Patent Publication Number: US-2015082691-A1

Title: Methods and Compositions for the Recombinant Biosynthesis of Fatty Acids and Esters

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application is a divisional of U.S. patent application Ser. No. 13/765,211, filed Feb. 12, 2013, which is a divisional of U.S. patent application Ser. No. 13/243,165, filed Sep. 23, 2011, which is a continuation of U.S. patent application Ser. No. 12/876,056, filed Sep. 3, 2010, which is a continuation-in-part of international application PCT/US/2009/035937, filed Mar. 3, 2009, which claims the benefit of earlier filed U.S. Provisional Patent Application No. 61/121,532, filed Dec. 10, 2008, U.S. Provisional Patent Application No. 61/033,411 filed Mar. 3, 2008, and U.S. Provisional Application No. 61/033,402, filed Mar. 3, 2008; this application also claims priority to U.S. Provisional Application 61/353,145, filed Jun. 9, 2010. The disclosures of each of these applications are incorporated hereinby reference, in their entirety, for all purposes. 
    
    
     REFERENCE TO SEQUENCE LISTING 
     The instant application contains a Sequence Listing which has been submitted via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Sep. 23, 2011, is named “19578_US_Sequence_Listing.txt”, lists 25 sequences, and is 91.4 kb in size. 
     FIELD OF THE INVENTION 
     The present disclosure relates to methods for conferring fatty acid and fatty acid ester-producing properties to a heterotrophic or photoautotrophic host, such that the modified host can be used in the commercial production of fuels and chemicals. 
     BACKGROUND OF THE INVENTION 
     Many existing photoautotrophic organisms (i.e., plants, algae, and photosynthetic bacteria) are poorly suited for industrial bioprocessing and have therefore not demonstrated commercial viability. Such organisms typically have slow doubling times (3-72 hrs) compared to industrialized heterotrophic organisms such as  Escherichia coli  (20 minutes), reflective of low total productivities. A need exists, therefore, for engineered photosynthetic microbes which produce increased yields of fatty acids and esters. 
     SUMMARY OF THE INVENTION 
     In one embodiment, the invention provides a method for producing fatty acid esters, comprising: (i) culturing an engineered photosynthetic microorganism in a culture medium, wherein said engineered photosynthetic microorganism comprises a recombinant thioesterase, a recombinant acyl-CoA synthetase, and a recombinant wax synthase; and (ii) exposing said engineered photosynthetic microorganism to light and carbon dioxide, wherein said exposure results in the incorporation of an alcohol into a fatty acid ester produced by said engineered photosynthetic microorganism. In a related embodiment, the engineered photosynthetic microorganism is an engineered  cyanobacterium . In another related embodiment, at least one of said fatty acid esters produced by the engineered  cyanobacterium  is selected from the group consisting of a tetradecanoic acid ester, a hexadecanoic acid ester, a heptadecanoic acid ester, a Δ9-octadecenoic acid ester, and an octadecanoic acid ester. In another related embodiment, the amount of said fatty acid esters produced by said engineered  cyanobacterium  is increased relative to the amount of fatty acid produced by an otherwise identical cell lacking said recombinant thioesterase, acyl-CoA synthetase or wax synthase. In certain embodiments, the incorporated alcohol is an exogenously added alcohol selected from the group consisting of methanol, ethanol, propanol, isopropanol, butanol, hexanol, cyclohexanol, and isoamyl alcohol. 
     In another related embodiment, the esters produce by the engineered cyanobacteria include a hexadecanoic acid ester and an octadecanoic acid ester. In another related embodiment, the amount of hexadecanoic acid ester produced is between 1.5 and 10 fold greater than the amount of octadecanoic acid ester. In yet another related embodiment, the amount of hexadecanoic acid ester produced is between 1.5 and 5 fold greater than the amount of octadecanoic acid ester produced. In yet another related embodiment, at least 50% of the esters produced by said engineered  cyanobacterium  are hexadecanoic acid esters. In yet another related embodiment, between 65% and 85% of the esters produced by said engineered  cyanobacterium  are hexadecanoic acid esters. 
     In a related embodiment of the method for producing fatty acid esters described above, the exogenously alcohol is butanol and fatty acid butyl esters are produced. In yet another related embodiment, the yield of fatty acid butyl esters is at least 5% dry cell weight. In yet another related embodiment, the yield of fatty acid butyl esters is at least 10% dry cell weight. In yet another related embodiment, exogenously added butanol is present in said culture at concentrations between 0.01 and 0.2% (vol/vol). In yet another related embodiment, the concentration of exogenously added butanol is about 0.05 to 0.075% (vol/vol). 
     In another related embodiment of the method for producing fatty acid esters described above, the exogenously added alcohol is ethanol. In yet another related embodiment, the yield of ethyl esters is at least 1% dry cell weight. 
     In another related embodiment of the method for producing fatty acid esters described above, the exogenously added alcohol is methanol. In yet another related embodiment, the yield of methyl esters is at least 0.01% dry cell weight. 
     In another related embodiment, said engineered  cyanobacterium  further comprises a recombinant resistance nodulation cell division type (“RND-type”) transporter, e.g., a TolC-AcrAB transporter. In another related embodiment, the expression of TolC is controlled by a promoter separate from the promoter that controls expression of AcrAB. In another related embodiment, the genes encoding the recombinant transporter are encoded by a plasmid. In another related embodiment, the fatty acid esters are secreted into the culture medium at increased levels relative to an otherwise identical  cyanobacterium  lacking the recombinant transporter. 
