leu Asn Asp Ala Gly Ser Val Gln Leu Lys Asn Gly Asp 85 90 95Pro Tyr Thr Leu Thr Ser Arg Pro Val Glu Cys Thr Glu Thr Ile Ser 100 105 110Ser Ile Ser Tyr Glu Tyr Leu Ala Asp Glu Val Pro Ser Gly Ala Arg 115 120 125Ile Leu Leu Asp Asp Gly Lys Leu Glu Met Leu Val Glu Glu Val Asp 130 135 140Thr Val Ala Arg Asp Leu His Cys Arg Val Ile Val Gly Gly Thr Leu145 150 155 160Ser Ser Asn Lys Gly Val Asn Phe Pro Gly Val Cys Leu Ser Val Lys 165 170 175Ala Met Thr Asp Lys Asp Lys Glu Asp Leu Met Phe Gly Leu Asp Gln 180 185 190Gly Val Asp Trp Val Ala Leu Ser Phe Val Arg Asn Pro Gln Asp Ile 195 200 205Asp Glu Ile Lys Gly Leu Ile Ala Ala Ala Gly Lys Ser Val Pro Val 210 215 220Ile Ala Lys Ile Glu Lys His Glu Ala Ile Lys Asp Met Gln Ala Val225 230 235 240Leu Glu Lys Cys Asp Gly Val Met Val Ala Arg Gly Asp Leu Gly Val 245 250 255Glu Leu Pro Ala Glu Asp Val Pro Ile Leu Gln Lys Lys Leu Ile Ala 260 265 270Thr Ala Asn Arg Leu Gly Ile Pro Val Ile Thr Ala Thr Gln Met Leu 275 280 285Asp Ser Met Val Asn Ser Pro Arg Pro Thr Arg Ala Glu Val Ser Asp 290 295 300Val Ala Asn Ala Ile Leu Asp Gly Thr Asp Ala Val Met Leu Ser Asn305 310 315 320Glu Thr Ala Ile Gly Lys Phe Pro Val Glu Ala Val Ala Ile Met Ala 325 330 335Lys Ile Ala Glu Arg Ile Glu Gln Glu Asp Ile Asn Pro Ser Gln Ala 340 345 350Glu Ala Ser Arg Thr Ser Ile Pro Asn Ala Ile Ser Ser Ala Val Ser 355 360 365Gln Ile Ala Glu Thr Leu Asn Ala Ala Ala Ile Met Ser Leu Thr Lys 370 375 380Thr Gly Ser Thr Ala Arg His Val Ser Lys Phe Arg Pro Lys Thr Pro385 390 395 400Ile Leu Ala Val Thr Pro His Val Asp Val Ser Arg Gln Leu Gln Leu 405 410 415Val Trp Gly Val Lys Pro Leu Leu Val Leu Asp Leu Pro Ser Thr Ser 420 425 430Gln Thr Phe Gln Ala Ala Ile Asn Val Ala Gln Glu Asn His Phe Leu 435 440 445Arg Asp Gly Asp Leu Val Val Met Thr Ala Gly Thr Leu Gln Gly Val 450 455 460Ala Gly Ser Thr Asp Leu Ile Lys Val Glu Val Val Lys Ala Ile Leu465 470 475 480Gly Arg Gly Val Gly Ile Gly Gln Gly Ala Val Ser Gly Arg Ala Arg 485 490 495Val Ala Ser Arg Pro Gln Ala Ile Ala Gln Phe Thr Gln Gly Glu Ile 500 505 510Leu Val Val Pro Ser Thr Asn Ala Asp Cys Val Asp Met Met Arg Arg 515 520 525Ala Ala Gly Ile Ile Thr Glu Glu Glu Ser Leu Thr Ser His Ala Ala 530 535 540Ile Ile Gly Leu Arg Leu Gly Val Pro Val Ile Val Gly Phe Lys Gly545 550 555 560Ala Thr Gln Lys Ile Arg Asp Gly Ala Ile Val Thr Ile Asp Ala Gln 565 570 575Lys Gly Leu Ile Tyr Ser Gly Ala Leu Pro Pro Val Ser Lys Gly 580 585 590412620DNAartificialinsert 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 Ser1 5 10 15Glu Glu Met Leu Ala Lys Met Leu Asp Ala Gly Met Asn Val Met Arg 20 25 30Leu Asn Phe Ser His Gly Asp Tyr Ala Glu His Gly Gln Arg Ile Gln 35 40 45Asn Leu Arg Asn Val Met Ser Lys Thr Gly Lys Thr Ala Ala Ile Leu 50 55 60Leu Asp Thr Lys Gly Pro Glu Ile Arg Thr Met Lys Leu Glu Gly Gly65 70 75 80Asn Asp Val Ser Leu Lys Ala Gly Gln Thr Phe Thr Phe Thr Thr Asp 85 90 95Lys Ser Val Ile Gly Asn Ser Glu Met Val Ala Val Thr Tyr Glu Gly 100 105 110Phe Thr Thr Asp Leu Ser Val Gly Asn Thr Val Leu Val Asp Asp Gly 115 120 125Leu Ile Gly Met Glu Val Thr Ala Ile Glu Gly Asn Lys Val Ile Cys 130 135 140Lys Val Leu Asn Asn Gly Asp Leu Asp Glu Asn Lys Gly Val Asn Leu145 150 155 160Pro Gly Val Ser Ile Ala Leu Pro Ala Leu Ala Glu Lys Asp Lys Gln 165 170 175Asp Leu Ile Phe Gly Cys Glu Gln Gly Val Asp Phe Val Ala Ala Ser 180 185 190Phe Ile Arg Lys Arg Ser Asp Val Ile Glu Ile Arg Glu His Leu Lys 195 200 205Ala His Gly Gly Glu Asn Ile His Ile Ile Ser Lys Ile Glu Asn Gln 210 215 220Glu Gly Leu Asn Asn Phe Asp Glu Ile Leu Glu Ala Ser Asp Gly Ile225 230 235 240Met Val Ala Arg Gly Asp Leu Gly Val Glu Ile Pro Val Glu Glu Val 245 250 255Ile Phe Ala Gln Lys Met Met Ile Glu Lys Cys Ile Arg Ala Arg Lys 260 265 270Val Val Ile Thr Ala Thr Gln Met Leu Asp Ser Met Ile Lys Asn Pro 275 280 285Arg Pro Thr Arg Ala Glu Ala Gly Asp Val Ala Asn Ala Ile Leu Asp 290 295 300Gly Thr Asp Ala Val Met Leu Ser Gly Glu Ser Ala Lys Gly Lys Tyr305 310 315 320Pro Leu Glu Ala Val Ser Ile Met Ala Thr Ile Cys Glu Arg Thr Asp 325 330 335Arg Val Met Asn Ser Arg Leu Glu Phe Asn Asn Asp Asn Arg Lys Leu 340 345 350Arg Ile Thr Glu Ala Val Cys Arg Gly Ala Val Glu Thr Ala Glu Lys 355 360 365Leu Asp Ala Pro Leu Ile Val Val Ala Thr Gln Gly Gly Lys Ser Ala 370 375 380Arg Ala Val Arg Lys Tyr Phe Pro Asp Ala Thr Ile Leu Ala Leu Thr385 390 395 400Thr Asn Glu Lys Thr Ala His Gln Leu Val Leu Ser Lys Gly Val Val 405 410 415Pro Gln Leu Val Lys Glu Ile Thr Ser Thr Asp Asp Phe Tyr Arg Leu 420 425 430Gly Lys Glu Leu Ala Leu Gln Ser Gly Leu Ala His Lys Gly Asp Val 435 440 445Val Val Met Val Ser Gly Ala Leu Val Pro Ser Gly Thr Thr Asn Thr 450 455 460Ala Ser Val His Val Leu465 47044429PRTZymomonas mobilis 44Met Thr Ala Ile Val Ser Ile His Gly Arg Gln Val Val Asp Ser Arg1 5 10 15Gly Asn Pro Thr Val Glu Val Asp Val Thr Leu Glu Asp Gly Ser Phe 20 25 30Gly Arg Ala Ala Val Pro Ser Gly Ala Ser Thr Gly Val His Glu Ala 35 40 45Val Glu Leu Arg Asp Gly Asp Lys Thr Arg Trp Gly Gly Lys Gly Val 50 55 60Thr Lys Ala Val His Ala Val Asn Asn Glu Ile Ala Asn Ala Ile Ile65 70 75 80Gly Leu Glu Ala Glu Asp Gln Glu Leu Ile Asp Gln Thr Met Ile Lys 85 90 95Leu Asp Gly Thr Pro Asn Lys Gly Lys Phe Gly Ala Asn Ala Ile Leu 100 105 110Gly Val Ser Leu Ala Val Ala Lys Ala Ala Ala Glu Ala Arg Gly Leu 115 120 125Pro Leu Tyr Arg Tyr Val Gly Gly Thr Ala Ala His Val Leu Pro Val 130 135 140Pro Met Met Asn Ile Val Asn Gly Gly Met His Ala Asp Asn Pro Ile145 150 155 160Asp Phe Gln Glu Phe Met Ile Ala Pro Val Gly Ala Ser Ser Ile Asn 165 170 175Glu Ala Val Arg Ile Gly Thr Glu Val Phe His Thr Leu Lys Lys Glu 180 185 190Leu Ser Ala Lys Gly Met Asn Thr Asn Val Gly Asp Glu Gly Gly Phe 195 200 205Ala Pro Ser Leu Asp Ser Ala Ser Ser Ala Leu Asp Phe Ile Val Asp 210 215 220Ser Ile Ser Lys Ala Gly Tyr Lys Pro Gly Glu Asp Val Phe Ile Ala225 230 235 240Leu Asp Ala Ala Ser Ser Glu Phe Tyr Asn Lys Asp Gln Asn Ile Tyr 245 250 255Asp Leu Lys Gly Glu Gly Arg Lys Leu Thr Ser Ala Gln Leu Val Asp 260 265 270Tyr Tyr Val Glu Leu Cys Gly Lys Tyr Pro Ile Tyr Ser Ile Glu Asp 275 280 285Gly Leu Ala Glu Asp Asp Phe Glu Gly Trp Lys Ile Leu Thr Glu Lys 290 295 300Leu Gly Asp Lys Val Gln Leu Val Gly Asp Asp Leu Phe Val Thr Asn305 310 315 320Val Lys Arg Leu Ser Asp Gly Ile Glu Arg Gly Ile Ala Asn Ser Leu 325 330 335Leu Val Lys Phe Asn Gln Ile Gly Ser Leu Ser Glu Thr Leu Ala Ala 340 345 350Val Asn Met Ala Asn Asp Ala Ser Tyr Thr Ala Val Met Ser His Arg 355 360 365Ser Gly Glu Thr Glu Asp Thr Thr Ile Ala Asp Leu Ala Val Ala Thr 370 375 380Asn Cys Gly Gln Ile Lys Thr Gly Ser Leu Cys Arg Ser Glu Arg Ile385 390 395 400Ala Lys Tyr Asn Gln Leu Met Arg Ile Glu Glu Glu Leu Gly Ser Val 405 410 415Ala Lys Tyr Ala Gly Arg Ser Val Leu Arg Lys Ala Lys 420 42545228PRTZymomonas mobilis 45Met Pro Thr Leu Val Leu Ser Arg His Gly Gln Ser Glu Trp Asn Leu1 5 10 15Glu Asn Arg Phe Thr Gly Trp Trp Asp Val Asn Leu Thr Glu Gln Gly 20 25 30Val Gln Glu Ala Thr Ala Gly Gly Lys Ala Leu Ala Glu Lys Gly Phe 35 40 45Glu Phe Asp Ile Ala Phe Thr Ser Val Leu Thr Arg Ala Ile Lys Thr 50 55 60Thr Asn Leu Ile Leu Glu Ala Gly Lys Thr Leu Trp Val Pro Thr Glu65 70 75 80Lys Asp Trp Arg Leu Asn Glu Arg His Tyr Gly Gly Leu Thr Gly Leu 85 90 95Asn Lys Ala Glu Thr Ala Ala Lys His Gly Glu Glu Gln Val His Ile 100 105 110Trp Arg Arg Ser Tyr Gly Val Pro Pro Pro Pro Met Glu Lys Gly Ser 115 120 125Lys Phe Asp Leu Ser Gly Asp Arg Arg Tyr Asp Gly Val Lys Ile Pro 130 135 140Glu Thr Glu Ser Leu Lys Asp Thr Val Ala Arg Val Leu Pro Tyr Trp145 150 155 160Glu Glu Arg Ile Ala Pro Glu Leu Lys Ala Gly Lys Arg Val Leu Ile 165 170 175Gly Ala His Gly Asn Ser Leu Arg Ala Leu Val Lys His Leu Ser Lys 180 185 190Leu Ser Asp Glu Glu Ile Val Lys Phe Glu Leu Pro Thr Gly Gln Pro 195 200 205Leu Val Tyr Glu Leu Asn Asp Asp Leu Thr Pro Lys Asp Arg Tyr Phe 210 215 220Leu Asn Glu Arg225464274DNAartificialinsert 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 Glu1 5 10 15Ile Leu Asp Ser Arg Gly Arg Pro Thr Ile Glu Ala Glu Val Arg Leu 20 25 30Glu Ser Gly Ala His Gly Ile Ala Gln Val Pro Ser Gly Ala Ser Thr 35 40 45Gly Ser Phe Glu Ala His Glu Leu Arg Asp Gly Asp Pro Lys Arg Tyr 50 55 60Asp Gly Lys Gly Val Glu Lys Ala Val Arg Asn Val Thr Glu Lys Ile65 70 75 80Ala Pro Val Val Glu Gly Leu Asp Ala Phe Asp Gln Met Ala Val Asp 85 90 95Gln Ala Met Ile Asp Arg Asp Gly Thr Asp Asn Lys Lys Glu Leu Gly 100 105 110Ala Asn Ala Ile Leu Gly Val Ser Leu Ala Thr Ala Lys Ala Ala Ala 115 120 125Ala Glu Leu Ala Ile Pro Leu Tyr Arg Tyr Leu Gly Gly Pro Leu Ala 130 135 140Asn Val Leu Pro Val Pro Met Met Asn Val Ile Asn Gly Gly Ala

His145 150 155 160Ala Asp Asn Asn Val Asp Phe Gln Glu Phe Met Ile Met Pro Val Gly 165 170 175Ala Glu Thr Phe Lys Glu Ala Leu Arg Trp Gly Ala Glu Val Phe Ala 180 185 190Val Leu Gly Lys Val Leu Lys Glu Arg Lys Leu Leu Ser Gly Gly Val 195 200 205Gly Asp Glu Gly Gly Tyr Ala Pro Asn Leu Thr Ser Asn Gln Gln Ala 210 215 220Leu Asp Ile Leu Ile Glu Ala Ile Glu Gln Ala Gly Tyr Lys Pro Gly225 230 235 240Ser Gln Ile Ala Leu Ala Met Asp Ile Ala Ala Ser Glu Phe Phe Lys 245 250 255Asn Gly Gln Tyr Glu Tyr Asp Gly Gly Ser His Ser Pro Gln Glu Phe 260 265 270Ile Asp Tyr Gln Ala Lys Leu Val Ser Gln Tyr Pro Ile Val Ser Ile 275 280 285Glu Asp Gly Leu His Glu Asp Asp Trp Glu Ser Trp Lys Gly Leu Thr 290 295 300Thr Ser Leu Gly Thr Lys Thr Gln Leu Val Gly Asp Asp Leu Met Val305 310 315 320Thr Asn Pro Val Arg Leu Gln Lys Ser Ile Asp Leu Gly Val Ala Asn 325 330 335Ala Ile Leu Ile Lys Leu Asn Gln Ile Gly Thr Leu Ser Glu Thr Leu 340 345 350Glu Thr Ile Ser Leu Ala Thr Arg His Ser Tyr Arg Ser Val Ile Ser 355 360 365His Arg Ser Gly Glu Thr Glu Asp Thr Thr Ile Ala Asp Leu Ala Val 370 375 380Ala Thr Arg Val Gly Gln Ile Lys Thr Gly Ser Leu Cys Arg Ser Glu385 390 395 400Arg Val Ala Lys Tyr Asn Arg Leu Leu Arg Ile Glu Asp Glu Leu Gly 405 410 415Asp Arg Ala Val Tyr Ala Pro Lys Ile Gly Leu Gly Pro Lys His Ser 420 425 430482026DNAartificialinsert 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 Asn1 5 10 15Ala Glu Lys Arg Ile Gln Gly Arg Ser Asn Leu Ser Val Leu Thr Asp 20 25 30Lys Gly Lys Ala Asp Ala Gln Lys Val Gly Gln Thr Leu Asn Ser Leu 35 40 45Ala Ile Asp Lys Ile Tyr Cys Ser Pro Leu Arg Arg Ala Lys Glu Thr 50 55 60Ala Gln Ile Ile Gln Ala Ser Phe Ala His Pro Pro Glu Leu Ile Pro65 70 75 80Ser Glu Asn Leu Leu Glu Val Asn Leu Pro Leu Trp Glu Lys Met Thr 85 90 95Lys Asp Asp Val Ala His Gln Tyr Pro Glu Gln Tyr Arg Leu Trp His 100 105 110Glu Ala Pro Asp Gln Leu Ala Met Thr Val Asp Gly Ala Glu Tyr Tyr 115 120 125Pro Val Ala Ala Leu Tyr Ala Gln Ala Gln Arg Phe Trp Gln Asp Val 130 135 140Leu Thr Asp Ala Ala Gly Gln Thr Leu Leu Ile Val Ala His Asn Gly145 150 155 160Ile Asn Arg Cys Leu Leu Met Ser Ala Ile Gly Met Pro Ala Ser His 165 170 175Tyr Gln Arg Leu Gln Gln Ser Asn Cys Asn Ile Asn Val Leu Asn Phe 180 185 190Ser Gly Gly Trp Gly Asp Pro Val Gln Leu Glu Ser Leu Asn Gln Thr 195 200 205Ala His Met Gly Val Pro Leu Pro Pro Pro Arg Lys Asp Asn Asn Arg 210 215 220Leu Arg Leu Leu Leu Ile Arg His Gly Glu Thr Gln Trp Asn Arg Glu225 230 235 240Gly Arg Phe Gln Gly Ile Arg Asp Ile Pro Leu Asn Asp Asn Gly Arg 245 250 255His Gln Ala Gln Lys Ala Ala Glu Phe Leu Lys Asp Val Pro Ile Asn 260 265 270Leu Gly Ile Ser Ser Pro Met Ala Arg Pro Lys Glu Thr Ala Glu Ile 275 280 285Ile Leu Gln Tyr His Pro Ser Ile Glu Leu Asp Leu Gln Pro Glu Leu 290 295 300Ala Glu Ile Cys His Gly Leu Trp Glu Gly Lys Leu Glu Thr Glu Ile305 310 315 320Glu Ala Glu Tyr Pro Gly Leu Leu Gln Gln Trp Lys Asp Ala Pro Ala 325 330 335Thr Val Gln Met Pro Glu Gly Glu Asn Leu Gln Gln Val Trp Asp Arg 340 345 350Ala Ile Ala Cys Trp Gln Asp Arg Val Lys Phe Tyr Ser Gln Gly Asp 355 360 365Gly Ser Thr Val Gly Ile Val Val Ala His Asp Ala Ile Asn Lys Val 370 375 380Ile Leu Ala Tyr Leu Leu Gly Leu Thr Pro Ala His Phe Trp Gln Val385 390 395 400Lys Gln Gly Asn Gly Gly Val Ser Val Ile Asp Tyr Pro Gln Gly Leu 405 410 415Asp Lys Pro Pro Val Ile Gln Ala Ile Asn Leu Met Gly His Leu Gly 420 425 430Thr Val Leu Asp Lys Thr Ala Ala Gly Ala Leu 435 440501706DNAartificialinsert 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 Ser1 5 10 15Met Val Thr Ser Pro Phe Ser Leu Ser Pro Phe Gly Gln Ala Arg Ser 20 25 30Thr Val Thr Gly Asn Pro Leu Asp Pro Thr Glu Leu Asn Gln Met His 35 40 45Gly Phe Trp Arg Ala Ala Asn Tyr Leu Ala Val Gly Met Ile Tyr Leu 50 55 60Arg Asp Asn Pro Leu Leu Arg Glu Pro Leu Gln Pro Glu Gln Ile Lys65 70 75 80His Arg Leu Leu Gly His Trp Gly Ser Ser Pro Gly Ile Ser Phe Leu 85 90 95Tyr Thr His Leu Asn Arg Ile Ile Arg Lys Phe Asp Gln Asp Met Leu 100 105 110Tyr Met Val Gly Pro Gly His Gly Ala Pro Gly Phe Leu Gly Pro Cys 115 120 125Tyr Leu Glu Gly Ser Tyr Ser Arg Phe Phe Ala Glu Cys Ser Glu Asp 130 135 140Glu Asp Gly Met Lys Arg Phe Phe Lys Gln Phe Ser Phe Pro Gly Gly145 150 155 160Ile Gly Ser His Cys Thr Pro Glu Thr Pro Gly Ser Ile His Glu Gly 165 170 175Gly Glu Leu Gly Tyr Cys Leu Ser His Ala Tyr Gly Ala Ala Phe Asp 180 185 190Asn Pro Asn Leu Ile Val Val Gly Leu Ala Gly Asp Gly Glu Ser Glu 195 200 205Thr Gly Pro Leu Ala Thr Ser Trp His Ser Asn Lys Phe Ile Asn Pro 210 215 220Ile Arg Asp Gly Ala Val Leu Pro Val Leu His Leu Asn Gly Tyr Lys225 230 235 240Ile Asn Asn Pro Ser Val Leu Ser Arg Ile Ser His Glu Glu Leu Lys 245 250 255Ala Leu Phe Glu Gly Tyr Gly Tyr Thr Pro Tyr Phe Val Glu Gly Ser 260 265 270Asp Pro Glu Ser Met His Gln Ala Met Ala Ala Thr Leu Asp His Cys 275 280 285Val Ser Glu Ile His Gln Ile Gln Gln Glu Ala Arg Ser Thr Gly Ile 290 295 300Ala Val Arg Pro Arg Trp Pro Met Val Val Met Arg Thr Pro Lys Gly305 310 315 320Trp Thr Gly Pro Asp Tyr Val Asp Gly His Lys Val Glu Gly Phe Trp 325 330 335Arg Ser His Gln Val Pro Met Gly Gly Met His Glu Asn Pro Ala His 340 345 350Leu Gln Gln Leu Glu Ala Trp Met Arg Ser Tyr Lys Pro Glu Glu Leu 355 360 365Phe Asp Glu Gln Gly Thr Leu Lys Pro Gly Phe Lys Ala Ile Ala Pro 370 375 380Glu Gly Asp Lys Arg Leu Gly Ser Thr Pro Tyr Ala Asn Gly Gly Leu385 390 395 400Leu Arg Arg Gly Leu Lys Met Pro Asp Phe Arg Gln Tyr Gly Ile Asp 405 410 415Val Asp Gln Pro Gly Thr Ile Glu Ala Pro Asn Thr Ala Pro Leu Gly 420 425 430Val Phe Leu Arg Asp Val Met Ala Asn Asn Met Thr Asn Phe Arg Leu 435 440 445Phe Gly Pro Asp Glu Asn Ser Ser Asn Lys Leu His Ala Val Tyr Glu 450 455 460Val Ser Lys Lys Phe Trp Ile Ala Glu Tyr Leu Glu Glu Asp Gln Asp465 470 475 480Gly Gly Glu Leu Ser Pro Asp Gly Arg Val Met Glu Met Leu Ser Glu 485 490 495His Thr Leu Glu Gly Trp Leu Glu Ala Tyr Leu Leu Thr Gly Arg His 500 505 510Gly Phe Phe Ala Thr Tyr Glu Ser Phe Ala His Val Ile Thr Ser Met 515 520 525Val Asn Gln His Ala Lys Trp Leu Asp Ile Cys Arg His Leu Asn Trp 530 535 540Arg Ala Asp Ile Ser Ser Leu Asn Ile Leu Met Thr Ser Thr Val Trp545 550 555 560Arg Gln Asp His Asn Gly Phe Thr His Gln Asp Pro Gly Phe Leu Asp 565 570 575Val Ile Leu Asn Lys Ser Pro Asp Val Val Arg Ile Tyr Leu Pro Pro 580 585 590Asp Val Asn Ser Leu Leu Ser Val Ala Asp His Cys Leu Gln Ser Lys 595 600 605Asn Tyr Ile Asn Ile Ile Val Cys Asp Lys Gln Ala His Leu Gln Tyr 610 615 620Gln Asp Met Thr Ser Ala Ile Arg Asn Cys Thr Lys Gly Val Asp Ile625 630 635 640Trp Glu Trp Ala Ser Asn Asp Ala Gly Thr Glu Pro Asp Val Val Met 645 650 655Ala Ala Ala Gly Asp Ile Pro Thr Lys Glu Ala Leu Ala Ala Thr Ala 660 665 670Met Leu Arg Gln Phe Phe Pro Asn Leu Arg Ile Arg Phe Val Ser Val 675 680 685Ile Asp Leu Leu Lys Leu Gln Pro Glu Ser Glu His Pro His Gly Leu 690 695 700Ser Asp Arg Asp Phe Asp Ser Leu Phe Thr Thr Asp Lys Pro Ile Ile705 710 715 720Phe Asn Phe His Ala Tyr Pro Trp Leu Ile His Arg Leu Thr Tyr Arg 725 730 735Arg Thr Asn His Gly Asn Leu His Val Arg Gly Tyr Lys Glu Lys Gly 740 745 750Asn Ile Asn Thr Pro Met Asp Leu Ala Ile Gln Asn Gln Ile Asp Arg 755 760 765Phe Ser Leu Ala Ile Asp Val Ile Asp Arg Leu Pro Gln Leu Arg Val 770 775 780Ala Gly Ala His Ile Lys Glu Met Leu Lys Asp Met Gln Ile Asp Cys785 790 795 800Thr Asn Tyr Ala Tyr Glu His Gly Ile Asp Met Pro Glu Ile Val Asn 805 810 815Trp Arg Trp Pro Leu 820543234DNAartificialinsert 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 Lys1 5 10 15Ser Leu Val Val Leu Gly Ile Leu Asp Leu Ile Leu Lys Lys Thr Thr 20 25 30Arg Ile Ala Tyr Phe Arg Pro Ile Ile Gln Asp Pro Val Asn Gly Lys 35 40 45His Asp Asn Asn Ile Ile Leu Val Leu Glu Asn Phe Arg Leu Gln Gln 50 55 60Thr Tyr Thr Asp Ser Phe Gly Leu Tyr Phe His Glu Ala Val Ser Leu65 70 75 80Ala Ser Asp Gly Ala Ile Asp Gln Val Leu Asp Arg Ile Leu Ala Lys 85 90 95Tyr Arg His Leu Ala Asp Gln Val Asp Phe Ile Leu Cys Glu Gly Ser 100 105 110Asp Tyr Leu Gly Glu Glu Ser Ala Phe Glu Phe Asp Leu Asn Thr Thr 115 120 125Ile Ala Lys Met Leu Asn Cys Pro Ile Leu Leu Leu Gly Asn Ala Met 130 135 140Gly Asn Thr Ile Ala Asp Ser Leu Gln Pro Ile Asp Met Ala Leu Asn145 150 155 160Ser Tyr Asp Gln Glu Ser Cys Gln Val Val Gly Val Ile Ile Asn Arg 165 170 175Val Gln Pro Glu Leu Ala Thr Glu Ile Gln Ala Gln Leu Glu Gln Arg 180 185 190Tyr Gly Asp Arg Pro Met Val Leu Gly Thr Ile Pro Gln Asp Ile Met 195 200 205Leu Lys Ser Leu Arg Leu Arg Glu Ile Val Ser Gly Leu Asn Ala Gln 210 215 220Val Leu Ser Gly Ala Asp Leu Leu Asp Asn Leu Val Tyr His His Leu225 230 235 240Val Val Ala Met His Ile Ala His Ala Leu His Trp Leu His Glu Lys 245 250 255Asn Thr Leu Ile Ile Thr Pro Gly Asp Arg Gly Asp Ile Ile Leu Gly 260 265 270Val Met Gln Ala His Arg Ser Leu Asn Tyr Pro Ser Ile Ala Gly Ile 275 280 285Leu Leu Thr Ala Asp Tyr His Pro Glu Pro Ala Ile Met Lys Leu Ile 290 295 300Glu Gly Leu Pro Asp Ala Pro Pro Leu Leu Leu Thr Ser Thr His Thr305 310 315 320His Glu Thr Ser Ala Arg Leu Glu Thr Leu His Pro Ala Leu Ser Pro 325 330 335Thr Asp Asn Tyr Lys Ile Arg His Ser Ile Ala Leu Phe Gln Gln Gln 340 345 350Ile Asp Gly Glu Lys Leu Leu Asn Tyr Leu Lys Thr Ile Arg Ser Lys 355 360 365Gly Ile Thr Pro Lys Leu Phe Leu Tyr Asn Leu Val Gln Ala Ala Thr 370 375 380Ala Ala Gln Arg His Ile Val Leu Pro Glu Gly Glu Glu Ile Arg Ile385 390 395 400Leu Lys Ala Ala Ala Ser Leu Ile Asn His Gly Ile Val Arg Leu Thr 405 410 415Leu Leu Gly Asn Ile Glu Ala Ile Glu Gln Thr Val Lys Ile Asn His 420 425 430Ile Asp Leu Asp Leu Ser Lys Val Arg Leu Ile Asn Pro Lys Thr Ser 435 440 445Pro Asp Arg Glu Arg Tyr Ala Glu Thr Tyr Tyr Gln Leu Arg Lys His 450 455 460Lys Gly Val Thr Leu Ala Met Ala Arg Asp Ile Leu Thr Asp Ile Ser465 470 475 480Tyr Phe Gly Thr Met Met Val His Leu Gly Glu Ala Asp Gly Met Val 485 490 495Ser Gly Ser Val Asn Thr Thr Gln His Thr Val Arg Pro Ala Leu Gln 500 505 510Ile Ile Lys Thr Gln Pro Gly Phe Ser Leu Val Ser Ser Val Phe Phe 515 520 525Met Cys Leu Glu Asp Arg Val Leu Val Tyr Gly Asp Cys Ala Val Asn 530 535 540Pro Asp Pro Asn Ala Glu Gln Leu Ala Glu Ile Ala Leu Thr Ser Ala545 550 555 560Ala Thr Ala Lys Asn Phe Gly Ile Glu Pro Arg Val Ala Leu Leu Ser 565 570 575Tyr Ser Ser Gly Ser Ser Gly Gln Gly Ala Asp Val Glu Lys Val Arg 580 585 590Gln Ala Thr Ala Ile Ala Lys Glu Arg Glu Pro Asp Leu Ala Leu Glu 595 600 605Gly Pro Ile Gln Tyr Asp Ala Ala Val Asp Ser Thr Val Ala Ala Gln 610 615 620Lys Met Pro Gly Ser Ala Val Ala Gly Lys Ala Thr Val Phe Ile Phe625 630 635 640Pro Asp Leu Asn Thr Gly Asn Asn Thr Tyr Lys Ala Val Gln Arg Glu 645

650 655Thr Lys Ala Ile Ala Ile Gly Pro Ile Leu Gln Gly Leu Asn Lys Pro 660 665 670Val Asn Asp Leu Ser Arg Gly Cys Leu Val Glu Asp Ile Ile Asn Thr 675 680 685Val Val Ile Thr Ala Leu Gln Val Lys 690 695562857DNAartificialinsert 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 Arg1 5 10 15Gln Gly Lys Thr Lys Ser Val Gln Asp Arg Leu Thr Ala Leu Ala Lys 20 25 30Leu Lys Thr Gln Ile Gln Ala Gln Glu Glu Glu Ile Ile Lys Ala Leu 35 40 45Lys Gln Asp Phe Gly Lys Pro Thr Phe Glu Ser Tyr Val Asn Glu Ile 50 55 60Leu Gly Val Ile Arg Glu Ile Asn Tyr Tyr Gln Lys His Leu Gln Gln65 70 75 80Trp Ser Lys Pro Gln Arg Val Gly Thr Asn Leu Met Val Phe Pro Ala 85 90 95Ser Ala Gln Leu Arg Pro Glu Pro Leu Gly Val Val Leu Ile Ile Ser 100 105 110Pro Trp Asn Tyr Pro Phe Tyr Leu Cys Leu Met Pro Leu Ile Gly Ala 115 120 125Ile Ala Ala Gly Asn Cys Val Val Val Lys Pro Ser Glu Tyr Thr Pro 130 135 140Ala Ile Ser Gly Val Ile Thr Arg Leu Ile Gln Asn Val Phe Ser Pro145 150 155 160Ala Trp Ala Thr Val Val Glu Gly Asp Glu Thr Ile Ser Gln Gln Leu 165 170 175Leu Gln Glu Lys Phe Asp His Ile Phe Phe Thr Gly Ser Pro Arg Val 180 185 190Gly Arg Leu Ile Met Ala Ala Ala Ala Glu Gln Leu Thr Pro Val Thr 195 200 205Leu Glu Leu Gly Gly Lys Ser Pro Cys Val Val Asp Arg Glu Ile Asn 210 215 220Leu Gln Glu Thr Ala Lys Arg Ile Met Trp Gly Lys Leu Val Asn Ala225 230 235 240Gly Gln Thr Cys Val Ala Pro Asp Tyr Leu Leu Val Glu Gln Ser Cys 245 250 255Leu Glu Gln Leu Leu Pro Ala Leu Gln Gln Ala Ile Gln Met Leu Phe 260 265 270Gly Glu Asn Pro Ala His Ser Pro Asp Tyr Thr Arg Ile Val Asn Gln 275 280 285Gln Gln Trp Ser Arg Leu Val Ser Leu Leu Ser His Gly Lys Val Ile 290 295 300Thr Arg Gly Asp His Asn Glu Gly Asp Arg Tyr Ile Ala Pro Thr Leu305 310 315 320Ile Ile Asp Pro Asp Leu Asn Ser Pro Leu Met Gln Glu Glu Ile Phe 325 330 335Gly Pro Ile Leu Pro Ile Leu Thr Tyr Gln Ser Leu Ser Glu Ala Ile 340 345 350Asp Phe Ile Asn Ile Lys Pro Lys Pro Leu Ala Leu Tyr Phe Phe Ser 355 360 365Asn Asn Arg Gln Lys Gln Glu Glu Ile Leu Gln Ser Thr Ser Ser Gly 370 375 380Ser Val Cys Leu Asn Asp Ile Leu Leu His Leu Thr Val Thr Asp Leu385 390 395 400Pro Phe Gly Gly Val Gly Glu Ser Gly Met Gly Arg Tyr His Gly Lys 405 410 415Ala Thr Phe Asp Thr Leu Ser Asn Tyr Lys Ser Ile Leu Arg Arg Pro 420 425 430Phe Trp Gly Glu Thr Asn Leu Arg Tyr Ser Pro Tyr Gly Lys Lys Met 435 440 445Asn Leu Ile Lys Lys Leu Phe Ser 450 455592089DNAartificialinsert 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 Gly1 5 10 15Asn Gly Asn Gly Ser Asn Ser Glu Glu Glu Ser Val Leu Tyr Gln Arg 20 25 30Leu Lys Met Val Glu Glu Leu Trp Glu Arg Val Leu Gln Ser Glu Cys 35 40 45Gly Gln Glu Leu Val Asp Leu Leu Thr Glu Leu Arg Leu Gln Gly Thr 50 55 60His Glu Ala Ile Thr Ser Glu Ile Ser Glu Glu Val Ile Met Gly Ile65 70 75 80Thr Gln Arg Ile Glu His Leu Glu Leu Asn Asp Ala Ile Arg Ala Ala 85 90 95Arg Ala Phe Ala Leu Tyr Phe Gln Leu Ile Asn Ile Val Glu Gln His 100 105 110Tyr Glu Gln Asn Glu Gln Gln Arg Asn Arg Trp Glu Ala Ser Gln Glu 115 120 125Thr Asn Phe Tyr Glu Gln Ala Gly Asn Glu Glu Glu Met Val Pro Pro 130 135 140Ser Arg Leu Gly Ala Ser Thr Glu Pro Leu Pro Val Gly Ile Asp Gln145 150 155 160Asn Glu Leu Gln Ala Ser Val Gly Thr Phe His Trp Leu Met Arg Glu 165 170 175Leu Lys Arg Leu Asn Val Pro Pro Gln His Ile Gln Asn Leu Leu Asp 180 185 190His Leu Asp Ile Arg Leu Val Ile Thr Ala His Pro Thr Glu Ile Val 195 200 205Arg His Thr Ile Arg Arg Lys Gln Arg Arg Val Asp Arg Ile Leu Arg 210 215 220Lys Leu Asp Gln Leu Gln Gly Ser Val Thr Gly Arg