     In certain embodiments of the methods for producing fatty acid esters described above, the recombinant thioesterase, wax synthase, and acyl-CoA synthetase are expressed as an operon under the control of a single promoter. In certain embodiments, the single promoter is an inducible promoter. In other embodiments of the methods described above, the expression of at least two of the genes selected from the group consisting of a recombinant thioesterase, wax synthase, and acyl-CoA synthetase is under the control of different promoters. One or more of the promoters can be an inducible promoter. In related embodiments, at least one of said recombinant genes is encoded on a plasmid. In yet other related embodiments, at least one of said recombinant genes is integrated into the chromosome of the engineered cyanobacteria. In yet other related embodiments, at least one of said recombinant genes is a gene that is native to the engineered cyanobacteria, but whose expression is controlled by a recombinant promoter. In yet other related embodiments, one or more promoters are selected from the group consisting of a cI promoter, a cpcB promoter, a lacI-Ptrc promoter, an EM7 promoter, an PaphII promoter, a NirA-type promoter, a PnrsA promoter, or a PnrsB promoter. 
     In another embodiment, the invention provides a method for producing fatty acid esters, comprising: (i) culturing an engineered  cyanobacterium  in a culture medium, wherein said engineered  cyanobacterium  comprises a recombinant acyl-CoA synthetase and a recombinant wax synthase; and (ii) exposing said engineered  cyanobacterium  to light and carbon dioxide, wherein said exposure results in the conversion of an alcohol by said engineered  cyanobacterium  into fatty acid esters, wherein at least one of said fatty acid esters is selected from the group consisting of a tetradecanoic acid ester, a hexadecanoic acid ester, a heptadecanoic acid ester, a Δ9-octadecenoic acid ester, and an octadecanoic acid ester, wherein the amount of said fatty acid esters produced by said engineered  cyanobacterium  is increased relative to the amount of fatty acid produced by an otherwise identical cell lacking said recombinant acyl-CoA synthetase or wax synthase. In a related embodiment, the alcohol is an exogenously added alcohol selected from the group consisting of methanol, ethanol, propanol, isopropanol, butanol, hexanol, cyclohexanol, and isoamyl alcohol. 
     In another embodiment, the invention provides a method for producing a fatty acid ester, comprising: (i) culturing an engineered  cyanobacterium  in a culture medium, wherein said engineered  cyanobacterium  comprises a recombinant RND-type transporter; and (ii) exposing said engineered  cyanobacterium  to light and carbon dioxide, wherein said exposure results in the production of a fatty acid ester by said engineered  cyanobacterium , and wherein said RND-type transporter secretes said fatty acid ester into said culture medium. In a related embodiment, said RND-type transporter is a TolC-AcrAB transporter. 
     In an embodiment related to the methods described above, the invention further comprises isolating said fatty acid ester from said engineered  cyanobacterium  or said culture medium. 
     In another embodiment, the invention also provides an engineered  cyanobacterium , wherein said  cyanobacterium  comprises a recombinant thioesterase, a recombinant acyl-CoA synthetase, and a recombinant wax synthase. In certain embodiments, the engineered  cyanobacterium  additionally comprises a recombinant RND-type transporter, e.g., a TolC-AcrAB transporter. 
     In a related embodiment, at least one of said recombinant enzymes is heterologous with respect to said engineered  cyanobacterium . In another embodiment, said  cyanobacterium  does not synthesize fatty acid esters in the absence of the expression of one or both of the recombinant enzymes. In another embodiment, at least one of said recombinant enzymes is not heterologous to said engineered  cyanobacterium.    
     In yet another related embodiment, the recombinant thioesterase, acyl-CoA synthetase and wax synthase are selected from the enzymes listed in Table 3A, Table 3B and Table 3C, respectively. In yet another related embodiment, the recombinant thioesterase has an amino acid sequence that is identical to SEQ ID NO: 1. In yet another related embodiment, the recombinant thioesterase has an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1. In yet another related embodiment, the recombinant acyl-CoA synthetase is identical to SEQ ID NO:2. In yet another related embodiment, the recombinant acyl-CoA synthetase has an amino acid sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 2. In yet another related embodiment, recombinant wax synthase is identical to SEQ ID NO: 3. In yet another related embodiment, the recombinant wax synthase has an amino acid sequence is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 3. In yet another related embodiment, the recombinant TolC transporter amino acid sequence is identical to SEQ ID NO: 7. In yet another related embodiment, the recombinant TolC transporter has an amino acid sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 7. In yet another related embodiment, the recombinant AcrA amino acid sequence is identical to SEQ ID NO: 8. In yet another related embodiment, the recombinant AcrA amino acid sequence is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 8. In yet another related embodiment, the recombinant AcrB amino acid sequence is identical to SEQ ID NO: 9. In yet another related embodiment, the recombinant AcrB amino acid sequence is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 9. 
     In related embodiments of the above-described embodiments, an engineered photosynthetic microorganism other than a  cyanobacterium  can be used. In other related embodiments, a thermophilic  cyanobacterium  can be used. 