Asp Trp Leu Asn225 230 235 240Thr Trp Asp Ala Lys Thr Ala Ile Ala Gln Leu Thr Glu Glu Ile Arg 245 250 255Phe Trp Trp Arg Thr Asp Glu Leu His Gln Phe Lys Pro Thr Val Leu 260 265 270Asp Glu Val Asp Tyr Ser Leu His Tyr Phe Asp Glu Val Leu Phe Asp 275 280 285Ala Val Pro Glu Leu Ser Lys Arg Leu Gly Gln Ala Ile Lys Glu Thr 290 295 300Phe Pro His Leu Arg Ala Pro Arg Ala Asn Phe Cys Tyr Phe Gly Ser305 310 315 320Trp Val Gly Gly Asp Arg Asp Gly Asn Pro Ser Val Thr Pro Glu Val 325 330 335Thr Trp Gln Thr Ala Cys Tyr Gln Arg Gly Leu Val Leu Gly Lys Tyr 340 345 350Leu Phe Ser Leu Gly Glu Leu Val Ala Ile Leu Ser Pro Ser Leu His 355 360 365Trp Cys Lys Val Ser Gln Glu Leu Leu Asp Ser Leu Glu Arg Asp Arg 370 375 380Ile Gln Leu Pro Glu Ile Tyr Glu Glu Leu Ser Leu Arg Tyr Arg Gln385 390 395 400Glu Pro Tyr Arg Met Lys Leu Ala Tyr Val Thr Lys Arg Leu Glu Asn 405 410 415Thr Leu Arg Arg Asn Asn Arg Leu Ala Asn Pro Glu Glu Arg Gln Thr 420 425 430Met Ile Thr Met Pro Ala Glu Asn His Tyr Arg Thr Gly Glu Glu Leu 435 440 445Leu Glu Glu Leu Arg Leu Ile Gln Arg Asn Leu Thr Glu Thr Gly Leu 450 455 460Thr Cys Leu Glu Leu Glu Asn Leu Ile Thr Gln Leu Glu Val Tyr Gly465 470 475 480Phe Asn Leu Ala Gln Leu Asp Phe Arg Gln Glu Ser Ser Arg His Ala 485 490 495Glu Ala Ile Ala Glu Ile Ala Glu Tyr Met Gly Val Leu Thr Thr Pro 500 505 510Tyr Glu Glu Met Ala Glu Glu Asp Lys Leu Ala Trp Leu Gly Val Glu 515 520 525Leu Gln Thr Arg Arg Pro Leu Ile Pro Gln Glu Met Pro Phe Ser Glu 530 535 540Arg Thr Arg Glu Thr Ile Glu Thr Leu Arg Thr Leu Arg His Leu Gln545 550 555 560Met Glu Phe Gly Val Asp Ile Cys Gln Thr Tyr Ile Ile Ser Met Thr 565 570 575Asn Asp Ala Ser Asp Val Leu Glu Val Leu Leu Leu Ala Lys Glu Ala 580 585 590Gly Leu Tyr Asp Pro Ala Thr Ala Ser Asn Ser Leu Arg Ile Val Pro 595 600 605Leu Phe Glu Thr Val Glu Asp Leu Lys Asn Ala Pro Gly Ile Met Asp 610 615 620Ser Leu Phe Ser Leu Pro Phe Tyr Arg Ala Thr Leu Ala Gly Ser Tyr625 630 635 640His Ser Leu Lys Glu Leu Gln Asn Gln Pro Pro Asp Tyr Tyr Gln Ile 645 650 655Pro Thr Thr Thr Ala Leu Leu Asn Pro Gly Asn Leu Gln Glu Ile Met 660 665 670Val Gly Tyr Ser Asp Ser Asn Lys Asp Ser Gly Phe Leu Ser Ser Asn 675 680 685Trp Glu Ile His Lys Ala Gln Lys Ser Leu Gln Ala Val Ala Gln Ser 690 695 700His Arg Val Ile Leu Arg Leu Phe His Gly Arg Gly Gly Ser Val Gly705 710 715 720Arg Gly Gly Gly Pro Ala Tyr Lys Ala Ile Leu Ala Gln Pro Ala Gly 725 730 735Thr Val Asp Gly Arg Ile Lys Ile Thr Glu Gln Gly Glu Val Leu Ala 740 745 750Ser Lys Tyr Ser Leu Pro Glu Leu Ala Leu Tyr Asn Leu Glu Thr Leu 755 760 765Thr Thr Ala Val Ile Gln Ala Ser Leu Leu Lys Ser Ser Phe Asp Phe 770 775 780Ile Glu Pro Trp Asn Arg Ile Met Glu Glu Leu Ala Cys Thr Ala Arg785 790 795 800Arg Ala Tyr Arg Ser Leu Ile Tyr Glu Glu Pro Asp Phe Leu Asp Phe 805 810 815Phe Leu Thr Val Thr Pro Ile Pro Glu Ile Ser Glu Leu Gln Ile Ser 820 825 830Ser Arg Pro Ala Arg Arg Lys Gly Gly Lys Ala Asp Leu Ser Ser Leu 835 840 845Arg Ala Ile Pro Trp Val Phe Ser Trp Thr Gln Thr Arg Phe Leu Leu 850 855 860Pro Ala Trp Tyr Gly Val Gly Thr Ala Leu Lys Ser Phe Val Asp Gln865 870 875 880Asp Pro Val Lys Asn Met Lys Leu Leu Arg Tyr Phe Tyr Phe Lys Trp 885 890 895Pro Phe Phe Asn Met Val Ile Ser Lys Val Glu Met Thr Leu Ser Lys 900 905 910Val Asp Leu Thr Ile Ala Ser His Tyr Val Gln Glu Leu Ser Lys Pro 915 920 925Glu Asp Arg Glu Arg Phe Asp Arg Leu Phe Gln Gln Ile Lys Gln Glu 930 935 940Tyr Gln Leu Thr Arg Asp Phe Ala Met Glu Ile Thr Ala His Pro His945 950 955 960Leu Leu Asp Gly Asp Arg Ser Leu Gln Arg Ser Val Leu Leu Arg Asn 965 970 975Arg Thr Ile Val Pro Leu Gly Leu Leu Gln Ile Ser Leu Leu Lys Arg 980 985 990Leu Arg Gln Val Thr Gln Glu Ala Glu Thr Ser Gly Val Arg Tyr Arg 995 1000 1005Arg Tyr Ser Lys Glu Glu Leu Leu Arg Gly Ala Leu Leu Thr Ile 1010 1015 1020Asn Gly Ile Ala Ala Gly Met Arg Asn Thr Gly 1025 1030613650DNAartificialinsert 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 Arg1 5 10 15Leu Thr Tyr Tyr Thr Pro Asp Tyr Thr Pro Lys Asp Thr Asp Leu Leu 20 25 30Ala Cys Phe Arg Met Thr Pro Gln Pro Gly Val Pro Ala Glu Glu Ala 35 40 45Ala Ala Ala Val Ala Ala Glu Ser Ser Thr Gly Thr Trp Thr Thr Val 50 55 60Trp Thr Asp Asn Leu Thr Asp Leu Asp Arg Tyr Lys Gly Arg Cys Tyr65 70 75 80Asp Leu Glu Ala Val Pro Asn Glu Asp Asn Gln Tyr Phe Ala Phe Ile 85 90 95Ala Tyr Pro Leu Asp Leu Phe Glu Glu Gly Ser Val Thr Asn Val Leu 100 105 110Thr Ser Leu Val Gly Asn Val Phe Gly Phe Lys Ala Leu Arg Ala Leu 115 120 125Arg Leu Glu Asp Ile Arg Phe Pro Val Ala Leu Ile Lys Thr Phe Gln 130 135 140Gly Pro Pro His Gly Ile Thr Val Glu Arg Asp Lys Leu Asn Lys Tyr145 150 155 160Gly Arg Pro Leu Leu Gly Cys Thr Ile Lys Pro Lys Leu Gly Leu Ser 165 170 175Ala Lys Asn Tyr Gly Arg Ala Val Tyr Glu Cys Leu Arg Gly Gly Leu 180 185 190Asp Phe Thr Lys Asp Asp Glu Asn Ile Asn Ser Gln Pro Phe Met Arg 195 200 205Trp Arg Asp Arg Phe Leu Phe Val Gln Glu Ala Ile Glu Lys Ala Gln 210 215 220Ala Glu Thr Asn Glu Met Lys Gly His Tyr Leu Asn Val Thr Ala Gly225 230 235 240Thr Cys Glu Glu Met Met Lys Arg Ala Glu Phe Ala Lys Glu Ile Gly 245 250 255Thr Pro Ile Ile Met His Asp Phe Phe Thr Gly Gly Phe Thr Ala Asn 260 265 270Thr Thr Leu Ala Arg Trp Cys Arg Asp Asn Gly Ile Leu Leu His Ile 275 280 285His Arg Ala Met His Ala Val Val Asp Arg Gln Lys Asn His Gly Ile 290 295 300His Phe Arg Val Leu Ala Lys Cys Leu Arg Leu Ser Gly Gly Asp His305 310 315 320Leu His Ser Gly Thr Val Val Gly Lys Leu Glu Gly Glu Arg Gly Ile 325 330 335Thr Met Gly Phe Val Asp Leu Met Arg Glu Asp Tyr Val Glu Glu Asp 340 345 350Arg Ser Arg Gly Ile Phe Phe Thr Gln Asp Tyr Ala Ser Met Pro Gly 355 360 365Thr Met Pro Val Ala Ser Gly Gly Ile His Val Trp His Met Pro Ala 370 375 380Leu Val Glu Ile Phe Gly Asp Asp Ser Cys Leu Gln Phe Gly Gly Gly385 390 395 400Thr Leu Gly His Pro Trp Gly Asn Ala Pro Gly Ala Thr Ala Asn Arg 405 410 415Val Ala Leu Glu Ala Cys Val Gln Ala Arg Asn Glu Gly Arg Asn Leu 420 425 430Ala Arg Glu Gly Asn Asp Val Ile Arg Glu Ala Cys Arg Trp Ser Pro 435 440 445Glu Leu Ala Ala Ala Cys Glu Leu Trp Lys Glu Ile Lys Phe Glu Phe 450 455 460Glu Ala Met Asp Thr Leu465 47067136PRTSynechocystis PCC 6803 67Met Gln Thr Lys His Ile Ala Gln Ala Thr Val Lys Val Leu Gln Ser1 5 10 15Tyr Leu Thr Tyr Gln Ala Val Leu Arg Ile Gln Ser Glu Leu Gly Glu 20 25