     In another embodiment, the invention provides a methods and compositions for producing fatty acids using an engineered photosynthetic microorganism. For example, in one embodiment, the invention provides a method for producing fatty acids, comprising: (a) culturing an engineered photosynthetic microorganism, wherein said engineered photosynthetic microorganism comprises a modification which reduces the expression of said microorganism&#39;s endogenous acyl-ACP synthetase; and (b) exposing said engineered photosynthetic microorganism to light and carbon dioxide, wherein said exposure results in the production of fatty acids by said engineered  cyanobacterium , wherein the amount of fatty acids produced is increased relative to the amount of fatty acids produced by an otherwise identical microorganism lacking said modification. In a related embodiment, the engineered microorganism is a thermophile. In another related embodiment, the engineered microorganism is a  cyanobacterium . In yet another related embodiment, the engineered microorganism is a thermophilic  cyanobacterium . In yet another related embodiment, the engineered microorganism is  Thermosynechococcus elongatus  BP-1. In yet another related embodiment of the method for producing fatty acids, the modification is a knock-out or deletion of the gene encoding said endogenous acyl-ACP synthetase. In yet another related embodiment, the gene encoding said acyl-ACP synthetase is the acyl-ACP synthetase or aas gene, e.g., GenBank accession number NP — 682091.1. In yet another related embodiment, the increase in fatty acid production is at least a 2 fold increase. In yet another related embodiment, the increase in fatty acid production is between 2 and 4.5 fold. In yet another related embodiment, the increase in fatty acid production includes an increase in fatty acids secreted into a culture media. In yet another related embodiment, most of said increase in fatty acid production arises from the increased production of myristic and oleic acid. In yet another related embodiment of the method for producing fatty acids, the engineered photosynthetic microorganism further comprises a TolC-AcrAB transporter. 
     In another embodiment, the invention provides an engineered photosynthetic microorganism, wherein said microorganism comprises a deletion or knock-out of an endogenous gene encoding a acyl-ACP synthetase or long-chain fatty acid ligase. In a related embodiment, engineered photosynthetic microorganism is a thermophile. In yet another related embodiment, the engineered photosynthetic microorganism is a  cyanobacterium  or a thermophilic  cyanobacterium . In yet another related embodiment, the  cyanobacterium  is  Thermosynechococcus elongatus  BP-1. In yet another related embodiment, the acyl-ACP synthetase is the aas gene of the thermophilic  cyanobacterium , e.g., GenBank accession number NP — 682091.1. In yet another related embodiment, the engineered photosynthetic microorganism further comprises a TolC-AcrAB transporter. 
     In yet another embodiment, the invention provides an engineered cyanbacterial strain selected from the group consisting of JCC723, JCC803, JCC1215, JCC803, JCC1132, and JCC1585. In yet another embodiment, the invention provides an engineered cyanobacterial strain selected from the group consisting of the engineered  Synechococcus  sp. PCC7002 strains JCC1648 (Δaas tesA, with tesA under control of P(nir07) on pAQ4), JCC1704 (Δaas fatB, with fatB inserted at aquI under the control of P(nir07)), JCC1705 (Δaas fatB1, with fatB1 inserted at aquI under the control of P(nir07)), JCC1706 (Δaas fatB2 with fatB2 inserted at aquI under the control of P(nir07)), JCC1751 (Δaas tesA, with tesA under control of P(nir07) on pAQ3), and JCC1755 (Δaas fatB_mat, with fatB_mat under control of P(nir07) on pAQ3). In yet another embodiment, the invention provides the engineered cyanobacterial strain JCC1862 ( Thermosynechococcus elongatus  BP-1 kan R  Δaas). 
     These and other embodiments of the invention are further described in the Figures, Description, Examples and Claims, herein. 
    
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
         FIG. 1  depicts a GC/MS chromatogram overlay comparing cell pellet extracts of JCC803 incubated with either methanol (top trace) or ethanol (bottom traces). The peaks due to methyl esters (MEs) or ethyl esters (EEs) are labeled. 
         FIG. 2  shows three stacked GC/FID chromatograms comparing cell pellet extracts of the indicated cyanobacterial strains when cultured in the presence of ethanol. The interval between tick marks on the FID response axis is 20,000. 
         FIG. 3  depicts stacks of GC/FID chromatograms comparing cell pellet extracts of JCC803 cultures incubated with different alcohols (indicated on respective chromatograms). Numbers indicate the respective fatty acid ester corresponding to the alcohol added (1=myristate; 2=palmitate; 3=oleate; 4=stearate). EA=ethyl arachidate. The interval between tick marks on the FID response axis is 400,000. 
         FIG. 4  depicts a GC/chromatogram of a cell pellet extract from a JCC803 culture incubated with ethanol. 1=ethyl myristate; 2=ethyl palmitoleate; 3=ethyl palmitate; 4=ethyl margarate; 5=ethyl oleate; 6=ethyl stearate. 
         FIG. 5  depicts a GC/chromatogram of a cell pellet extract from a JCC803 culture incubated with butanol. 1=butyl myristate, 2=butyl palmitoleate, 3=butyl palmitate, 4=butyl margarate, 5=butyl oleate, 6=butyl stearate. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular. Generally, nomenclatures used in connection with, and techniques of, biochemistry, enzymology, molecular and cellular biology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art. 
     The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. See, e.g., Sambrook et al.,  Molecular Cloning: A Laboratory Manual,  2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989); Ausubel et al.,  Current Protocols in Molecular Biology , Greene Publishing Associates (1992, and Supplements to 2002); Harlow and Lane,  Antibodies: A Laboratory Manual , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1990); Taylor and Drickamer,  Introduction to Glycobiology,  Oxford Univ. Press (2003);  Worthington Enzyme Manual , Worthington Biochemical Corp., Freehold, N.J.;  Handbook of Biochemistry: Section A Proteins , Vol I, CRC Press (1976); Handbook of Biochemistry: Section A Proteins, Vol II, CRC Press (1976);  Essentials of Glycobiology , Cold Spring Harbor Laboratory Press (1999). 
     All publications, patents and other references mentioned herein are hereby incorporated by reference in their entireties. 
     The following terms, unless otherwise indicated, shall be understood to have the following meanings: 
     The term “polynucleotide” or “nucleic acid molecule” refers to a polymeric form of nucleotides of at least 10 bases in length. The term includes DNA molecules (e.g., cDNA or genomic or synthetic DNA) and RNA molecules (e.g., mRNA or synthetic RNA), as well as analogs of DNA or RNA containing non-natural nucleotide analogs, non-native internucleoside bonds, or both. The nucleic acid can be in any topological conformation. For instance, the nucleic acid can be single-stranded, double-stranded, triple-stranded, quadruplexed, partially double-stranded, branched, hairpinned, circular, or in a padlocked conformation. 
     Unless otherwise indicated, and as an example for all sequences described herein under the general format “SEQ ID NO:”, “nucleic acid comprising SEQ ID NO:1” refers to a nucleic acid, at least a portion of which has either (i) the sequence of SEQ ID NO:1, or (ii) a sequence complementary to SEQ ID NO:1. The choice between the two is dictated by the context. For instance, if the nucleic acid is used as a probe, the choice between the two is dictated by the requirement that the probe be complementary to the desired target. 
     An “isolated” RNA, DNA or a mixed polymer is one which is substantially separated from other cellular components that naturally accompany the native polynucleotide in its natural host cell, e.g., ribosomes, polymerases and genomic sequences with which it is naturally associated. 
     As used herein, an “isolated” organic molecule (e.g., a fatty acid or a fatty acid ester) is one which is substantially separated from the cellular components (membrane lipids, chromosomes, proteins) of the host cell from which it originated, or from the medium in which the host cell was cultured. The term does not require that the biomolecule has been separated from all other chemicals, although certain isolated biomolecules may be purified to near homogeneity. 
     The term “recombinant” refers to a biomolecule, e.g., a gene or protein, that (1) has been removed from its naturally occurring environment, (2) is not associated with all or a portion of a polynucleotide in which the gene is found in nature, (3) is operatively linked to a polynucleotide which it is not linked to in nature, or (4) does not occur in nature. The term “recombinant” can be used in reference to cloned DNA isolates, chemically synthesized polynucleotide analogs, or polynucleotide analogs that are biologically synthesized by heterologous systems, as well as proteins and/or mRNAs encoded by such nucleic acids. 
     As used herein, an endogenous nucleic acid sequence in the genome of an organism (or the encoded protein product of that sequence) is deemed “recombinant” herein if a heterologous sequence is placed adjacent to the endogenous nucleic acid sequence, such that the expression of this endogenous nucleic acid sequence is altered. In this context, a heterologous sequence is a sequence that is not naturally adjacent to the endogenous nucleic acid sequence, whether or not the heterologous sequence is itself endogenous (originating from the same host cell or progeny thereof) or exogenous (originating from a different host cell or progeny thereof). By way of example, a promoter sequence can be substituted (e.g., by homologous recombination) for the native promoter of a gene in the genome of a host cell, such that this gene has an altered expression pattern. This gene would now become “recombinant” because it is separated from at least some of the sequences that naturally flank it. 
     A nucleic acid is also considered “recombinant” if it contains any modifications that do not naturally occur to the corresponding nucleic acid in a genome. For instance, an endogenous coding sequence is considered “recombinant” if it contains an insertion, deletion or a point mutation introduced artificially, e.g., by human intervention. A “recombinant nucleic acid” also includes a nucleic acid integrated into a host cell chromosome at a heterologous site and a nucleic acid construct present as an episome. 
     As used herein, the phrase “degenerate variant” of a reference nucleic acid sequence encompasses nucleic acid sequences that can be translated, according to the standard genetic code, to provide an amino acid sequence identical to that translated from the reference nucleic acid sequence. The term “degenerate oligonucleotide” or “degenerate primer” is used to signify an oligonucleotide capable of hybridizing with target nucleic acid sequences that are not necessarily identical in sequence but that are homologous to one another within one or more particular segments. 
     The term “percent sequence identity” or “identical” in the context of nucleic acid sequences refers to the residues in the two sequences which are the same when aligned for maximum correspondence. The length of sequence identity comparison may be over a stretch of at least about nine nucleotides, usually at least about 20 nucleotides, more usually at least about 24 nucleotides, typically at least about 28 nucleotides, more typically at least about 32 nucleotides, and preferably at least about 36 or more nucleotides. There are a number of different algorithms known in the art which can be used to measure nucleotide sequence identity. For instance, polynucleotide sequences can be compared using FASTA, Gap or Bestfit, which are programs in Wisconsin Package Version 10.0, Genetics Computer Group (GCG), Madison, Wis. FASTA provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences. Pearson, Methods Enzymol. 183:63-98 (1990) (hereby incorporated by reference in its entirety). For instance, percent sequence identity between nucleic acid sequences can be determined using FASTA with its default parameters (a word size of 6 and the NOPAM factor for the scoring matrix) or using Gap with its default parameters as provided in GCG Version 6.1, herein incorporated by reference. Alternatively, sequences can be compared using the computer program, BLAST (Altschul et al., J. Mol. Biol. 215:403-410 (1990); Gish and States, Nature Genet. 3:266-272 (1993); Madden et al., Meth. Enzymol. 266:131-141 (1996); Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997); Zhang and Madden, Genome Res. 7:649-656 (1997)), especially blastp or tblastn (Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997)). 