30Thr Asn Pro Pro Gln Ala Ile Trp Leu Asn Gln Tyr Leu Ala Ser His 35 40 45Ser Ile Gln Asn Gly Glu Thr Phe Leu Thr Glu Leu Leu Asp Glu Asn 50 55 60Lys Glu Leu Val Leu Arg Ile Leu Ala Val Arg Glu Asp Ile Ala Glu65 70 75 80Ser Val Leu Asp Phe Leu Pro Gly Met Thr Arg Asn Ser Leu Ala Glu 85 90 95Ser Asn Ile Ala His Arg Arg His Leu Leu Glu Arg Leu Thr Arg Thr 100 105 110Val Ala Glu Val Asp Asn Phe Pro Ser Glu Thr Ser Asn Gly Glu Ser 115 120 125Asn Asn Asn Asp Ser Pro Pro Ser 130 13568113PRTSynechocystis PCC 6803 68Met Lys Thr Leu Pro Lys Glu Arg Arg Tyr Glu Thr Leu Ser Tyr Leu1 5 10 15Pro Pro Leu Thr Asp Gln Gln Ile Ala Lys Gln Val Glu Phe Leu Leu 20 25 30Asp Gln Gly Phe Ile Pro Gly Val Glu Phe Glu Glu Asp Pro Gln Pro 35 40 45Glu Thr His Phe Trp Thr Met Trp Lys Leu Pro Phe Phe Gly Gly Ala 50 55 60Thr Ala Asn Glu Val Leu Ala Glu Val Arg Glu Cys Arg Ser Glu Asn65 70 75 80Pro Asn Cys Tyr Ile Arg Val Ile Gly Phe Asp Asn Ile Lys Gln Cys 85 90 95Gln Thr Val Ser Phe Ile Val His Lys Pro Asn Gln Asn Gln Gly Arg 100 105 110Tyr696267DNAartificialvector 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 Gln1 5 10 15Ile Gly Leu Lys His His Phe Ala Val Ala Gly Asp Tyr Asn Leu Val 20 25 30Leu Leu Asp Asn Leu Leu Leu Asn Lys Asn Met Glu Gln Val Tyr Cys 35 40 45Cys Asn Glu Leu Asn Cys Gly Phe Ser Ala Glu Gly Tyr Ala Arg Ala 50 55 60Lys Gly Ala Ala Ala Ala Val Val Thr Tyr Ser Val Gly Ala Leu Ser65 70 75 80Ala Phe Asp Ala Ile Gly Gly Ala Tyr Ala Glu Asn Leu Pro Val Ile 85 90 95Leu Ile Ser Gly Ala Pro Asn Asn Asn Asp His Ala Ala Gly His Val 100 105 110Leu His His Ala Leu Gly Lys Thr Asp Tyr His Tyr Gln Leu Glu Met 115 120 125Ala Lys Asn Ile Thr Ala Ala Ala Glu Ala Ile Tyr Thr Pro Glu Glu 130 135 140Ala Pro Ala Lys Ile Asp His Val Ile Lys Thr Ala Leu Arg Glu Lys145 150 155 160Lys Pro Val Tyr Leu Glu Ile Ala Cys Asn Ile Ala Ser Met Pro Cys 165 170 175Ala Ala Pro Gly Pro Ala Ser Ala Leu Phe Asn Asp Glu Ala Ser Asp 180 185 190Glu Ala Ser Leu Asn Ala Ala Val Glu Glu Thr Leu Lys Phe Ile Ala 195 200 205Asn Arg Asp Lys Val Ala Val Leu Val Gly Ser Lys Leu Arg Ala Ala 210 215 220Gly Ala Glu Glu Ala Ala Val Lys Phe Ala Asp Ala Leu Gly Gly Ala225 230 235 240Val Ala Thr Met Ala Ala Ala Lys Ser Phe Phe Pro Glu Glu Asn Pro 245 250 255His Tyr Ile Gly Thr Ser Trp Gly Glu Val Ser Tyr Pro Gly Val Glu 260 265 270Lys Thr Met Lys Glu Ala Asp Ala Val Ile Ala Leu Ala Pro Val Phe 275 280 285Asn Asp Tyr Ser Thr Thr Gly Trp Thr Asp Ile Pro Asp Pro Lys Lys 290 295 300Leu Val Leu Ala Glu Pro Arg Ser Val Val Val Asn Gly Val Arg Phe305 310 315 320Pro Ser Val His Leu Lys Asp Tyr Leu Thr Arg Leu Ala Gln Lys Val 325 330 335Ser Lys Lys Thr Gly Ala Leu Asp Phe Phe Lys Ser Leu Asn Ala Gly 340 345 350Glu Leu Lys Lys Ala Ala Pro Ala Asp Pro Ser Ala Pro Leu Val Asn 355 360 365Ala Glu Ile Ala Arg Gln Val Glu Ala Leu Leu Thr Pro Asn Thr Thr 370 375 380Val Ile Ala Glu Thr Gly Asp Ser Trp Phe Asn Ala Gln Arg Met Lys385 390 395 400Leu Pro Asn Gly Ala Arg Val Glu Tyr Glu Met Gln Trp Gly His Ile 405 410 415Gly Trp Ser Val Pro Ala Ala Phe Gly Tyr Ala Val Gly Ala Pro Glu 420 425 430Arg Arg Asn Ile Leu Met Val Gly Asp Gly Ser Phe Gln Leu Thr Ala 435 440 445Gln Glu Val Ala Gln Met Val Arg Leu Lys Leu Pro Val Ile Ile Phe 450 455 460Leu Ile Asn Asn Tyr Gly Tyr Thr Ile Glu Val Met Ile His Asp Gly465 470 475 480Pro Tyr Asn Asn Ile Lys Asn Trp Asp Tyr Ala Gly Leu Met Glu Val 485 490 495Phe Asn Gly Asn Gly Gly Tyr Asp Ser Gly Ala Gly Lys Gly Leu Lys 500 505 510Ala Lys Thr Gly Gly Glu Leu Ala Glu Ala Ile Lys Val Ala Leu Ala 515 520 525Asn Thr Asp Gly Pro Thr Leu Ile Glu Cys Phe Ile Gly Arg Glu Asp 530 535 540Cys Thr Glu Glu Leu Val Lys Trp Gly Lys Arg Val Ala Ala Ala Asn545 550 555 560Ser Arg Lys Pro Val Asn Lys Leu Leu 56575383PRTZymomonas mobilis 75Met Ala Ser Ser Thr Phe Tyr Ile Pro Phe Val Asn Glu Met Gly Glu1 5 10 15Gly Ser Leu Glu Lys Ala Ile Lys Asp Leu Asn Gly Ser Gly Phe Lys 20 25 30Asn Ala Leu Ile Val Ser Asp Ala Phe Met Asn Lys Ser Gly Val Val 35 40 45Lys Gln Val Ala Asp Leu Leu Lys Ala Gln Gly Ile Asn Ser Ala Val 50 55 60Tyr Asp Gly Val Met Pro Asn Pro Thr Val Thr Ala Val Leu Glu Gly65 70 75 80Leu Lys Ile Leu Lys Asp Asn Asn Ser Asp Phe Val Ile Ser Leu Gly 85 90 95Gly Gly Ser Pro His Asp Cys Ala Lys Ala Ile Ala Leu Val Ala Thr 100 105 110Asn Gly Gly Glu Val Lys Asp Tyr Glu Gly Ile Asp Lys Ser Lys Lys 115 120 125Pro Ala Leu Pro Leu Met Ser Ile Asn Thr Thr Ala Gly Thr Ala Ser 130 135 140Glu Met Thr Arg Phe Cys Ile Ile Thr Asp Glu Val Arg His Val Lys145 150 155 160Met Ala Ile Val Asp Arg His Val Thr Pro Met Val Ser Val Asn Asp 165 170 175Pro Leu Leu Met Val Gly Met Pro Lys Gly Leu Thr Ala Ala Thr Gly 180 185 190Met Asp Ala Leu Thr His Ala Phe Glu Ala Tyr Ser Ser Thr Ala Ala 195 200 205Thr Pro Ile Thr Asp Ala Cys Ala Leu Lys Ala Ala Ser Met Ile Ala 210 215 220Lys Asn Leu Lys Thr Ala Cys Asp Asn Gly Lys Asp Met Pro Ala Arg225 230 235 240Glu Ala Met Ala Tyr Ala Gln Phe Leu Ala Gly Met Ala Phe Asn Asn 245 250 255Ala Ser Leu Gly Tyr Val His Ala Met Ala His Gln Leu Gly Gly Tyr 260 265 270Tyr Asn Leu Pro His Gly Val Cys Asn Ala Val Leu Leu Pro His Val 275 280 285Leu Ala Tyr Asn Ala Ser Val Val Ala Gly Arg Leu Lys Asp Val Gly 290 295 300Val Ala Met Gly Leu Asp Ile Ala Asn Leu Gly Asp Lys Glu Gly Ala305 310 315 320Glu Ala Thr Ile Gln Ala Val Arg Asp Leu Ala Ala Ser Ile Gly Ile 325 330 335Pro Ala Asn Leu Thr Glu Leu Gly Ala Lys Lys Glu Asp Val Pro Leu 340 345 350Leu Ala Asp His Ala Leu Lys Asp Ala Cys Ala Leu Thr Asn Pro Arg 355 360 365Gln Gly Asp Gln Lys Glu Val Glu Glu Leu Phe Leu Ser Ala Phe 370 375 38076614DNASynechocystis 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 Asp1 5 10 15Thr Lys Leu Arg Pro Leu Lys Tyr Gly Glu Ala Leu Leu Glu Met Glu 20 25 30Tyr Cys Gly Val Cys His Thr Asp Leu His Val Lys Asn Gly Asp Phe 35 40 45Gly Asp Glu Thr Gly Arg Ile Thr Gly His Glu Gly Ile Gly Ile Val 50 55 60Lys Gln Val Gly Glu Gly Val Thr Ser Leu Lys Val Gly Asp Arg Ala65 70 75 80Ser Val Ala Trp Phe Phe Lys Gly Cys Gly His Cys Glu Tyr Cys Val 85 90 95Ser Gly Asn Glu Thr Leu Cys Arg Asn Val Glu Asn Ala Gly Tyr Thr 100 105 110Val Asp Gly Ala Met Ala Glu Glu Cys Ile Val Val Ala Asp Tyr Ser 115 120 125Val Lys Val Pro Asp Gly Leu Asp Pro Ala Val Ala Ser Ser Ile Thr 130 135 140Cys Ala Gly Val Thr Thr Tyr Lys Ala Val Lys Val Ser Gln Ile Gln145 150 155 160Pro Gly Gln Trp Leu Ala Ile Tyr Gly Leu Gly Gly Leu Gly Asn Leu 165 170 175Ala Leu Gln Tyr Ala Lys Asn Val Phe Asn Ala Lys Val Ile Ala Ile 180 185 190Asp Val Asn Asp Glu Gln Leu Ala Phe Ala Lys Glu Leu Gly Ala Asp 195 200 205Met Val Ile Asn Pro Lys Asn Glu Asp Ala Ala Lys Ile Ile Gln Glu 210 215 220Lys Val Gly Gly Ala His Ala Thr Val Val Thr Ala Val Ala Lys Ser225 230 235 240Ala Phe Asn Ser Ala Val Glu Ala Ile Arg Ala Gly Gly Arg Val Val 245 250 255Ala Val Gly Leu Pro Pro Glu Lys Met Asp Leu Ser Ile Pro Arg Leu 260 265 270Val Leu Asp Gly Ile Glu Val Leu Gly Ser Leu Val Gly Thr Arg Glu 275 280 285Asp Leu Lys Glu Ala Phe Gln Phe Ala Ala Glu Gly Lys Val Lys Pro 290 295 300Lys Val Thr Lys Arg Lys Val Glu Glu Ile Asn Gln Ile Phe Asp Glu305 310 315 320Met Glu His Gly Lys Phe Thr Gly Arg Met Val Val Asp Phe Thr His 325 330 335His951144DNASynechocystis 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 Pro1 5 10 15Phe Glu Tyr Asp Pro Gly Ala Leu Gly Ala Asn Glu Val Glu Ile Glu 20 25 30Val Gln Tyr Cys Gly Val Cys His Ser Asp Leu Ser Met Ile Asn Asn 35 40 45Glu Trp Gly Ile Ser Asn Tyr Pro Leu Val Pro Gly His Glu Val Val 50 55 60Gly Thr Val Ala Ala Met Gly Glu Gly Val Asn His Val Glu Val Gly65 70 75 80Asp Leu Val Gly Leu Gly Trp His Ser Gly Tyr Cys Met Thr Cys His 85 90 95Ser Cys Leu Ser Gly Tyr His Asn Leu Cys Ala Thr Ala Glu Ser Thr 100 105 110Ile Val Gly His Tyr Gly Gly Phe Gly Asp Arg Val Arg Ala Lys Gly 115 120 125Val Ser Val Val Lys Leu Pro Lys Gly Ile Asp Leu Ala Ser Ala Gly 130 135 140Pro Leu Phe Cys Gly Gly Ile Thr Val Phe Ser Pro Met Val Glu Leu145 150 155 160Ser Leu Lys Pro Thr Ala Lys Val Ala Val Ile Gly Ile Gly Gly Leu 165 170 175Gly His Leu Ala Val Gln Phe Leu Arg Ala Trp Gly Cys Glu Val Thr 180 185 190Ala Phe Thr Ser Ser Ala Arg Lys Gln Thr Glu Val Leu Glu Leu Gly 195 200 205Ala His His Ile Leu Asp Ser Thr Asn Pro Glu Ala Ile Ala Ser Ala 210 215 220Glu Gly Lys Phe Asp Tyr Ile Ile Ser Thr Val Asn Leu Lys Leu Asp225 230 235 240Trp Asn Leu Tyr Ile Ser Thr Leu Ala Pro Gln Gly His Phe His Phe 245 250 255Val Gly Val Val Leu Glu Pro Leu Asp Leu Asn Leu Phe Pro Leu Leu 260 265 270Met Gly Gln Arg Ser Val Ser Ala Ser Pro Val Gly Ser Pro Ala Thr 275 280 285Ile Ala Thr Met Leu Asp Phe Ala Val Arg His Asp Ile Lys Pro Val 290 295 300Val Glu Gln Phe Ser Phe Asp Gln Ile Asn Glu Ala Ile Ala His Leu305 310 315 320Glu Ser Gly Lys Ala His Tyr Arg Val Val Leu Ser His Ser Lys Asn 325 330 335972781DNAE. 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 Glu1 5 10 15Arg Val Lys Lys Ala Gln Arg Glu Tyr Ala Ser Phe Thr Gln Glu Gln 20 25 30Val Asp Lys Ile Phe Arg Ala Ala Ala Leu Ala Ala Ala Asp Ala Arg 35 40 45Ile Pro Leu Ala Lys Met Ala Val Ala Glu Ser Gly Met Gly Ile Val 50 55 60Glu Asp Lys Val Ile Lys Asn His Phe Ala Ser Glu Tyr Ile Tyr Asn65 70 75 80Ala Tyr Lys Asp Glu Lys Thr Cys Gly Val Leu Ser Glu Asp Asp Thr 85 90 95Phe Gly Thr Ile Thr Ile Ala Glu Pro Ile Gly Ile Ile Cys Gly Ile 100 105 110Val Pro Thr Thr Asn Pro Thr Ser Thr Ala Ile Phe Lys Ser Leu Ile 115 120 125Ser Leu Lys Thr Arg Asn Ala Ile Ile Phe Ser Pro His Pro Arg Ala 130 135 140Lys Asp Ala Thr Asn Lys Ala Ala Asp Ile Val Leu Gln Ala Ala Ile145 150 155 160Ala Ala Gly Ala Pro Lys Asp Leu Ile Gly Trp Ile Asp Gln Pro Ser 165 170 175Val Glu Leu Ser Asn Ala Leu Met His His Pro Asp Ile Asn Leu Ile 180 185 190Leu Ala Thr Gly Gly Pro Gly Met Val Lys Ala Ala Tyr Ser Ser Gly 195 200 205Lys Pro Ala Ile Gly Val Gly Ala Gly Asn Thr Pro Val Val Ile Asp 210 215 220Glu Thr Ala Asp Ile Lys Arg Ala Val Ala Ser Val Leu Met Ser Lys225 230 235 240Thr Phe Asp Asn Gly Val Ile Cys Ala Ser Glu Gln Ser Val Val Val 245 250 255Val Asp Ser Val Tyr Asp Ala Val Arg Glu Arg Phe Ala Thr His Gly 260 265 270Gly Tyr Leu Leu Gln Gly Lys Glu Leu Lys Ala Val Gln Asp Val Ile 275 280 285Leu Lys Asn Gly Ala Leu Asn Ala Ala Ile Val Gly Gln Pro Ala Tyr 290 295 300Lys Ile Ala Glu Leu Ala Gly Phe Ser Val Pro Glu Asn Thr Lys Ile305 310 315 320Leu Ile Gly Glu Val Thr Val Val Asp Glu Ser Glu Pro Phe Ala His 325 330 335Glu Lys Leu Ser Pro Thr Leu Ala Met Tyr Arg Ala Lys Asp Phe Glu 340 345 350Asp Ala Val Glu Lys Ala Glu Lys Leu Val Ala Met Gly Gly Ile Gly 355 360 365His Thr Ser Cys Leu Tyr Thr Asp Gln Asp Asn Gln Pro Ala Arg Val 370 375 380Ser Tyr Phe Gly Gln Lys Met Lys Thr Ala Arg Ile Leu Ile Asn Thr385 390 395 400Pro Ala Ser Gln Gly Gly Ile Gly Asp Leu Tyr Asn Phe Lys Leu Ala 405 410 415Pro Ser Leu Thr Leu Gly Cys Gly Ser Trp Gly Gly Asn Ser Ile Ser 420 425 430Glu Asn Val Gly Pro Lys His Leu Ile Asn Lys Lys Thr Val Ala Lys 435 440 445Arg Ala Glu Asn Met Leu Trp His Lys Leu Pro Lys Ser Ile Tyr Phe 450 455 460Arg Arg Gly Ser Leu Pro Ile Ala Leu Asp Glu Val Ile Thr Asp Gly465 470 475 480His Lys Arg Ala Leu Ile Val Thr Asp Arg Phe Leu Phe Asn Asn Gly 485 490 495Tyr Ala Asp Gln Ile Thr Ser Val Leu Lys Ala Ala Gly Val Glu Thr 500 505 510Glu Val Phe Phe Glu Val Glu Ala Asp Pro Thr Leu Ser Ile Val Arg 515 520 525Lys Gly Ala Glu Leu Ala Asn Ser Phe Lys Pro Asp Val Ile Ile Ala 530 535 540Leu Gly Gly Gly Ser Pro Met Asp Ala Ala Lys Ile Met Trp Val Met545 550 555 560Tyr Glu His Pro Glu Thr His Phe Glu Glu Leu Ala Leu Arg Phe Met 565 570 575Asp Ile Arg Lys Arg Ile Tyr Lys Phe Pro Lys Met Gly Val Lys Ala 580 585 590Lys Met Ile Ala Val Thr Thr Thr Ser Gly Thr Gly Ser Glu Val Thr 595 600 605Pro Phe Ala Val Val Thr Asp Asp Ala Thr Gly Gln Lys Tyr Pro Leu 610 615 620Ala Asp Tyr Ala Leu Thr Pro Asp Met Ala Ile Val Asp Ala Asn Leu625 630 635 640Val Met Asp Met Pro Lys Ser Leu Cys Ala Phe Gly Gly Leu Asp Ala 645 650 655Val Thr His Ala Met Glu Ala Tyr Val Ser Val Leu Ala Ser Glu Phe 660 665 670Ser Asp Gly Gln Ala Leu Gln Ala Leu Lys Leu Leu Lys Glu Tyr Leu 675 680 685Pro Ala Ser Tyr His Glu Gly Ser Lys Asn Pro Val Ala Arg Glu Arg 690 695 700Val His Ser Ala Ala Thr Ile Ala Gly Ile Ala Phe Ala Asn Ala Phe705 710 715 720Leu Gly Val Cys His Ser Met Ala His Lys Leu Gly Ser Gln Phe His 725 730 735Ile Pro His Gly Leu Ala Asn Ala Leu Leu Ile Cys Asn Val Ile Arg 740 745 750Tyr Asn Ala Asn Asp Asn Pro Thr Lys Gln Thr Ala Phe Ser Gln Tyr 755 760 765Asp Arg Pro Gln Ala Arg Arg Arg Tyr Ala Glu Ile Ala Asp His Leu 770 775 780Gly Leu Ser Ala Pro Gly Asp Arg Thr Ala Ala Lys Ile Glu Lys Leu785 790 795 800Leu Ala Trp Leu Glu Thr Leu Lys Ala Glu Leu Gly Ile Pro Lys Ser 805 810 815Ile Arg Glu Ala Gly Val Gln Glu Ala Asp Phe Leu Ala Asn Val Asp 820 825 830Lys Leu Ser Glu Asp Ala Phe Asp Asp Gln Cys Thr Gly Ala Asn Pro 835 840 845Arg Tyr Pro Leu Ile Ser Glu Leu Lys Gln Ile Leu Leu Asp Thr Tyr 850 855 860Tyr Gly Arg Asp Tyr Val Glu Gly Glu Thr Ala Ala Lys Lys Glu Ala865 870 875 880Ala Pro Ala Lys Ala Glu Lys Lys Ala Lys Lys Ser Ala 885 890992747DNAThermosynechococcus 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 Ala1 5 10 15Asp Leu Glu Gly Leu Ile Glu Arg Val Gln Arg Ala Gln Ser Gln Tyr 20 25 30Ala Gln Phe Thr Gln Glu Gln Val Asp His Ile Phe His Glu Ala Ala 35 40 45Met Ala Ala Asn Gln Ala Arg Ile Pro Leu Ala Lys Gln Ala Val Ala 50 55 60Glu Thr Gly Met Gly Val Val Glu Asp Lys Val Ile Lys Asn His Phe65 70 75 80Ala Ser Glu Tyr Ile Tyr Asn Lys Tyr Lys Asn Glu Lys Thr Cys Gly 85 90 95Val Ile Glu Asp Asp Pro Ile Phe Gly Ile Gln Lys Ile Ala Glu Pro 100 105 110Val Gly Ile Ile Ala Gly Val Val Pro Val Thr Asn Pro Thr Ser Thr 115 120 125Thr Ile Phe Lys Ala Leu Ile