     The term “substantial homology” or “substantial similarity,” when referring to a nucleic acid or fragment thereof, indicates that, when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 76%, 80%, 85%, preferably at least about 90%, and more preferably at least about 95%, 96%, 97%, 98% or 99% of the nucleotide bases, as measured by any well-known algorithm of sequence identity, such as FASTA, BLAST or Gap, as discussed above. 
     Alternatively, substantial homology or similarity exists when a nucleic acid or fragment thereof hybridizes to another nucleic acid, to a strand of another nucleic acid, or to the complementary strand thereof, under stringent hybridization conditions. “Stringent hybridization conditions” and “stringent wash conditions” in the context of nucleic acid hybridization experiments depend upon a number of different physical parameters. Nucleic acid hybridization will be affected by such conditions as salt concentration, temperature, solvents, the base composition of the hybridizing species, length of the complementary regions, and the number of nucleotide base mismatches between the hybridizing nucleic acids, as will be readily appreciated by those skilled in the art. One having ordinary skill in the art knows how to vary these parameters to achieve a particular stringency of hybridization. 
     In general, “stringent hybridization” is performed at about 25° C. below the thermal melting point (T m ) for the specific DNA hybrid under a particular set of conditions. “Stringent washing” is performed at temperatures about 5° C. lower than the T m  for the specific DNA hybrid under a particular set of conditions. The T m  is the temperature at which 50% of the target sequence hybridizes to a perfectly matched probe. See Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), page 9.51, hereby incorporated by reference. For purposes herein, “stringent conditions” are defined for solution phase hybridization as aqueous hybridization (i.e., free of formamide) in 6×SSC (where 20×SSC contains 3.0 M NaCl and 0.3 M sodium citrate), 1% SDS at 65° C. for 8-12 hours, followed by two washes in 0.2×SSC, 0.1% SDS at 65° C. for 20 minutes. It will be appreciated by the skilled worker that hybridization at 65° C. will occur at different rates depending on a number of factors including the length and percent identity of the sequences which are hybridizing. 
     The nucleic acids (also referred to as polynucleotides) of this present invention may include both sense and antisense strands of RNA, cDNA, genomic DNA, and synthetic forms and mixed polymers of the above. They may be modified chemically or biochemically or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those of skill in the art. Such modifications include, for example, labels, methylation, substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), pendent moieties (e.g., polypeptides), intercalators (e.g., acridine, psoralen, etc.), chelators, alkylators, and modified linkages (e.g., alpha anomeric nucleic acids, etc.) Also included are synthetic molecules that mimic polynucleotides in their ability to bind to a designated sequence via hydrogen bonding and other chemical interactions. Such molecules are known in the art and include, for example, those in which peptide linkages substitute for phosphate linkages in the backbone of the molecule. Other modifications can include, for example, analogs in which the ribose ring contains a bridging moiety or other structure such as the modifications found in “locked” nucleic acids. 
     The term “mutated” when applied to nucleic acid sequences means that nucleotides in a nucleic acid sequence may be inserted, deleted or changed compared to a reference nucleic acid sequence. A single alteration may be made at a locus (a point mutation) or multiple nucleotides may be inserted, deleted or changed at a single locus. In addition, one or more alterations may be made at any number of loci within a nucleic acid sequence. A nucleic acid sequence may be mutated by any method known in the art including but not limited to mutagenesis techniques such as “error-prone PCR” (a process for performing PCR under conditions where the copying fidelity of the DNA polymerase is low, such that a high rate of point mutations is obtained along the entire length of the PCR product; see, e.g., Leung et al.,  Technique,  1:11-15 (1989) and Caldwell and Joyce,  PCR Methods Applic.  2:28-33 (1992)); and “oligonucleotide-directed mutagenesis” (a process which enables the generation of site-specific mutations in any cloned DNA segment of interest; see, e.g., Reidhaar-Olson and Sauer,  Science  241:53-57 (1988)). 
     The term “attenuate” as used herein generally refers to a functional deletion, including a mutation, partial or complete deletion, insertion, or other variation made to a gene sequence or a sequence controlling the transcription of a gene sequence, which reduces or inhibits production of the gene product, or renders the gene product non-functional. In some instances a functional deletion is described as a knockout mutation. Attenuation also includes amino acid sequence changes by altering the nucleic acid sequence, placing the gene under the control of a less active promoter, down-regulation, expressing interfering RNA, ribozymes or antisense sequences that target the gene of interest, or through any other technique known in the art. In one example, the sensitivity of a particular enzyme to feedback inhibition or inhibition caused by a composition that is not a product or a reactant (non-pathway specific feedback) is lessened such that the enzyme activity is not impacted by the presence of a compound. In other instances, an enzyme that has been altered to be less active can be referred to as attenuated. 
     Deletion: The removal of one or more nucleotides from a nucleic acid molecule or one or more amino acids from a protein, the regions on either side being joined together. 
     Knock-out: A gene whose level of expression or activity has been reduced to zero. In some examples, a gene is knocked-out via deletion of some or all of its coding sequence. In other examples, a gene is knocked-out via introduction of one or more nucleotides into its open reading frame, which results in translation of a non-sense or otherwise non-functional protein product. 