Ala Leu Lys Thr Arg Asn Gly Ile Ile 130 135 140Phe Ser Pro His Pro Arg Ala Lys Ala Cys Thr Val Ala Ala Ala Lys145 150 155 160Val Val Leu Asp Ala Ala Val Ala Ala Gly Ala Pro Pro Asp Ile Ile 165 170 175Gly Trp Ile Asp Glu Pro Thr Ile Glu Leu Ser Gln Ala Leu Met Gln 180 185 190His Pro Gln Ile Lys Leu Ile Leu Ala Thr Gly Gly Pro Gly Met Val 195 200 205Lys Ala Ala Tyr Ser Ser Gly His Pro Ala Ile Gly Val Gly Ala Gly 210 215 220Asn Thr Pro Val Leu Ile Asp Ala Thr Ala Asp Ile Pro Thr Ala Val225 230 235 240Ser Ser Ile Leu Leu Ser Lys Ala Phe Asp Asn Gly Met Ile Cys Ala 245 250 255Ser Glu Gln Ala Val Ile Val Val Asp Glu Ile Tyr Asp Ala Leu Lys 260 265 270Ala Glu Phe Gln Arg Arg Gly Ala Tyr Leu Leu Ser Pro Glu Glu Arg 275 280 285Gln Gln Val Ala Gln Leu Leu Leu Lys Asp Gly Arg Leu Asn Ala Ala 290 295 300Ile Val Gly Gln Ser Ala Ala Thr Ile Ala Ala Met Ala Asn Ile Gln305 310 315 320Val Pro Pro Glu Thr Arg Val Leu Ile Gly Glu Val Ser Glu Val Gly 325 330 335Pro Gln Glu Pro Phe Ser Tyr Glu Lys Leu Cys Pro Val Leu Ala Leu 340 345 350Tyr Arg Ala Pro Gln Phe His Lys Gly Val Glu Ile Ala Ala Gln Leu 355 360 365Val Asn Phe Gly Gly Lys Gly His Thr Ser Val Leu Tyr Thr Asp Pro 370 375 380Arg Asn Gln Asp Asp Ile Ala Tyr Phe Lys Tyr Arg Met Gln Thr Ala385 390 395 400Arg Val Leu Ile Asn Thr Pro Ser Ser Gln Gly Ala Ile Gly Asp Leu 405 410 415Tyr Asn Phe Lys Leu Asp Pro Ser Leu Thr Leu Gly Cys Gly Thr Trp 420 425 430Gly Gly Asn Val Thr Ser Glu Asn Val Gly Pro Arg His Leu Leu Asn 435 440 445Ile Lys Thr Val Ser Asp Arg Arg Glu Asn Met Leu Trp Phe Arg Val 450 455 460Pro Pro Lys Ile Tyr Phe Lys Pro Gly Cys Leu Pro Ile Ala Leu Arg465 470 475 480Glu Leu Ala Gly Lys Lys Arg Ala Phe Leu Val Thr Asp Lys Pro Leu 485 490 495Phe Asp Leu Gly Ile Thr Glu Pro Ile Val His Thr Leu Glu Glu Leu 500 505 510Gly Ile Lys Tyr Asp Ile Phe His Glu Val Glu Pro Asp Pro Thr Leu 515 520 525Ser Thr Val Asn Arg Gly Leu Gly Leu Leu Arg Gln Tyr Gln Pro Asp 530 535 540Val Ile Val Ala Val Gly Gly Gly Ser Pro Met Asp Ala Ala Lys Val545 550 555 560Met Trp Leu Leu Tyr Glu His Pro Glu Val Glu Phe Asp Gly Leu Ala 565 570 575Met Arg Phe Met Asp Ile Arg Lys Arg Val Tyr Gln Leu Pro Pro Leu 580 585 590Gly Gln Lys Ala Ile Leu Val Ala Ile Pro Thr Thr Ser Gly Thr Gly 595 600 605Ser Glu Val Thr Pro Phe Ala Val Val Thr Asp Asp Arg Val Gly Ile 610 615 620Lys Tyr Pro Leu Ala Asp Tyr Ala Leu Thr Pro Thr Met Ala Ile Val625 630 635 640Asp Pro Asp Leu Val Leu His Met Pro Lys Lys Leu Thr Ala Tyr Gly 645 650 655Gly Ile Asp Ala Leu Thr His Ala Leu Glu Ala Tyr Val Ser Val Leu 660 665 670Ser Thr Glu Phe Thr Glu Gly Leu Ala Leu Glu Ala Ile Lys Leu Leu 675 680 685Phe Thr Tyr Leu Pro Arg Ala Tyr Arg Leu Gly Ala Ala Asp Pro Glu 690 695 700Ala Arg Glu Lys Val His Tyr Ala Ala Thr Ile Ala Gly Met Ala Phe705 710 715 720Ala Asn Ala Phe Leu Gly Val Cys His Ser Leu Ala His Lys Leu Gly 725 730 735Ser Thr Phe His Val Pro His Gly Leu Ala Asn Ala Leu Met Ile Ser 740 745 750His Val Ile Arg Tyr Asn Ala Thr Asp Ala Pro Leu Lys Gln Ala Ile 755 760 765Phe Pro Gln Tyr Lys Tyr Pro Gln Ala Lys Glu Arg Tyr Ala Gln Ile 770 775 780Ala Asp Phe Leu Glu Leu Gly Gly Thr Thr Pro Glu Glu Lys Val Glu785 790 795 800Arg Leu Ile Ala Ala Ile Glu Asp Leu Lys Ala Gln Leu Glu Ile Pro 805 810 815Ala Thr Ile Lys Glu Ala Leu Asn Ser Glu Asp Gln Ala Phe Tyr Glu 820 825 830Gln Val Glu Ser Met Ala Glu Leu Ala Phe Asp Asp Gln Cys Thr Gly 835 840 845Ala Asn Pro Arg Tyr Pro Leu Ile Gln Asp Leu Lys Glu Leu Tyr Ile 850 855 860Leu Ala Tyr Met Gly Cys Arg Arg Asp Ala Ala Ala Tyr Tyr Gly Gly865 870 875 880Glu Ala Thr Gly Ser 8851011680DNAZymobacter 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 Gly1 5 10 15Leu Lys His His Phe Ala Val Ala Gly Asp Tyr Asn Leu Val Leu Leu 20 25 30Asp Gln Leu Leu Leu Asn Lys Asp Met Glu Gln Val Tyr Cys Cys Asn 35 40 45Glu Leu Asn Cys Gly Phe Ser Ala Glu Gly Tyr Ala Arg Ala Arg Gly 50 55 60Ala Ala Ala Ala Ile Val Thr Phe Ser Val Gly Ala Ile Ser Ala Met65 70 75 80Asn Ala Ile Gly Gly Ala Tyr Ala Glu Asn Leu Pro Val Ile Leu Ile 85 90 95Ser Gly Ser Pro Asn Thr Asn Asp Tyr Gly Thr Gly His Ile Leu His 100 105 110His Thr Ile Gly Thr Thr Asp Tyr Asn Tyr Gln Leu Glu Met Val Lys 115 120 125His Val Thr Cys Ala Arg Glu Ser Ile Val Ser Ala Glu Glu Ala Pro 130 135 140Ala Lys Ile Asp His Val Ile Arg Thr Ala Leu Arg Glu Arg Lys Pro145 150 155 160Ala Tyr Leu Glu Ile Ala Cys Asn Val Ala Gly Ala Glu Cys Val Arg 165 170 175Pro Gly Pro Ile Asn Ser Leu Leu Arg Glu Leu Glu Val Asp Gln Thr 180 185 190Ser Val Thr Ala Ala Val Asp Ala Ala Val Glu Trp Leu Gln Asp Arg 195 200 205Gln Asn Val Val Met Leu Val Gly Ser Lys Leu Arg Ala Ala Ala Ala 210 215 220Glu Lys Gln Ala Val Ala Leu Ala Asp Arg Leu Gly Cys Ala Val Thr225 230 235 240Ile Met Ala Ala Glu Lys Gly Phe Phe Pro Glu Asp His Pro Asn Phe 245 250 255Arg Gly Leu Tyr Trp Gly Glu Val Ser Ser Glu Gly Ala Gln Glu Leu 260 265 270Val Glu Asn Ala Asp Ala Ile Leu Cys Leu Ala Pro Val Phe Asn Asp 275 280 285Tyr Ala Thr Val Gly Trp Asn Ser Trp Pro Lys Gly Asp Asn Val Met 290 295 300Val Met Asp Thr Asp Arg Val Thr Phe Ala Gly Gln Ser Phe Glu Gly305 310 315 320Leu Ser Leu Ser Thr Phe Ala Ala Ala Leu Ala Glu Lys Ala Pro Ser 325 330 335Arg Pro Ala Thr Thr Gln Gly Thr Gln Ala Pro Val Leu Gly Ile Glu 340 345 350Ala Ala Glu Pro Asn Ala Pro Leu Thr Asn Asp Glu Met Thr Arg Gln 355 360 365Ile Gln Ser Leu Ile Thr Ser Asp Thr Thr Leu Thr Ala Glu Thr Gly 370 375 380Asp Ser Trp Phe Asn Ala Ser Arg Met Pro Ile Pro Gly Gly Ala Arg385 390 395 400Val Glu Leu Glu Met Gln Trp Gly His Ile Gly Trp Ser Val Pro Ser 405 410 415Ala Phe Gly Asn Ala Val Gly Ser Pro Glu Arg Arg His Ile Met Met 420 425 430Val Gly Asp Gly Ser Phe Gln Leu Thr Ala Gln Glu Val Ala Gln Met 435 440 445Ile Arg Tyr Glu Ile Pro Val Ile Ile Phe Leu Ile Asn Asn Arg Gly 450 455 460Tyr Val Ile Glu Ile Ala Ile His Asp Gly Pro Tyr Asn Tyr Ile Lys465 470 475 480Asn Trp Asn Tyr Ala Gly Leu Ile Asp Val Phe Asn Asp Glu Asp Gly 485 490 495His Gly Leu Gly Leu Lys Ala Ser Thr Gly Ala Glu Leu Glu Gly Ala 500 505 510Ile Lys Lys Ala Leu Asp Asn Arg Arg Gly Pro Thr Leu Ile Glu Cys 515 520 525Asn Ile Ala Gln Asp Asp Cys Thr Glu Thr Leu Ile Ala Trp Gly Lys 530 535 540Arg Val Ala Ala Thr Asn Ser Arg Lys Pro Gln Ala545 550 5551035711DNAartificialpSK10 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 Pro1 5 10 15Phe Glu Tyr Asp Pro Gly Ala Leu Gly Ala Asn Glu Val Glu Ile Glu 20 25 30Val Gln Tyr Cys Gly Val Cys His Ser Asp Leu Ser Met Ile Asn Asn 35 40 45Glu Trp Gly Ile Ser Asn Tyr Pro Leu Val Pro Gly His Glu Val Val 50 55 60Gly Thr Val Ala Ala Met Gly Glu Gly Val Asn His Val Glu Val Gly65 70 75 80Asp Leu Val Gly Leu Gly Trp His Ser Gly Tyr Cys Met Thr Cys His 85 90 95Ser Cys Leu Ser Gly Tyr His Asn Leu Cys Ala Thr Ala Glu Ser Thr 100 105 110Ile Val Gly His Tyr Gly Gly Phe Gly Asp Arg Val Arg Ala Lys Gly 115 120 125Val Ser Val Val Lys Leu Pro Lys Gly Ile Asp Leu Ala Ser Ala Gly 130 135 140Pro Leu Phe Cys Gly Gly Ile Thr Val Phe Ser Pro Met Val Glu Leu145 150 155 160Ser Leu Lys Pro Thr Ala Lys Val Ala Val Ile Gly Ile Gly Gly Leu 165 170 175Gly His Leu Ala Val Gln Phe Leu Arg Ala Trp Gly Cys Glu Val Thr 180 185 190Ala Phe Thr Ser Ser Ala Arg Lys Gln Thr Glu Val Leu Glu Leu Gly 195 200 205Ala His His Ile Leu Asp Ser Thr Asn Pro Glu Ala Ile Ala Ser Ala 210 215 220Glu Gly Lys Phe Asp Tyr Ile Ile Ser Thr Val Asn Leu Lys Leu Asp225 230 235 240Trp Asn Leu Tyr Ile Ser Thr Leu Ala Pro Gln Gly His Phe His Phe 245 250 255Val Gly Val Val Leu Glu Pro Leu Asp Leu Asn Leu Phe Pro Leu Leu 260 265 270Met Gly Gln Arg Ser Val Ser Ala Ser Pro Val Gly Ser Pro Ala Thr 275 280 285Ile Ala Thr Met Leu Asp Phe Ala Val Arg His Asp Ile Lys Pro Val 290 295 300Val Glu Gln Phe Ser Phe Asp Gln Ile Asn Glu Ala Ile Ala His Leu305 310 315 320Glu Ser Gly Lys Ala His Tyr Arg Val Val Leu Ser His Ser Lys Asn 325 330 335109341PRTOceanobacter sp. RED65 109Met Ile Lys Ala Phe Ala Ala Asp Thr Ala Lys Gly Glu Leu Lys Pro1 5 10 15Phe Glu Tyr Glu Val Gly Glu Leu Gly Ser Gln Glu Val Glu Ile Glu 20 25 30Val His Tyr Cys Gly Val Cys His Ser Asp Ile Ser Met Leu Asp Asn 35 40 45Glu Trp Gly Met Thr Gln Tyr Pro Phe Val Pro Gly His Glu Val Ala 50 55 60Gly Leu Ile Lys Gln Val Gly Ala Glu Val Asn His Leu Lys Val Gly65 70 75 80Asp Arg Val Gly Leu Gly Trp Gln Ser Gly Tyr Cys Asn His Cys Glu 85 90 95Asn Cys Met Ser Gly Asp His Asn Leu Cys Gly Thr Ala Glu Met Thr 100 105 110Ile Val Gly Arg His Gly Gly Phe Ala Asp His Val Arg Ala Gln Ala 115 120 125Ser Ser Val Val Lys Leu Pro Asp Asp Ile His Met Ala Asp Ala Gly 130 135 140Pro Leu Phe Cys Gly Gly Val