     The term “vector” as used herein is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid,” which generally refers to a circular double stranded DNA loop into which additional DNA segments may be ligated, but also includes linear double-stranded molecules such as those resulting from amplification by the polymerase chain reaction (PCR) or from treatment of a circular plasmid with a restriction enzyme. Other vectors include cosmids, bacterial artificial chromosomes (BAC) and yeast artificial chromosomes (YAC). Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome (discussed in more detail below). Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., vectors having an origin of replication which functions in the host cell). Other vectors can be integrated into the genome of a host cell upon introduction into the host cell, and are thereby replicated along with the host genome. Moreover, certain preferred vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “recombinant expression vectors” (or simply “expression vectors”). 
     “Operatively linked” or “operably linked” expression control sequences refers to a linkage in which the expression control sequence is contiguous with the gene of interest to control the gene of interest, as well as expression control sequences that act in trans or at a distance to control the gene of interest. 
     The term “expression control sequence” as used herein refers to polynucleotide sequences which are necessary to affect the expression of coding sequences to which they are operatively linked. Expression control sequences are sequences which control the transcription, post-transcriptional events and translation of nucleic acid sequences. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (e.g., ribosome binding sites); sequences that enhance protein stability; and when desired, sequences that enhance protein secretion. The nature of such control sequences differs depending upon the host organism; in prokaryotes, such control sequences generally include promoter, ribosomal binding site, and transcription termination sequence. The term “control sequences” is intended to include, at a minimum, all components whose presence is essential for expression, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences. 
     Promoters useful for expressing the recombinant genes described herein include both constitutive and inducible/repressible promoters. Examples of inducible/repressible promoters include nickel-inducible promoters (e.g., PnrsA, PnrsB; see, e.g., Lopez-Mauy et al.,  Cell  (2002) v. 43:247-256, incorporated by reference herein) and urea repressible promoters such as PnirA (described in, e.g., Qi et al.,  Applied and Environmental Microbiology  (2005) v. 71: 5678-5684, incorporated by reference herein). In other embodiments, a PaphII and/or a lacIq-Ptrc promoter can used to control expression. Where multiple recombinant genes are expressed in an engineered cyanobacteria of the invention, the different genes can be controlled by different promoters or by identical promoters in separate operons, or the expression of two or more genes may be controlled by a single promoter as part of an operon. 
     The term “recombinant host cell” (or simply “host cell”), as used herein, is intended to refer to a cell into which a recombinant vector has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term “host cell” as used herein. A recombinant host cell may be an isolated cell or cell line grown in culture or may be a cell which resides in a living tissue or organism. 
     The term “peptide” as used herein refers to a short polypeptide, e.g., one that is typically less than about 50 amino acids long and more typically less than about 30 amino acids long. The term as used herein encompasses analogs and mimetics that mimic structural and thus biological function. 
     The term “polypeptide” encompasses both naturally-occurring and non-naturally-occurring proteins, and fragments, mutants, derivatives and analogs thereof. A polypeptide may be monomeric or polymeric. Further, a polypeptide may comprise a number of different domains each of which has one or more distinct activities. 
     The term “isolated protein” or “isolated polypeptide” is a protein or polypeptide that by virtue of its origin or source of derivation (1) is not associated with naturally associated components that accompany it in its native state, (2) exists in a purity not found in nature, where purity can be adjudged with respect to the presence of other cellular material (e.g., is free of other proteins from the same species) (3) is expressed by a cell from a different species, or (4) does not occur in nature (e.g., it is a fragment of a polypeptide found in nature or it includes amino acid analogs or derivatives not found in nature or linkages other than standard peptide bonds). Thus, a polypeptide that is chemically synthesized or synthesized in a cellular system different from the cell from which it naturally originates will be “isolated” from its naturally associated components. A polypeptide or protein may also be rendered substantially free of naturally associated components by isolation, using protein purification techniques well known in the art. As thus defined, “isolated” does not necessarily require that the protein, polypeptide, peptide or oligopeptide so described has been physically removed from its native environment. 
     The term “polypeptide fragment” as used herein refers to a polypeptide that has a deletion, e.g., an amino-terminal and/or carboxy-terminal deletion compared to a full-length polypeptide. In a preferred embodiment, the polypeptide fragment is a contiguous sequence in which the amino acid sequence of the fragment is identical to the corresponding positions in the naturally-occurring sequence. Fragments typically are at least 5, 6, 7, 8, 9 or 10 amino acids long, preferably at least 12, 14, 16 or 18 amino acids long, more preferably at least 20 amino acids long, more preferably at least 25, 30, 35, 40 or 45, amino acids, even more preferably at least 50 or 60 amino acids long, and even more preferably at least 70 amino acids long. 
     A “modified derivative” refers to polypeptides or fragments thereof that are substantially homologous in primary structural sequence but which include, e.g., in vivo or in vitro chemical and biochemical modifications or which incorporate amino acids that are not found in the native polypeptide. Such modifications include, for example, acetylation, carboxylation, phosphorylation, glycosylation, ubiquitination, labeling, e.g., with radionuclides, and various enzymatic modifications, as will be readily appreciated by those skilled in the art. A variety of methods for labeling polypeptides and of substituents or labels useful for such purposes are well known in the art, and include radioactive isotopes such as  125 I,  32 P,  35 S, and  3 H, ligands which bind to labeled antiligands (e.g., antibodies), fluorophores, chemiluminescent agents, enzymes, and antiligands which can serve as specific binding pair members for a labeled ligand. The choice of label depends on the sensitivity required, ease of conjugation with the primer, stability requirements, and available instrumentation. Methods for labeling polypeptides are well known in the art. See, e.g., Ausubel et al.,  Current Protocols in Molecular Biology,  Greene Publishing Associates (1992, and Supplements to 2002) (hereby incorporated by reference). 
     The term “fusion protein” refers to a polypeptide comprising a polypeptide or fragment coupled to heterologous amino acid sequences. Fusion proteins are useful because they can be constructed to contain two or more desired functional elements from two or more different proteins. A fusion protein comprises at least 10 contiguous amino acids from a polypeptide of interest, more preferably at least 20 or 30 amino acids, even more preferably at least 40, 50 or 60 amino acids, yet more preferably at least 75, 100 or 125 amino acids. Fusions that include the entirety of the proteins of the present invention have particular utility. The heterologous polypeptide included within the fusion protein of the present invention is at least 6 amino acids in length, often at least 8 amino acids in length, and usefully at least 15, 20, and 25 amino acids in length. Fusions that include larger polypeptides, such as an IgG Fc region, and even entire proteins, such as the green fluorescent protein (“GFP”) chromophore-containing proteins, have particular utility. Fusion proteins can be produced recombinantly by constructing a nucleic acid sequence which encodes the polypeptide or a fragment thereof in frame with a nucleic acid sequence encoding a different protein or peptide and then expressing the fusion protein. Alternatively, a fusion protein can be produced chemically by crosslinking the polypeptide or a fragment thereof to another protein. 
     As used herein, the term “antibody” refers to a polypeptide, at least a portion of which is encoded by at least one immunoglobulin gene, or fragment thereof, and that can bind specifically to a desired target molecule. The term includes naturally-occurring forms, as well as fragments and derivatives. 
     Fragments within the scope of the term “antibody” include those produced by digestion with various proteases, those produced by chemical cleavage and/or chemical dissociation and those produced recombinantly, so long as the fragment remains capable of specific binding to a target molecule. Among such fragments are Fab, Fab′, Fv, F(ab′).sub.2, and single chain Fv (scFv) fragments. 
     Derivatives within the scope of the term include antibodies (or fragments thereof) that have been modified in sequence, but remain capable of specific binding to a target molecule, including: interspecies chimeric and humanized antibodies; antibody fusions; heteromeric antibody complexes and antibody fusions, such as diabodies (bispecific antibodies), single-chain diabodies, and intrabodies (see, e.g.,  Intracellular Antibodies: Research and Disease Applications , (Marasco, ed., Springer-Verlag New York, Inc., 1998), the disclosure of which is incorporated herein by reference in its entirety). 
     As used herein, antibodies can be produced by any known technique, including harvest from cell culture of native B lymphocytes, harvest from culture of hybridomas, recombinant expression systems and phage display. 
     The term “non-peptide analog” refers to a compound with properties that are analogous to those of a reference polypeptide. A non-peptide compound may also be termed a “peptide mimetic” or a “peptidomimetic.” See, e.g., Jones,  Amino Acid and Peptide Synthesis,  Oxford University Press (1992); Jung,  Combinatorial Peptide and Nonpeptide Libraries: A Handbook , John Wiley (1997); Bodanszky et al.,  Peptide Chemistry—A Practical Textbook,  Springer Verlag (1993);  Synthetic Peptides: A Users Guide , (Grant, ed., W. H. Freeman and Co., 1992); Evans et al.,  J. Med. Chem.  30:1229 (1987); Fauchere,  J. Adv. Drug Res.  15:29 (1986); Veber and Freidinger,  Trends Neurosci.,  8:392-396 (1985); and references sited in each of the above, which are incorporated herein by reference. Such compounds are often developed with the aid of computerized molecular modeling. Peptide mimetics that are structurally similar to useful peptides of the present invention may be used to produce an equivalent effect and are therefore envisioned to be part of the present invention. 
     A “polypeptide mutant” or “mutein” refers to a polypeptide whose sequence contains an insertion, duplication, deletion, rearrangement or substitution of one or more amino acids compared to the amino acid sequence of a native or wild-type protein. A mutein may have one or more amino acid point substitutions, in which a single amino acid at a position has been changed to another amino acid, one or more insertions and/or deletions, in which one or more amino acids are inserted or deleted, respectively, in the sequence of the naturally-occurring protein, and/or truncations of the amino acid sequence at either or both the amino or carboxy termini. A mutein may have the same but preferably has a different biological activity compared to the naturally-occurring protein. 
     A mutein has at least 85% overall sequence homology to its wild-type counterpart. Even more preferred are muteins having at least 90% overall sequence homology to the wild-type protein. 
     In an even more preferred embodiment, a mutein exhibits at least 95% sequence identity, even more preferably 98%, even more preferably 99% and even more preferably 99.9% overall sequence identity. 
     Sequence homology may be measured by any common sequence analysis algorithm, such as Gap or Bestfit. 
     Amino acid substitutions can include those which: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter binding affinity or enzymatic activity, and (5) confer or modify other physicochemical or functional properties of such analogs. 
     As used herein, the twenty conventional amino acids and their abbreviations follow conventional usage. See  Immunology—A Synthesis  (Golub and Gren eds., Sinauer Associates, Sunderland, Mass., 2 nd  ed. 1991), which is incorporated herein by reference. Stereoisomers (e.g., D-amino acids) of the twenty conventional amino acids, unnatural amino acids such as α-, α-disubstituted amino acids, N-alkyl amino acids, and other unconventional amino acids may also be suitable components for polypeptides of the present invention. Examples of unconventional amino acids include: 4-hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). In the polypeptide notation used herein, the left-hand end corresponds to the amino terminal end and the right-hand end corresponds to the carboxy-terminal end, in accordance with standard usage and convention. 