Thr Val Tyr Asn Pro Met Lys Gln Phe145 150 155 160Asp Leu Lys Pro Thr Ala Lys Val Ala Val Ile Gly Ile Gly Gly Leu 165 170 175Gly His Met Ala Leu Gln Phe Leu Asn Ser Trp Gly Cys Glu Val Thr 180 185 190Ala Phe Thr Ser Thr Glu Glu Lys Arg Lys Glu Ala Ile Ala Leu Gly 195 200 205Ala His Lys Thr Leu Asn Ser Arg Asp Glu Gly Glu Leu Lys Gly Ala 210 215 220Ala Gly Ser Phe Asp Met Ile Ile Ser Thr Val Asn Val Ser Leu Asn225 230 235 240Trp Glu Ala Tyr Ile Asn Thr Leu Lys Ala Lys Gly Arg Leu His Phe 245 250 255Val Gly Ala Val Leu Glu Pro Ile Gln Val Gly Val Phe Pro Leu Met 260 265 270Met Gly Gln Arg Ser Ile Ser Ala Ser Pro Val Gly Ser Pro Ser Thr 275 280 285Ile Ser Gln Met Leu Glu Phe Thr Ala Arg His Gln Ile Lys Pro Gln 290 295 300Val Glu Leu Phe Gln Lys Asp Gln Ile Asn Asp Ala Ile Asn His Val305 310 315 320Arg Glu Gly Lys Ala Arg Tyr Arg Ala Val Ile Gln Phe Lys Ala Thr 325 330 335Ser Asp Asn Ser Ala 340110337PRTLimnobacter sp. MED105 110Met Glu Leu Ile Met Ile Asn Ala Tyr Ala Ala Phe Glu Ala Lys Gly1 5 10 15Pro Leu Lys Pro Phe Gln Tyr Asp Pro Gly Glu Leu Asn Ala Phe Asp 20 25 30Ile Glu Ile Asp Val Asp His Cys Gly Ile Cys His Ser Asp Val Ser 35 40 45Met Leu Asp Asn Asp Trp Gly Arg Ala Lys Tyr Pro Met Val Ala Gly 50 55 60His Glu Ile Ile Gly Arg Val Ser Gln Val Gly Ser His Val Ser His65 70 75 80Leu Ala Ile Gly Asp Val Val Gly Leu Gly Trp His Ser Gly Tyr Cys 85 90 95Glu Ser Cys Arg Met Cys Met Gly Gly Asp His Asn Leu Cys Ser Thr 100 105 110Ala Lys Gly Thr Ile Val Gly Arg His Gly Gly Phe Ala Asp Lys Val 115 120 125Arg Ala Gln Ala Val Ser Ala Val Lys Ile Pro Ala Gly Val Asn Pro 130 135 140Ala Thr Ala Gly Pro Leu Leu Cys Gly Gly Ile Thr Val Tyr Asn Pro145 150 155 160Leu Val Gln Phe Asn Ile Ser Pro Gln Ser Lys Val Ala Val Ile Gly 165 170 175Val Gly Gly Leu Gly His Met Ala Val Met Phe Leu Lys Ala Trp Gly 180 185 190Cys Glu Val Thr Ala Phe Ser Ser Asn Val Ser Lys Thr Asp Glu Leu 195 200 205Leu Gly Met Gly Ala His His Val Leu Asn Ser Lys Asp Pro Asp Ala 210 215 220Leu Lys Lys Ala Ala Gly Ser Phe Asp Leu Ile Leu Ser Thr Val Asn225 230 235 240Val Lys Leu Asp Trp Asn Ala Tyr Ile Gly Thr Leu Ala Pro Lys Gly 245 250 255Arg Leu His Phe Leu Gly Ala Val Leu Glu Pro Leu Asp Ile Gly Val 260 265 270Phe Gly Leu Met Gly Gln Gln Arg Ser Ile Ser Ser Ser Pro Val Gly 275 280 285Ser Pro Arg Val Ile Ala Asp Met Leu Lys Phe Ala Ala Leu His Asn 290 295 300Ile Gln Pro Ile Val Glu Thr Tyr Ser Phe Asp Gln Ile Asn Glu Ala305 310 315 320Val Asp Lys Val Arg Asn Gly Ser Pro Arg Phe Arg Val Val Leu Ser 325 330 335Arg111333PRTPsychrobacter cryohalolentis K5 111Met Ile Asn Ala Tyr Ala Ala Lys Glu Lys Gly Gly Glu Phe Val Pro1 5 10 15Tyr Gln Tyr Asp Pro Gly Thr Leu Gly Asp His Glu Val Glu Ile Glu 20 25 30Val His Ser Cys Gly Ile Cys His Ser Asp Leu Ser Met Trp Gln Asn 35 40 45Glu Trp Gly Met Thr Gln Tyr Pro Phe Val Gly Gly His Glu Val Ala 50 55 60Gly Lys Val Leu Ala Lys Gly Lys His Val Lys His Leu Glu Leu Gly65 70 75 80Asp Lys Val Gly Leu Gly Trp His Lys Gly Tyr Cys Asn Val Cys Asp 85 90 95Leu Cys Ile Gly Gly Asp His Asn Leu Cys Pro Glu Gln Glu Gly Thr 100 105 110Ile Ile Gly Asn His Gly Gly Phe Ala Asp Lys Val Arg Ala Lys Asp 115 120 125Thr Ser Val Ile Lys Ile Pro Glu Gly Leu Asp Phe Asn Ala Val Gly 130 135 140Pro Leu Leu Cys Gly Gly Val Thr Val Phe Asn Pro Leu Met Gln Tyr145 150 155 160Asp Ile Thr Pro Thr Ser Arg Val Ala Val Ile Gly Ile Gly Gly Leu 165 170 175Gly His Leu Ala Leu Gln Phe Ala Asn Ala Trp Gly Cys Glu Val Thr 180 185 190Ala Phe Thr Ser Glu Ser Lys Met Glu Glu Ala Lys Glu Met Gly Ala 195 200 205His His Ser Leu Asn Ser Arg Glu Asp Ser Glu Ile Glu Lys Ala Ala 210 215 220Gly Ser Phe Asp Leu Ile Ile Ser Thr Val Asn Val Asp Met Asn Trp225 230 235 240Asp Val Val Ile Lys Thr Leu Arg Pro Lys Gly Lys Leu His Phe Val 245 250 255Gly Leu Leu Glu Ala Pro Leu Glu Ile Ser Ala Ala Pro Met Ile Met 260 265 270Ala Gln Asn Ser Leu Ser Gly Ser Pro Val Gly Ser Pro Ser Thr Leu 275 280 285Arg Lys Met Leu Asp Phe Ala Ala Arg His Asn Ile Gln Pro Val Thr 290 295 300Glu Thr Tyr Lys Met Ser Glu Ile Asn Glu Ala Phe Glu Arg Leu Glu305 310 315 320Ser Gly Asn Ala Arg Tyr Arg Val Val Leu Glu Arg Asp 325 330112333PRTVerrucomicrobiae bacterium DG1235 112Met Ile Lys Ala Tyr Ala Thr His Thr Pro Gly Gly Lys Leu Glu Pro1 5 10 15Phe Glu Tyr Asp Pro Gly Glu Leu Ala Pro Thr Asp Val Glu Ile Asn 20 25 30Val Glu His Cys Gly Ile Cys His Ser Asp Leu Ser Met Leu Asn Asn 35 40 45Glu Trp Gly Met Thr Thr Tyr Pro Phe Val Pro Gly His Glu Val Val 50 55 60Gly Thr Ile Gly Ala Ile Gly Ser Asp Val Lys Asn Leu Ala Pro Gly65 70 75 80Gln Arg Val Gly Leu Gly Trp His Ser Ser Tyr Cys Thr Thr Cys Pro 85 90 95Ser Cys Leu Ser Gly Asp His Asn Leu Cys Gln Ala Ala Ala Gly Thr 100 105 110Ile Val Gly Arg His Gly Gly Phe Ala Asp Lys Val Arg Ala Ser Ala 115 120 125Leu Ser Val Ile Pro Leu Pro Asp Ser Ile Asp Ala Ala Lys Ala Gly 130 135 140Pro Leu Phe Cys Gly Gly Ile Thr Val Phe Asn Pro Leu Ile Gln Tyr145 150 155 160Glu Val Ser Pro Thr Ala Lys Val Ala Val Ile Gly Ile Gly Gly Leu 165 170 175Gly His Met Ala Leu Ala Phe Leu Asn Ala Trp Gly Cys Glu Val Thr 180 185 190Ala Phe Thr Thr Ser Glu Ala Lys Arg Gln Glu Ala Leu Lys Leu Gly 195 200 205Ala His His Thr Leu Asn Ser Arg Asp Ala Ala Glu Ile Glu Ala Ala 210 215 220Ala Gly Arg Phe Asp Leu Ile Leu Ser Thr Val Asn Val Gly Leu Asp225 230 235 240Trp Asn Gly Tyr Val Asn Thr Leu Lys Pro Lys Gly Arg Leu His Phe 245 250 255Leu Gly Ala Ala Leu Glu Pro Ile Gln Ile Gly Ala Phe Ser Leu Ile 260 265 270Met Ala Gln Arg Gln Ile Ser Gly Ser Pro Val Gly Ser Pro Ala Thr 275 280 285Ile Ala Lys Met Ile Glu Phe Ala Ala Leu His Lys Ile Glu Pro Val 290 295 300Thr Glu His Phe Lys Phe Asp Gln Ala Asn Glu Ala Leu Ala His Leu305 310 315 320Glu Ser Gly Gln Ala Arg Tyr Arg Ile Val Leu Ser His 325 330113334PRTSaccharophagus degradans 2-40 113Met Ile Lys Ala Tyr Ala Ala Met Glu Pro Gly Ala Ala Leu Val Pro1 5 10 15Phe Glu Tyr Glu Pro Gly Pro Leu Ala Asn Asn Glu Val Glu Leu Lys 20 25 30Val Glu Ser Cys Gly Ile Cys His Ser Asp Leu Ser Met Leu Asp Asn 35 40 45Glu Trp Gly Phe Thr Gln Tyr Pro Phe Val Gly Gly His Glu Val Ile 50 55 60Gly Ile Val Glu Ala Val Gly Ser Ser Val Asn Asn Val Ala Val Gly65 70 75 80Gln Arg Val Gly Leu Gly Trp His Ser Gly Tyr Cys Asn Thr Cys Ala 85 90 95Ser Cys Gln Ser Gly Asp Gln Asn Leu Cys Asn Ser Ala Gln Pro Thr 100 105 110Ile Ala Gly His His Gly Gly Phe Ala Asp Lys Val Arg Ala Asp Ala 115 120 125Asn Ala Val Val Ala Leu Pro Glu Gly Val Asn Pro Asp Ser Ala Gly 130 135 140Pro Leu Phe Cys Gly Gly Ile Thr Val Phe Asn Pro Leu Val Gln Phe145 150 155 160Gly Ile Lys Pro Thr Ser Lys Val Gly Val Ile Gly Ile Gly Gly Leu 165 170 175Gly His Ile Ala Leu Gln Phe Leu Asn Ala Trp Gly Cys Glu Val Thr 180 185 190Ala Phe Thr Ser Ser Glu Ser Lys Lys Glu Glu Ala Leu Lys Leu Gly 195 200 205Ala His His Val Leu Asn Ser Ser Asp Ala Ala Gln Leu Glu Ala Ala 210 215 220Ala Gly Arg Phe Asp Phe Ile Ile Ser Thr Val Asn Val Lys Leu Asp225 230 235 240Trp Asn Glu Tyr Leu Ala Thr Leu Ala Pro Lys Gly Arg Leu His Phe 245 250 255Val Gly Ala Thr Leu Ala Pro Leu Asp Ile Asn Val Phe Gln Leu Ile 260 265 270Gly Ser Gln Arg Glu Ile Ser Gly Ser Pro Val Gly Ser Pro Gly Thr 275 280 285Ile Ser Gln Met Leu Asp Phe Ala Ala Leu His Asn Ile Gln Pro Val 290 295 300Thr Glu Tyr Phe Arg Phe Asp Gln Ile Asn Glu Ala Leu Thr Lys Leu305 310 315 320Arg Glu Gly Lys Ala His Tyr Arg Ile Val Leu Thr Asn Lys 325 330114333PRTAlteromonas macleodii 'Deep ecotype' 114Met Ile Tyr Ala Tyr Ala Ala Lys Glu Ala Gly Gly Lys Leu Glu Lys1 5 10 15Phe Glu Tyr Asp Pro Gly Glu Leu Gly Ala His Asp Val Glu Ile Asp 20 25 30Val Glu Ser Cys Gly Ile Cys His Ser Asp Leu Ser Met Leu Asp Asn 35 40 45Glu Trp Gly Ile Thr Glu Phe Pro Phe Val Pro Gly His Glu Val Val 50 55 60Gly Thr Val Ser Lys Ile Gly Asp His Val Thr Ser Leu Lys Val Gly65 70 75 80Gln Arg Val Gly Leu Gly Trp His Ala Ser Tyr Cys Asn Ser Cys Arg 85 90 95Thr Cys Glu Ala Gly Asp His Asn Leu Cys Ala Gly Ala Thr Met Thr 100 105 110Ile Gly Gly Arg His Gly Gly Phe Ala Asp Lys Val Arg Ala Gln Ala 115 120 125Arg Ala Val Ile Pro Leu Pro Glu Ser Ile Asp Ser Thr Lys Ala Gly 130 135 140Pro Leu Phe Cys Gly Gly Ile Thr Val Phe Asn Pro Leu Val Gln Phe145 150 155 160Asn Ile Ser Pro Thr Ser Glu Val Gly Val Val Gly Ile Gly Gly Leu 165 170 175Gly His Leu Ala Leu Gln Phe Leu Asn Ala Trp Gly Cys Lys Val Val 180 185 190Ala Phe Thr Ser Ser Glu Ser Lys Glu Lys Glu Ala Leu Ser Leu Gly 195 200 205Ala Ser Glu Thr Ile Asn Ser Arg Asp Glu Asp Glu Ile Lys Lys Ala 210 215 220Gln Gly Arg Phe Asp Leu Ile Ile Ser Thr Val Asn Val Lys Leu Asp225 230 235 240Trp Asn Leu Tyr Leu Ser Thr Leu Ala Pro Lys Gly Arg Leu His Phe 245 250 255Val Gly Ala Thr Leu Glu Pro Leu Asp Ile Gly Ala Phe Asn Leu Ile 260 265 270Gly Gly Gln Lys Ser Val Ser Gly Ser Pro Val Gly Ser Pro Ala Thr 275 280 285Ile Lys Thr Met Leu Asp Phe Ala Ala His His Asp Ile Glu Pro Val 290 295 300Thr Glu Thr Phe Lys Phe Glu Asp Val Asn Lys Ala Ile Asp Arg Leu305 310 315 320Arg Glu Gly Lys Ala His Tyr Arg Ile Val Leu Thr Arg 325 330115332PRTAcaryochloris marina MBIC11017 115Met Val Asn Ala Tyr Ala Ala Phe Glu Gln Gly Gly Val Leu Gln Pro1 5 10 15Phe Glu Tyr Asp Pro Gly Pro Leu Gly Arg Gln Gln Val Asp Ile Gln 20 25 30Val Glu Tyr Cys Gly Ile Cys His Ser Asp Leu Ser Met Ile Lys Asn 35 40 45Glu Trp Gly Met Thr Gln Tyr Pro Phe Val Pro Gly His Glu Ile Val 50 55 60Gly Ile Val Ala Glu Ile Gly Ser Glu Val Thr Thr Leu Arg Val Gly65 70 75 80Gln Arg Val Gly Leu Gly Trp Tyr Ser Ser Ser Cys Met His Cys Glu 85 90 95Trp Cys Met Gly Gly Asp His His Leu Cys Leu Ser Ala Glu Gly Thr 100 105 110Ile Val Gly Arg Pro Gly Gly Phe Ala Asp Gln Val Arg Ala Asp Gln 115 120 125Ser Trp Ile Val Pro Ile Pro Glu Ser Ile Asp Ser Ala Val Ala Gly 130 135 140Pro Leu Phe Cys Ala Gly Ile Thr Val Phe Gln Pro Ile Ile Gln Cys145 150 155 160Gly Val Gln Pro Thr Asp Arg Val Ala Val Ile Gly Ile Gly Gly Leu 165 170 175Gly His Leu Ala Leu Gln Phe Leu Asn Ala Trp Gly Cys Glu Val Thr 180 185 190Ala Leu Ser Thr Gln Pro Asp Lys Glu Ala Glu Ala Arg Arg Leu Gly 195 200 205Ala His His Phe Val Asn Thr Arg Asp Pro Ala Ala Leu Gln Ala Ile 210 215 220Ala Asn Ser Cys Asp Tyr Ile Ile Ser Thr Val Asn Val Ser Leu Glu225 230 235 240Trp Ser Ile Tyr Leu Asn Ala Leu Arg Pro Lys Gly Arg Leu His Leu 245 250 255Val Gly Val Ala Pro Asp Leu Ser Leu Pro Val Phe Pro Leu Leu Ala 260 265 270Gly Gln Arg Ser Ile Ser Gly Ser Pro Val Gly Ser Pro Ala Thr Ile 275 280 285Thr Lys Met Leu Asn Phe Val Ala Arg His Gly Leu Ala Pro Gln Thr 290 295 300Glu Val Phe Pro Leu Ala Gln Val Asn Glu Ala Leu Glu Lys Leu Arg305 310 315 320Ser Gln His Pro Pro Tyr Arg Leu Ala Leu Lys Cys 325 330116335PRTCyanothece sp. PCC 7424 116Met Ile Arg Ala Tyr Ala Ala His Glu Pro Gly Gly Lys Leu Glu Pro1 5 10 15Phe Glu Tyr Glu Pro Gly Ser Leu Gly Asp Glu Glu Val Asp Ile Lys 20 25 30Val Glu Tyr Cys Gly Ile Cys His Ser Asp Leu Ser Met Leu Lys Asn 35 40 45Asp Trp Gly Met Thr Gln Tyr Pro Phe Val Pro Gly His Glu Val Val 50 55 60Gly Val Val Glu Ala Val Gly Ser Lys Val Lys Asn Leu Gln Ile Gly65 70 75 80Gln Lys Val Gly Leu Gly Trp Tyr Ser Arg Ser Cys Met Thr Cys Glu 85 90 95Phe Cys Met Ser Gly Asn His Asn Leu Cys Gln Asp Ala Glu Gly Thr 100 105 110Ile Val Gly Arg Tyr Gly Gly Phe Ala Glu Lys Val Arg Ala His Gln 115 120 125Gly Trp Val Ile Pro Leu Pro Glu Gly Val Asn Pro Val Thr Ala Gly 130 135 140Pro Leu Phe Cys Gly Gly Ile Thr Val Phe Asn Pro Ile Val Gln Phe145 150 155 160Asn Ile Lys Pro Thr Asp Gln Val Gly Val Ile Gly Ile Gly Gly Leu 165 170 175Gly His Met Ala Leu Gly Phe Leu Arg Ala Trp Gly Cys Glu Ile Thr 180 185 190Ala Phe Ser Thr Ser Pro Asp Lys Glu Ala Glu Ala Lys Ala Leu Gly 195 200 205Ala Thr His Phe Val Asn Ser Arg Asp Pro Glu Ala Leu Lys Ala Leu 210 215 220Thr Asn Ser Phe Asp Val Ile Leu Ser Thr Val Asn Ala Asp Leu Asp225 230 235

240Trp Pro Thr Tyr Ile Lys Leu Leu Arg Pro Gln Gly Arg Leu His Leu 245 250 255Val Gly Val Ile Pro Asn Pro Leu Ser Val Pro Ile Phe Pro Met Ile 260 265 270Leu Gly Gln Lys Ser Val Ser Ala Ser Pro Leu Gly Ser Pro Thr Thr 275 280 285Ile Ala Gln Met Leu Asn Phe Ala Gly Arg His His Leu Glu Pro Ile 290 295 300Val Glu Phe Phe Pro Leu Glu Gln Val Asn Glu Ala Leu Glu Arg Leu305 310 315 320Gln Ser Asn Lys Ala Arg Tyr Arg Ile Ile Leu Lys Met Asp His 325 330 335117335PRTCyanothece sp. PCC 7424 117Met Ile Arg Ala Tyr Ala Ala His Glu Pro Gly Gly Lys Leu Glu Pro1 5 10 15Phe Glu Tyr Glu Pro Gly Ser Leu Gly Asp Glu Glu Val Asp Ile Lys 20 25 30Val Glu Tyr Cys Gly Ile Cys His Ser Asp Leu Ser Met Leu Lys Asn 35 40 45Asp Trp Gly Met Thr Gln Tyr Pro Phe Val Pro Gly His Glu Val Val 50 55 60Gly Val Val Glu Ala Val Gly Ser Lys Val Lys Asn Leu Gln Ile Gly65 70 75 80Gln Lys Val Gly Leu Gly Trp Tyr Ser Arg Ser Cys Met Thr Cys Glu 85 90 95Phe Cys Met Ser Gly Asn His Asn Leu Cys Gln Asp Ala Glu Gly Thr 100 105 110Ile Val Gly Arg Tyr Gly Gly Phe Ala Glu Lys Val Arg Ala His Gln 115 120 125Gly Trp Val Ile Pro Leu Pro Glu Gly Val Asn Pro Val Thr Ala Gly 130 135 140Pro Leu Phe Cys Gly Gly Ile Thr Val Phe Asn Pro Ile Val Gln Phe145 150 155 160Asn Ile Lys Pro Thr Asp Gln Val Gly Val Ile Gly Ile Gly Gly Leu 165 170 175Gly His Met Ala Leu Gly Phe Leu Arg Ala Trp Gly Cys Glu Ile Thr 180 185 190Ala Phe Ser Thr Ser Pro Asp Lys Glu Ala Glu Ala Lys Ala Leu Gly 195 200 205Ala Thr His Phe Val Asn Ser Arg Asp Pro Glu Ala Leu Lys Ala Leu 210 215 220Thr Asn Ser Phe Asp Val Ile Leu Ser Thr Val Asn Ala Asp Leu Asp225 230 235 240Trp Pro Thr Tyr Ile Lys Leu Leu Arg Pro Gln Gly Arg Leu His Leu 245 250 255Val Gly Val Ile Pro Asn Pro Leu Ser Val Pro Ile Phe Pro Met Ile 260 265 270Leu Gly Gln Lys Ser Val Ser Ala Ser Pro Leu Gly Ser Pro Thr Thr 275 280 285Ile Ala Gln Met Leu Asn Phe Ala Gly Arg His His Leu Glu Pro Ile 290 295 300Val Glu Phe Phe Pro Leu Glu Gln Val Asn Glu Ala Leu Glu Arg Leu305 310 315 320Gln Ser Asn Lys Ala Arg Tyr Arg Ile Ile Leu Lys Met Asp His 325 330 335118336PRTCyanothece sp. PCC 7822 118Met Ile Arg Ala Tyr Ala Ala His Glu Pro Gly Gly Lys Leu Glu Pro1 5 10 15Phe Glu Tyr Asp Pro Gly Ser Leu Gly Asp Glu Asp Val Glu Ile Gln 20 25 30Val Glu Tyr Cys Gly Ile Cys His Ser Asp Leu Ser Met Leu Asn Asn 35 40 45Glu Trp Gly Met Thr Arg Tyr Pro Phe Val Pro Gly His Glu Val Val 50 55 60Gly Thr Ile Asn Ala Val Gly Glu Arg Val Lys His Leu Gln Val Gly65 70 75 80Gln Arg Val Gly Leu Gly Trp Tyr Ser Arg Ser Cys Met Thr Cys Glu 85 90 95Trp Cys Leu Ser Gly Asn Gln Asn Leu Cys Pro Gln Ala Glu Gly Thr 100 105 110Ile Val Gly Arg Tyr Gly Gly Phe Ala Glu Lys Val Arg Ala His Gln 115 120 125Gly Trp Val Leu Pro Leu Pro Glu Lys Leu Asn Pro Leu Thr Ala Gly 130 135 140Pro Leu Phe Cys Gly Gly Ile Thr Val Phe Asn Pro Ile Val Gln Phe145 150 155 160Asp Val Lys Pro Thr Asp Arg Val Gly Val Ile Gly Ile Gly Gly Leu 165 170 175Gly His Met Ala Leu Gly Phe Leu Ala Ala Trp Gly Cys Glu Ile Thr 180 185 190Ala Phe Ser Thr Ser Pro Asp Lys Glu Ile Glu Ala Lys Asn Leu Gly 195 200 205Ala Asn His Phe Val Asn Ser Arg Asp Pro Gln Ala Leu Lys Ala Leu 210 215 220Ala Asn Ser Leu Asp Leu Ile Leu Ser Thr Val Asn Ala Asp Leu Asp225 230 235 240Trp Asp Thr Tyr Ile Ser Leu Leu Arg Pro Lys Gly Arg Leu His Phe 245 250 255Val Gly Val Ile Pro Asn Pro Leu Ser Val Gln Leu Phe Pro Leu Ile 260 265 270Gly Gly Gln Lys Ser Val Ser Gly Ser Pro Leu Gly Ser Pro Val Thr 275 280 285Leu Ala Gln Met Leu Asn Phe Ala Gly Arg His His Val Glu Pro Val 290 295 300Val Glu Phe Tyr Pro Ile Glu Gln Val Asn Glu Ala Met Glu Arg Leu305 310 315 320Lys Ala Asn Lys Ala Arg Tyr Arg Ile Val Leu Thr Phe Lys Asn Ser 325 330 335119333PRTCyanothece sp. PCC 8801 119Met Ile Lys Ala Tyr Ala Ala Ser Glu Pro Gly Lys Glu Leu Asn Ser1 5 10 15Phe Glu Tyr Asp Pro Gly Leu Leu Gly Glu Glu Asp Val Glu Ile Asn 20 25 30Val Gln Tyr Cys Gly Ile Cys His Ser Asp Leu Ser Met Leu Asp Asn 35 40 45Glu Trp Gly Ile Thr Gln Tyr Pro Phe Val Pro Gly His Glu Val Val 50 55 60Gly Thr Ile Gly Ala Val Gly Ser Lys Val Thr Thr Phe Gln Val Gly65 70 75 80Gln Thr Val Gly Leu Gly Trp Phe Ser Arg Ser Cys Phe Asp Cys Glu 85 90 95Trp Cys Leu Ser Gly Asp Gln Asn Leu Cys Gln Thr Ala Glu Gly Thr 100 105 110Ile Val Gly Arg Pro Gly Gly Phe Ala Asp Lys Val Arg Ala His His 115 120 125Arg Trp Val Val Pro Leu Pro Ser Gly Val Asn Pro Glu Thr Ala Gly 130 135 140Pro Leu Phe Cys Gly Gly Ile Thr Val Phe Asn Pro Ile Ile Gln Cys145 150 155 160Gly Val Lys Ser Thr Asp Arg Val Gly Val Ile Gly Ile Gly Gly Leu 165 170 175Gly His Leu Ala Ile Glu Phe Leu His Ala Trp Gly Cys Glu Val Thr 180 185 190Ala Phe Ser Ser Asn Pro Glu Lys Glu Ser Glu Val Lys Gln Leu Gly 195 200 205Ala Asp Tyr Phe Val Asn Ser Arg Asp Pro Glu Ala Ile Lys Ala Val 210 215 220Glu Asn Ser Phe Asp Phe Ile Ile Ser Thr Val Asn Val Ser Leu Asp225 230 235 240Trp Asn Ser Tyr Ile Leu Ala Leu Arg Pro Arg Gly Thr Leu His Phe 245 250 255Val Gly Ala Val Leu Asn Pro Ile Ser Thr Gln Ile Phe Pro Leu Leu 260 265 270Met Gly Gln Lys Thr Ile Ser Gly Ser Pro Thr Gly Ser Pro Thr Thr 275 280 285Ile Ala Gln Met Leu Asp Phe Ala Ala Arg His Gln Ile Glu Pro Val 290 295 300Thr Glu Ile Phe Pro Phe Glu Gln Val Asn Glu Ala Ile Asp Lys Leu305 310 315 320Arg His Gly Gln Pro Arg Tyr Arg Leu Val Leu Lys Met 325 330120362PRTCyanothece sp. PCC 8801 120Met Arg Gly Glu Arg Ile Val Arg Ser Gly Val Lys Glu Asp Ile Leu1 5 10 15Cys Asn Asn Ala Ile Asn Thr Thr Ile Glu Val Lys Val Val Ile Lys 20 25 30Ala Tyr Ala Ala Ser Glu Pro Gly Lys Glu Leu Asn Ser Phe Glu Tyr 35 40 45Asp Pro Gly Leu Leu Gly Glu Glu Asp Val Glu Ile Asn Val Gln Tyr 50 55 60Cys Gly Ile Cys His Ser Asp Leu Ser Met Leu Asp Asn Glu Trp Gly65 70 75 80Ile Thr Gln Tyr Pro Phe Val Pro Gly His Glu Val Val Gly Thr Ile 85 90 95Gly Ala Val Gly Ser Lys Val Thr Thr Phe Gln Val Gly Gln Thr Val 100 105 110Gly Leu Gly Trp Phe Ser Arg Ser Cys Phe Asp Cys Glu Trp Cys Leu 115 120 125Ser Gly Asp Gln Asn Leu Cys Gln Thr Ala Glu Gly Thr Ile Val Gly 130 135 140Arg Pro Gly Gly Phe Ala Asp Lys Val Arg Ala His His Arg Trp Val145 150 155 160Val Pro Leu Pro Ser Gly Val Asn Pro Glu Thr Ala Gly Pro Leu Phe 165 170 175Cys Gly Gly Ile Thr Val Phe Asn Pro Ile Ile Gln Cys Gly Val Lys 180 185 190Ser Thr Asp Arg Val Gly Val Ile Gly Ile Gly Gly Leu Gly His Leu 195 200 205Ala Ile Glu Phe Leu His Ala Trp Gly Cys Glu Val Thr Ala Phe Ser 210 215 220Ser Asn Pro Glu Lys Glu Ser Glu Val Lys Gln Leu Gly Ala Asp Tyr225 230 235 240Phe Val Asn Ser Arg Asp Pro Glu Ala Ile Lys Ala Val Glu Asn Ser 245 250 255Phe Asp Phe Ile Ile Ser Thr Val Asn Val Ser Leu Asp Trp Asn Ser 260 265 270Tyr Ile Leu Ala Leu Arg Pro Arg Gly Thr Leu His Phe Val Gly Ala 275 280 285Val Leu Asn Pro Ile Ser Thr Gln Ile Phe Pro Leu Leu Met Gly Gln 290 295 300Lys Thr Ile Ser Gly Ser Pro Thr Gly Ser Pro Thr Thr Ile Ala Gln305 310 315 320Met Leu Asp Phe Ala Ala Arg His Gln Ile Glu Pro Val Thr Glu Ile 325 330 335Phe Pro Phe Glu Gln Val Asn Glu Ala Ile Asp Lys Leu Arg His Gly 340 345 350Gln Pro Arg Tyr Arg Leu Val Leu Lys Met 355 360121362PRTCyanothece sp. PCC 8802 121Met Arg Gly Glu Arg Ile Val Arg Ser Gly Val Lys Glu Asp Ile Leu1 5 10 15Cys Asn Asn Ala Ile Asn Thr Thr Ile Glu Val Lys Val Val Ile Lys 20 25 30Ala Tyr Ala Ala Ser Glu Pro Gly Lys Glu Leu Asn Ser Phe Glu Tyr 35 40 45Asp Pro Gly Leu Leu Gly Glu Glu Asp Val Glu Ile Asn Val Gln Tyr 50 55 60Cys Gly Ile Cys His Ser Asp Leu Ser Met Leu Asp Asn Glu Trp Gly65 70 75 80Ile Thr Gln Tyr Pro Phe Val Pro Gly His Glu Val Val Gly Thr Ile 85 90 95Gly Ala Val Gly Ser Lys Val Thr Thr Phe Gln Val Gly Gln Thr Val 100 105 110Gly Leu Gly Trp Phe Ser Arg Ser Cys Phe Asp Cys Glu Trp Cys Leu 115 120 125Ser Gly Asp Gln Asn Leu Cys Gln Thr Ala Glu Gly Thr Ile Val Gly 130 135 140Arg Pro Gly Gly Phe Ala Asp Lys Val Arg Ala His His Arg Trp Val145 150 155 160Val Pro Leu Pro Ser Gly Val Asn Pro Glu Thr Ala Gly Pro Leu Phe 165 170 175Cys Gly Gly Ile Thr Val Phe Asn Pro Ile Ile Gln Cys Gly Val Lys 180 185 190Ser Thr Asp Arg Val Gly Val Ile Gly Ile Gly Gly Leu Gly His Leu 195 200 205Ala Ile Glu Phe Leu His Ala Trp Gly Cys Glu Val Thr Ala Phe Ser 210 215 220Ser Asn Pro Glu Lys Glu Ser Glu Val Lys Gln Leu Gly Ala Asp Tyr225 230 235 240Phe Val Asn Ser Arg Asp Pro Glu Ala Ile Lys Ala Val Glu Asn Ser 245 250 255Phe Asp Phe Ile Ile Ser Thr Val Asn Val Ser Leu Asp Trp Asn Ser 260 265 270Tyr Ile Leu Ala Leu Arg Pro Arg Gly Thr Leu His Phe Val Gly Ala 275 280 285Val Leu Asn Pro Ile Ser Thr Gln Ile Phe Pro Leu Leu Met Gly Gln 290 295 300Lys Thr Ile Ser Gly Ser Pro Thr Gly Ser Pro Thr Thr Ile Ala Gln305 310 315 320Met Leu Asp Phe Ala Ala Arg His Gln Ile Glu Pro Val Thr Glu Ile 325 330 335Phe Pro Phe Glu Gln Val Asn Glu Ala Ile Asp Lys Leu Arg His Gly 340 345 350Gln Pro Arg Tyr Arg Leu Val Leu Lys Met 355 360122334PRTMicrocoleus chthonoplastes PCC 7420 122Met Ile Lys Ala Tyr Ala Ala His Glu Pro Gly Gly Gln Leu Gln Pro1 5 10 15Phe Glu Tyr Asp Pro Gly Thr Leu Gly Asp Glu Glu Val Glu Ile Lys 20 25 30Val Glu Tyr Cys Gly Ile Cys His Ser Asp Leu Ser Met Leu Asp Asn 35 40 45Glu Trp Gly Met Thr Asp Tyr Pro Phe Val Pro Gly His Glu Val Val 50 55 60Gly Thr Ile Ala Ala Leu Gly Asp Lys Val Thr Thr Leu Asn Leu Gly65 70 75 80Gln Arg Val Gly Leu Gly Trp Phe Ser Gly Ser Cys Met Thr Cys Glu 85 90 95Trp Cys Met Ser Gly Asn His Asn Leu Cys Ser Asn Ala Glu Gly Thr 100 105 110Ile Val Ser Arg His Gly Gly Phe Ala Asp Lys Val Arg Ala Asp Tyr 115 120 125Ser Trp Val Val Pro Leu Pro Asp Gly Ile Asn Pro Ala Thr Ala Gly 130 135 140Pro Leu Phe Cys Gly Gly Ile Thr Val Phe Asn Pro Ile Val Gln Phe145 150 155 160Asp Ile Lys Pro Ser Asp Arg Val Gly Val Ile Gly Ile Gly Gly Leu 165 170 175Gly His Ile Ala Leu Gly Phe Leu Gln Ala Trp Gly Cys Glu Ile Thr 180 185 190Ala Phe Ser Ser Ser Pro Asp Lys Glu Ala Glu Ala Arg Glu Leu Gly 195 200 205Ala Thr His Phe Ile Asn Ser Gly Asp Val Asn Ala Leu Glu Ser Val 210 215 220Gln Asn Ser Phe Asp Phe Ile Leu Ala Thr Ala Asn Ala Asp Leu Asp225 230 235 240Trp Asn Ala Tyr Ile Ala Ala Leu Arg Pro Lys Gly Arg Leu His Phe 245 250 255Val Gly Val Ile Pro Asn Pro Leu Ser Thr Pro Ile Phe Pro Leu Ile 260 265 270Leu Gly Gln Lys Ser Ile Ser Ala Ser Pro Val Gly Ser Pro Ala Thr 275 280 285Ile Ser Gln Met Ile Asn Phe Ala Ala Arg Gln Gly Val Glu Pro Ile 290 295 300Thr Glu Thr Phe Ser Phe Glu Gln Val Asn Glu Ala Met Glu Lys Leu305 310 315 320Arg His Gly Lys Pro Arg Tyr Arg Leu Val Leu Lys His Ser 325 330123334PRTMicrocystis aeruginosa NIES-843 123Met Ile Arg Ala Tyr Ala Ala Arg Glu Lys Gly Gly Lys Leu Glu Pro1 5 10 15Phe Asp Tyr Asp Pro Gly Ile Leu Ala Asp Glu Asp Val Glu Ile Ala 20 25 30Val Glu Tyr Cys Gly Ile Cys His Ser Asp Leu Ser Met Leu Asp Asn 35 40 45Asp Trp Gly Leu Thr Thr Tyr Pro Phe Val Pro Gly His Glu Val Val 50 55 60Gly Thr Ile Ala Ala Leu Gly Ala Lys Val Lys Glu Leu Lys Leu Gly65 70 75 80Gln Arg Val Gly Leu Gly Trp Phe Ser Arg Ser Cys Ser Thr Cys Glu 85 90 95Thr Cys Met Ser Gly Asp Gln Asn Leu Cys Ala Thr Ala Glu Gly Thr 100 105 110Ile Val Gly Arg His Gly Gly Phe Ala Asp Arg Val Arg Ala His His 115 120 125Ser Trp Leu Val Pro Leu Gly Asn Gln Leu Asp Ala Ala Lys Ala Gly 130 135 140Pro Leu Phe Cys Gly Gly Ile Thr Val Phe Asn Pro Ile Val Gln Phe145 150 155 160Asn Ile Lys Pro Thr Ala Arg Val Gly Val Ile Gly Ile Gly Gly Leu 165 170 175Gly His Ile Ala Leu Lys Phe Leu Lys Ala Trp Gly Cys Glu Val Thr 180 185 190Ala Phe Ser Ser Ser Pro Asp Lys Glu Thr Glu Ala Lys Glu Leu Gly 195 200 205Ala Thr His Phe Ile Asn Ser Arg Asp Pro Glu Ala Leu Gln Ser Val 210 215 220Gln Asn Tyr Phe Asp Phe Ile Ile Ser Thr Val Asn Val Asn Leu Asp225 230 235 240Trp Gly Leu Tyr Ile Ala Cys Leu Arg Pro Lys Gly Arg Leu His Ile 245 250 255Val Gly Ala Val Leu Glu Pro Met Ala Thr Tyr Ala Phe Pro Leu Ile 260 265 270Met Gly Gln Lys Ser Ile Ser Gly Ser Pro Leu Gly Ser Pro Ser Thr 275

280 285Ile Asn Lys Met Ile Glu Phe Ala Ser Arg His Gly Ile Glu Pro Val 290 295 300Thr Glu Ile Tyr Pro Ile Ser Gln Val Asn Glu Ala Met Glu Lys Leu305 310 315 320Arg Thr Gly Gln Pro Lys Tyr Arg Leu Val Leu Gln Ile Lys 325 330124334PRTMicrocystis aeruginosa PCC 7806 124Met Ile Arg Ala Tyr Ala Ala Gln Glu Lys Gly Gly Lys Leu Glu Pro1 5 10 15Phe Asp Tyr Asp Pro Gly Ile Leu Ala Asp Glu Asp Val Glu Ile Ala 20 25 30Val Glu Tyr Cys Gly Ile Cys His Ser Asp Leu Ser Met Leu Asp Asn 35 40 45Asp Trp Gly Leu Thr Thr Tyr Pro Phe Val Pro Gly His Glu Val Val 50 55 60Gly Thr Ile Ala Ala Leu Gly Ala Lys Val Lys Glu Leu Lys Leu Gly65 70 75 80Gln Arg Val Gly Leu Gly Trp Phe Ser Arg Ser Cys Ser Thr Cys Glu 85 90 95Thr Cys Met Ser Gly Asp Gln Asn Leu Cys Ala Thr Ala Glu Gly Thr 100 105 110Ile Val Gly Arg His Gly Gly Phe Ala Glu Arg Val Arg Ala His His 115 120 125Ser Trp Leu Val Pro Leu Pro Asp Gln Leu Asp Ala Ala Lys Ala Gly 130 135 140Pro Leu Phe Cys Gly Gly Ile Thr Val Phe Asn Pro Ile Val Gln Phe145 150 155 160Asn Ile Lys Pro Thr Ala Arg Val Gly Val Ile Gly Ile Gly Gly Leu 165 170 175Gly His Ile Ala Leu Lys Phe Leu Lys Ala Trp Gly Cys Glu Val Thr 180 185 190Ala Phe Ser Ser Ser Pro Asp Lys Glu Thr Glu Ala Lys Glu Leu Gly 195 200 205Ala Thr His Phe Ile Asn Ser Arg Asp Pro Glu Ala Leu Gln Ser Val 210 215 220Gln Asn Tyr Phe Asp Phe Ile Ile Ser Thr Val Asn Val Asn Leu Asp225 230 235 240Trp Gly Leu Tyr Ile Ala Cys Leu Arg Pro Lys Gly Arg Leu His Ile 245 250 255Val Gly Ala Val Leu Glu Pro Met Ala Thr Tyr Ala Phe Pro Leu Ile 260 265 270Met Gly Gln Lys Ser Ile Ser Gly Ser Pro Leu Gly Ser Pro Ser Thr 275 280 285Val Ser Lys Met Ile Glu Phe Ala Ser Arg His Gly Ile Glu Pro Val 290 295 300Thr Glu Thr Tyr Pro Ile Ser Arg Val Asn Glu Ala Met Glu Lys Leu305 310 315 320Arg Thr Gly Gln Pro Lys Tyr Arg Leu Val Leu Gln Ile Lys 325 330125333PRTSynechococcus sp. WH 5701 125Met Gln Ile Thr Val Trp Gln Ala Leu Ala Lys Gly Gly Arg Leu Glu1 5 10 15Arg Ser Gln Ala Thr Leu Leu Asp Pro Gly Pro Asp Glu Val Leu Leu 20 25 30Glu Val Leu His Cys Gly Leu Cys His Ser Asp Leu Ser Met Leu Asp 35 40 45Asn Ser Trp Gly Ile Ser Thr Tyr Pro Leu Val Pro Gly His Glu Val 50 55 60Val Gly Arg Val Ala Ala Val Gly Ala Gly Val Asp Ser Gly Leu Leu65 70 75 80Gly Ser Ile Gln Gly Leu Gly Trp Ile Ala Gly Ser Cys Arg His Cys 85 90 95Asp Trp Cys Leu Gly Gly Asn Ala Asn Leu Cys Pro Ser Leu Glu Ala 100 105 110Ser Val Val Gly Arg His Gly Gly Phe Ala Ser His Val Met Ala His 115 120 125Gln Asp Trp Ile Val Ala Ile Pro Asp Gly Val Ser Ala Ala Asp Ala 130 135 140Gly Pro Leu Phe Cys Gly Gly Ile Thr Val Phe Ala Pro Leu Phe Asp145 150 155 160Glu Ala Val Ser Pro Thr Ser Arg Val Ala Val Ile Gly Ile Gly Gly 165 170 175Leu Gly His Met Ala Leu Gln Phe Ala Arg Ala Trp Gly Cys Glu Val 180 185 190Thr Ala Val Thr Thr Ser Pro Ala Lys Ala Asp Glu Ala Arg Arg Leu 195 200 205Gly Ala His Arg Val Leu Ala Leu Ser Glu Leu Gly Asp His Pro Gly 210 215 220Val Phe Asp Leu Ile Ile Asn Thr Ser Asn His Asp Leu Asp Trp Pro225 230 235 240Ala Leu Ile Gly Ser Leu Ala Pro Leu Gly Arg Leu His Gln Leu Gly 245 250 255Val Pro Leu Ser Pro Leu Gln Ile Pro Ala Phe Pro Leu Ile Ala Gly 260 265 270Arg Arg Ser Val Thr Gly Ser Pro Thr Ser Ser Pro Ala Ser Leu Arg 275 280 285Arg Met Val Glu Phe Cys Ala Arg His Gly Ile Ala Pro Leu Val Glu 290 295 300His Leu Pro Met Ala Glu Ile Asn Thr Ala Ile Glu Arg Leu Arg Gln305 310 315 320Gly Asp Val Arg Tyr Arg Phe Val Leu Asp Gly Pro Ala 325 330126336PRTSynechococcus sp. RS9917 126Met Val Val Thr Ile Thr Val Trp Gln Ala Arg Glu Ala Gly Ala Pro1 5 10 15Leu Glu Arg Ala Glu Arg Ala Met Leu Glu Pro Ala Ala Gly Glu Leu 20 25 30Val Leu Glu Val Leu His Cys Gly Leu Cys His Ser Asp Leu Ser Met 35 40 45Leu Asp Asn Asn Trp Gly Leu Ser Ala Tyr Pro Leu Val Pro Gly His 50 55 60Glu Val Val Gly Arg Val Val Arg Val Gly Glu Gly Val Asp Pro Gly65 70 75 80Val Ile Gly Glu Leu Arg Gly Leu Gly Trp Ile Ser Gly Ser Cys Met 85 90 95His Cys Ala Leu Cys Leu Gly Gly Thr Ala Asn Leu Cys Gly Ser Leu 100 105 110Glu Ala Thr Ile Val Gly Arg Gln Gly Gly Phe Ala Ser His Val Thr 115 120 125Ala Arg Gln Asp Trp Ala Ile Arg Leu Pro Glu Gly Met Asp Pro Ala 130 135 140Ala Ala Gly Pro Leu Phe Cys Gly Gly Ile Thr Val Phe Ala Pro Leu145 150 155 160Val Asp Glu Val Val Ser Pro Thr Ala His Val Ala Val Ile Gly Ile 165 170 175Gly Gly Leu Gly His Met Ala Leu Gln Phe Ala Arg Ala Trp Gly Cys 180 185 190Glu Val Thr Ala Leu Thr Thr His Leu Ala Lys Ala Glu Glu Ala Lys 195 200 205Arg Phe Gly Ala His His Val Glu Ser Leu Glu Glu Leu Pro Asp Leu 210 215 220Ala Gly Arg Phe Asp Leu Val Ile Asn Thr Val Asn His Ala Leu Asp225 230 235 240Trp Gly Ala Val Met Gly Ser Leu Ala Pro Leu Gly Arg Leu His Gln 245 250 255Leu Gly Ala Val Leu Glu Pro Leu Gln Val Ser Ala Phe Asp Leu Ile 260 265 270Met Ala Arg Arg Ser Ile Thr Gly Ser Pro Thr Ser Ser Pro Ala Ser 275 280 285Leu Met Lys Met Val Glu Phe Cys Val Arg His Asn Ile Arg Pro Gln 290 295 300Val Glu His Leu Pro Met Asp Arg Leu Asn Glu Ala Ile Asp Arg Leu305 310 315 320Arg Arg Gly Asp Val Arg Tyr Arg Phe Val Leu Asp Ser Val Ala Asp 325 330 335127333PRTSynechococcus sp. WH 5701 127Met Gln Ile Thr Val Trp Gln Ala Leu Ala Lys Gly Gly Arg Leu Glu1 5 10 15Arg Ser Gln Ala Thr Leu Leu Asp Pro Gly Pro Asp Glu Val Leu Leu 20 25 30Glu Val Leu His Cys Gly Leu Cys His Ser Asp Leu Ser Met Leu Asp 35 40 45Asn Ser Trp Gly Ile Ser Thr Tyr Pro Leu Val Pro Gly His Glu Val 50 55 60Val Gly Arg Val Ala Ala Val Gly Ala Gly Val Asp Ser Gly Leu Leu65 70 75 80Gly Ser Ile Gln Gly Leu Gly Trp Ile Ala Gly Ser Cys Arg His Cys 85 90 95Asp Trp Cys Leu Gly Gly Asn Ala Asn Leu Cys Pro Ser Leu Glu Ala 100 105 110Ser Val Val Gly Arg His Gly Gly Phe Ala Ser His Val Met Ala His 115 120 125Gln Asp Trp Ile Val Ala Ile Pro Asp Gly Val Ser Ala Ala Asp Ala 130 135 140Gly Pro Leu Phe Cys Gly Gly Ile Thr Val Phe Ala Pro Leu Phe Asp145 150 155 160Glu Ala Val Ser Pro Thr Ser Arg Val Ala Val Ile Gly Ile Gly Gly 165 170 175Leu Gly His Met Ala Leu Gln Phe Ala Arg Ala Trp Gly Cys Glu Val 180 185 190Thr Ala Val Thr Thr Ser Pro Ala Lys Ala Asp Glu Ala Arg Arg Leu 195 200 205Gly Ala His Arg Val Leu Ala Leu Ser Glu Leu Gly Asp His Pro Gly 210 215 220Val Phe Asp Leu Ile Ile Asn Thr Ser Asn His Asp Leu Asp Trp Pro225 230 235 240Ala Leu Ile Gly Ser Leu Ala Pro Leu Gly Arg Leu His Gln Leu Gly 245 250 255Val Pro Leu Ser Pro Leu Gln Ile Pro Ala Phe Pro Leu Ile Ala Gly 260 265 270Arg Arg Ser Val Thr Gly Ser Pro Thr Ser Ser Pro Ala Ser Leu Arg 275 280 285Arg Met Val Glu Phe Cys Ala Arg His Gly Ile Ala Pro Leu Val Glu 290 295 300His Leu Pro Met Ala Glu Ile Asn Thr Ala Ile Glu Arg Leu Arg Gln305 310 315 320Gly Asp Val Arg Tyr Arg Phe Val Leu Asp Gly Pro Ala 325 330128330PRTSynechococcus sp. WH 7803 128Met Ile Ser Val Trp Gln Ala Pro Ser Ala Gly Ala Pro Leu Glu Cys1 5 10 15Gly Gln Arg Pro Ala Pro Glu Pro Ala Ala Asp Glu Leu Val Leu Glu 20 25 30Val Met His Cys Gly Leu Cys His Ser Asp Leu Ser Met Ile Gly Asn 35 40 45His Trp Gly Val Ser Arg Tyr Pro Leu Val Pro Gly His Glu Val Ile 50 55 60Gly Arg Val Thr Ala Val Gly Glu Gly Val Asp Pro Gly Leu Ile Gly65 70 75 80Asp Val Arg Gly Leu Gly Trp Ile Ser Gly Ser Cys Asn His Cys Ser 85 90 95Leu Cys Leu Gly Gly Asp Gln Asn Leu Cys Thr Ser Leu Glu Ala Thr 100 105 110Ile Val Gly Arg Gln Gly Gly Phe Ala Ser His Val Val Ala Arg Gln 115 120 125Asp Trp Ala Ile Pro Leu Pro Pro Gly Leu Asp Pro Ala Asp Ala Gly 130 135 140Pro Leu Phe Cys Gly Gly Ile Thr Val Phe Ala Pro Leu Val Asp Glu145 150 155 160Ala Val Ser Pro Thr Ala His Val Ala Val Val Gly Ile Gly Gly Leu 165 170 175Gly His Ile Ala Leu Gln Phe Ala Arg Ala Trp Gly Cys Glu Val Thr 180 185 190Ala Ile Thr Thr Asn Leu Ala Lys Ala Glu Gln Ala Arg Arg Phe Gly 195 200 205Ala His His Val Glu Glu Leu Glu Met Leu Pro Asp Leu Gln Ser Arg 210 215 220Phe Asp Leu Val Ile Asn Thr Val Asn His Pro Leu Asp Trp Ser Ala225 230 235 240Val Met Ala Ser Leu Arg Pro Arg Gly Arg Leu His Gln Leu Gly Ala 245 250 255Val Leu Glu Pro Ile Gln Val Gly Ala Phe Asp Leu Ile Pro Ala Arg 260 265 270Arg Ser Ile Thr Gly Ser Pro Thr Ser Ser Pro Ala Ser Leu Gln Lys 275 280 285Met Val Glu Phe Cys Val Arg His Asn Ile Leu Pro Leu Val Glu His 290 295 300Leu Pro Met Asp Gln Val Asn Val Ala Ile Gln Arg Leu Ala Lys Gly305 310 315 320Asp Val Arg Tyr Arg Phe Val Leu Asp Ala 325 330129330PRTSynechococcus sp. WH 7805 129Met Ile Ser Val Trp Gln Ala Pro Ser Ala Gly Ala Pro Leu Glu Cys1 5 10 15Ala Gln Arg Pro Ala Leu Gln Pro Val Ala Asp Glu Leu Val Leu Glu 20 25 30Val Met His Cys Gly Leu Cys His Ser Asp Leu Ser Met Ile Gly Asn 35 40 45His Trp Gly Val Ser Arg Tyr Pro Leu Val Pro Gly His Glu Val Ile 50 55 60Gly Arg Val Thr Ala Val Gly Glu Gly Val Asp Pro Gly Val Ile Gly65 70 75 80Glu Val Arg Gly Leu Gly Trp Ile Ser Gly Ser Cys Asn His Cys Ser 85 90 95Leu Cys Leu Gly Gly Asp Gln Asn Leu Cys Ser Ser Leu Glu Ala Thr 100 105 110Ile Val Gly Arg Gln Gly Gly Phe Ala Ser His Val Val Ala Arg Gln 115 120 125Asp Trp Thr Ile Pro Leu Pro Thr Gly Leu Asp Pro Ala Glu Ala Gly 130 135 140Pro Leu Phe Cys Gly Gly Val Thr Val Phe Ala Pro Leu Val Asp Glu145 150 155 160Ala Val Ser Pro Thr Ala His Val Ala Val Val Gly Ile Gly Gly Leu 165 170 175Gly His Ile Ala Leu Gln Phe Ala Arg Ala Trp Gly Cys Glu Val Thr 180 185 190Ala Ile Thr Thr Asn Pro Ala Lys Thr Glu Gln Ala Arg Arg Phe Gly 195 200 205Ala His His Val Glu Glu Leu Glu Ala Leu Ser Asp Leu Gln Arg Arg 210 215 220Phe Asp Leu Val Ile Asn Thr Val Asn His Pro Leu Asp Trp Ser Ala225 230 235 240Val Met Ala Ser Leu Lys Pro Arg Gly Arg Leu His Gln Leu Gly Ala 245 250 255Val Leu Glu Pro Ile Gln Val Gly Ala Phe Asp Leu Ile Ser Ala Arg 260 265 270Arg Ser Ile Thr Gly Ser Pro Thr Ser Ser Pro Ala Ser Leu Leu Lys 275 280 285Met Val Glu Phe Cys Val Arg His Asn Ile Leu Pro Leu Val Glu His 290 295 300Leu Pro Met Asp Gln Val Asn Val Ala Ile Glu Arg Leu Ala Lys Gly305 310 315 320Asp Val Arg Tyr Arg Phe Val Leu Asp Ala 325 3301301692DNASaccharomyces 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 Gln1 5 10 15Val Asn Val Asn Thr Val Phe Gly Leu Pro Gly Asp Phe Asn Leu Ser 20 25 30Leu Leu Asp Lys Ile Tyr Glu Val Glu Gly Met Arg Trp Ala Gly Asn 35 40 45Ala Asn Glu Leu Asn Ala Ala Tyr Ala Ala Asp Gly Tyr Ala Arg Ile 50 55 60Lys Gly Met Ser Cys Ile Ile Thr Thr Phe Gly Val Gly Glu Leu Ser65 70 75 80Ala Leu Asn Gly Ile Ala Gly Ser Tyr Ala Glu His Val Gly Val Leu 85 90 95His Val Val Gly Val Pro Ser Ile Ser Ala Gln Ala Lys Gln Leu Leu 100

105 110Leu His His Thr Leu Gly Asn Gly Asp Phe Thr Val Phe His Arg Met 115 120 125Ser Ala Asn Ile Ser Glu Thr Thr Ala Met Ile Thr Asp Ile Ala Thr 130 135 140Ala Pro Ala Glu Ile Asp Arg Cys Ile Arg Thr Thr Tyr Val Thr Gln145 150 155 160Arg Pro Val Tyr Leu Gly Leu Pro Ala Asn Leu Val Asp Leu Asn Val 165 170 175Pro Ala Lys Leu Leu Gln Thr Pro Ile Asp Met Ser Leu Lys Pro Asn 180 185 190Asp Ala Glu Ser Glu Lys Glu Val Ile Asp Thr Ile Leu Ala Leu Val 195 200 205Lys Asp Ala Lys Asn Pro Val Ile Leu Ala Asp Ala Cys Cys Ser Arg 210 215 220His Asp Val Lys Ala Glu Thr Lys Lys Leu Ile Asp Leu Thr Gln Phe225 230 235 240Pro Ala Phe Val Thr Pro Met Gly Lys Gly Ser Ile Asp Glu Gln His 245 250 255Pro Arg Tyr Gly Gly Val Tyr Val Gly Thr Leu Ser Lys Pro Glu Val 260 265 270Lys Glu Ala Val Glu Ser Ala Asp Leu Ile Leu Ser Val Gly Ala Leu 275 280 285Leu Ser Asp Phe Asn Thr Gly Ser Phe Ser Tyr Ser Tyr Lys Thr Lys 290 295 300Asn Ile Val Glu Phe His Ser Asp His Met Lys Ile Arg Asn Ala Thr305 310 315 320Phe Pro Gly Val Gln Met Lys Phe Val Leu Gln Lys Leu Leu Thr Thr 325 330 335Ile Ala Asp Ala Ala Lys Gly Tyr Lys Pro Val Ala Val Pro Ala Arg 340 345 350Thr Pro Ala Asn Ala Ala Val Pro Ala Ser Thr Pro Leu Lys Gln Glu 355 360 365Trp Met Trp Asn Gln Leu Gly Asn Phe Leu Gln Glu Gly Asp Val Val 370 375 380Ile Ala Glu Thr Gly Thr Ser Ala Phe Gly Ile Asn Gln Thr Thr Phe385 390 395 400Pro Asn Asn Thr Tyr Gly Ile Ser Gln Val Leu Trp Gly Ser Ile Gly 405 410 415Phe Thr Thr Gly Ala Thr Leu Gly Ala Ala Phe Ala Ala Glu Glu Ile 420 425 430Asp Pro Lys Lys Arg Val Ile Leu Phe Ile Gly Asp Gly Ser Leu Gln 435 440 445Leu Thr Val Gln Glu Ile Ser Thr Met Ile Arg Trp Gly Leu Lys Pro 450 455 460Tyr Leu Phe Val Leu Asn Asn Asp Gly Tyr Thr Ile Glu Lys Leu Ile465 470 475 480His Gly Pro Lys Ala Gln Tyr Asn Glu Ile Gln Gly Trp Asp His Leu 485 490 495Ser Leu Leu Pro Thr Phe Gly Ala Lys Asp Tyr Glu Thr His Arg Val 500 505 510Ala Thr Thr Gly Glu Trp Asp Lys Leu Thr Gln Asp Lys Ser Phe Asn 515 520 525Asp Asn Ser Lys Ile Arg Met Ile Glu Ile Met Leu Pro Val Phe Asp 530 535 540Ala Pro Gln Asn Leu Val Glu Gln Ala Lys Leu Thr Ala Ala Thr Asn545 550 555 560Ala Lys Gln1321047DNASaccharomyces 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 His1 5 10 15Gly Lys Leu Glu Tyr Lys Asp Ile Pro Val Pro Lys Pro Lys Ala Asn 20 25 30Glu Leu Leu Ile Asn Val Lys Tyr Ser Gly Val Cys His Thr Asp Leu 35 40 45His Ala Trp His Gly Asp Trp Pro Leu Pro Val Lys Leu Pro Leu Val 50 55 60Gly Gly His Glu Gly Ala Gly Val Val Val Gly Met Gly Glu Asn Val65 70 75 80Lys Gly Trp Lys Ile Gly Asp Tyr Ala Gly Ile Lys Trp Leu Asn Gly 85 90 95Ser Cys Met Ala Cys Glu Tyr Cys Glu Leu Gly Asn Glu Ser Asn Cys 100 105 110Pro His Ala Asp Leu Ser Gly Tyr Thr His Asp Gly Ser Phe Gln Gln 115 120 125Tyr Ala Thr Ala Asp Ala Val Gln Ala Ala His Ile Pro Gln Gly Thr 130 135 140Asp Leu Ala Gln Val Ala Pro Ile Leu Cys Ala Gly Ile Thr Val Tyr145 150 155 160Lys Ala Leu Lys Ser Ala Asn Leu Met Ala Gly His Trp Val Ala Ile 165 170 175Ser Gly Ala Ala Gly Gly Leu Gly Ser Leu Ala Val Gln Tyr Ala Lys 180 185 190Ala Met Gly Tyr Arg Val Leu Gly Ile Asp Gly Gly Glu Gly Lys Glu 195 200 205Glu Leu Phe Arg Ser Ile Gly Gly Glu Val Phe Ile Asp Phe Thr Lys 210 215 220Glu Lys Asp Ile Val Gly Ala Val Leu Lys Ala Thr Asp Gly Gly Ala225 230 235 240His Gly Val Ile Asn Val Ser Val Ser Glu Ala Ala Ile Glu Ala Ser 245 250 255Thr Arg Tyr Val Arg Ala Asn Gly Thr Thr Val Leu Val Gly Met Pro 260 265 270Ala Gly Ala Lys Cys Cys Ser Asp Val Phe Asn Gln Val Val Lys Ser 275 280 285Ile Ser Ile Val Gly Ser Tyr Val Gly Asn Arg Ala Asp Thr Arg Glu 290 295 300Ala Leu Asp Phe Phe Ala Arg Gly Leu Val Lys Ser Pro Ile Lys Val305 310 315 320Val Gly Leu Ser Thr Leu Pro Glu Ile Tyr Glu Lys Met Glu Lys Gly 325 330 335Gln Ile Val Gly Arg Tyr Val Val Asp Thr Ser Lys 340 345134232DNAChlamydomonas 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

Claims

1. 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, whereinthe promoter can be induced by nutrient starvation, oxidative stress, darkness, heat shock, cold shock, salt stress or stationary growth of the host cell.

2. The genetically modified host cell according to claim 1, whereinthe promoter is selected from a group of promoters consisting of:ntcA, nblA, isiA, petJ, petE, sigB, lrtA, htpG, ggpS, psaA, psbA2, hspA, clpB1, hliB and crhC, psbA.

3. The genetically modified host cell according to claim 1, wherein the promoter is a heterologous promoter.

4. The genetically modified host cell according to claim 1, wherein the promoter is an endogenous promoter.

5. The genetically modified host cell according to claim 1, wherein the promoter is synechocystis PpetJ.

6. The genetically modified host cell according to claim 1, wherein the promoter is Anabena PpetE.

7. The genetically modified host cell according to claim 1, wherein the promoter is a heterologous promoter and one overexpressed enzyme for the formation of ethanol is a gene encoding an alcohol dehydrogenase, which directly converts acetyl-CoA to ethanol, wherein the gene is under the transcriptional control of the heterologous promoter.

8. The genetically modified host cell according to claim 1, wherein the promoter is PpetJ and induction occurs at night time.

9. 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, anda gene encoding at least one overexpressed enzyme for the formation of ethanol under the transcriptional control of the promoter.

10. The construct according to claim 8, whereinthe 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 sequence into the host genome.

Images