     A protein has “homology” or is “homologous” to a second protein if the nucleic acid sequence that encodes the protein has a similar sequence to the nucleic acid sequence that encodes the second protein. Alternatively, a protein has homology to a second protein if the two proteins have “similar” amino acid sequences. (Thus, the term “homologous proteins” is defined to mean that the two proteins have similar amino acid sequences.) As used herein, homology between two regions of amino acid sequence (especially with respect to predicted structural similarities) is interpreted as implying similarity in function. 
     When “homologous” is used in reference to proteins or peptides, it is recognized that residue positions that are not identical often differ by conservative amino acid substitutions. A “conservative amino acid substitution” is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of homology may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. See, e.g., Pearson, 1994 , Methods Mol. Biol.  24:307-31 and 25:365-89 (herein incorporated by reference). 
     The following six groups each contain amino acids that are conservative substitutions for one another: 1) Serine (S), Threonine (T); 2) Aspartic Acid (D), Glutamic Acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Alanine (A), Valine (V), and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W). 
     Sequence homology for polypeptides, which is also referred to as percent sequence identity, is typically measured using sequence analysis software. See, e.g., the Sequence Analysis Software Package of the Genetics Computer Group (GCG), University of Wisconsin Biotechnology Center, 910 University Avenue, Madison, Wis. 53705. Protein analysis software matches similar sequences using a measure of homology assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions. For instance, GCG contains programs such as “Gap” and “Bestfit” which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild-type protein and a mutein thereof. See, e.g., GCG Version 6.1. 
     A preferred algorithm when comparing a particular polypeptide sequence to a database containing a large number of sequences from different organisms is the computer program BLAST (Altschul et al.,  J. Mol. Biol.  215:403-410 (1990); Gish and States,  Nature Genet.  3:266-272 (1993); Madden et al.,  Meth. Enzymol.  266:131-141 (1996); Altschul et al.,  Nucleic Acids Res.  25:3389-3402 (1997); Zhang and Madden,  Genome Res.  7:649-656 (1997)), especially blastp or tblastn (Altschul et al.,  Nucleic Acids Res.  25:3389-3402 (1997)). 
     Preferred parameters for BLASTp are: Expectation value: 10 (default); Filter: seg (default); Cost to open a gap: 11 (default); Cost to extend a gap: 1 (default); Max. alignments: 100 (default); Word size: 11 (default); No. of descriptions: 100 (default); Penalty Matrix: BLOWSUM62. 
     Preferred parameters for BLASTp are: Expectation value: 10 (default); Filter: seg (default); Cost to open a gap: 11 (default); Cost to extend a gap: 1 (default); Max. alignments: 100 (default); Word size: 11 (default); No. of descriptions: 100 (default); Penalty Matrix: BLOWSUM62. The length of polypeptide sequences compared for homology will generally be at least about 16 amino acid residues, usually at least about 20 residues, more usually at least about 24 residues, typically at least about 28 residues, and preferably more than about 35 residues. When searching a database containing sequences from a large number of different organisms, it is preferable to compare amino acid sequences. Database searching using amino acid sequences can be measured by algorithms other than blastp known in the art. For instance, polypeptide sequences can be compared using FASTA, a program in GCG Version 6.1. FASTA provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences. Pearson,  Methods Enzymol.  183:63-98 (1990) (incorporated by reference herein). For example, percent sequence identity between amino acid sequences can be determined using FASTA with its default parameters (a word size of 2 and the PAM250 scoring matrix), as provided in GCG Version 6.1, herein incorporated by reference. 
     “Specific binding” refers to the ability of two molecules to bind to each other in preference to binding to other molecules in the environment. Typically, “specific binding” discriminates over adventitious binding in a reaction by at least two-fold, more typically by at least 10-fold, often at least 100-fold. Typically, the affinity or avidity of a specific binding reaction, as quantified by a dissociation constant, is about 10 −7  M or stronger (e.g., about 10 −8  M, 10 −9  M or even stronger). 
     “Percent dry cell weight” refers to a production measurement of esters of fatty acids or fatty acids obtained as follows: a defined volume of culture is centrifuged to pellet the cells. Cells are washed then dewetted by at least one cycle of microcentrifugation and aspiration. Cell pellets are lyophilized overnight, and the tube containing the dry cell mass is weighed again such that the mass of the cell pellet can be calculated within ±0.1 mg. At the same time cells are processed for dry cell weight determination, a second sample of the culture in question is harvested, washed, and dewetted. The resulting cell pellet, corresponding to 1-3 mg of dry cell weight, is then extracted by vortexing in approximately 1 ml acetone plus butylated hydroxytoluene (BHT) as antioxidant and an internal standard, e.g., ethyl arachidate. Cell debris is then pelleted by centrifugation and the supernatant (extractant) is taken for analysis by GC. For accurate quantitation of the molecules, flame ionization detection (FID) was used as opposed to MS total ion count. The concentrations of the esters or fatty acids in the biological extracts were calculated using calibration relationships between GC-FID peak area and known concentrations of authentic standards. Knowing the volume of the extractant, the resulting concentrations of the products in the extractant, and the dry cell weight of the cell pellet extracted, the percentage of dry cell weight that comprised the esters or fatty acids can be determined. 
     The term “region” as used herein refers to a physically contiguous portion of the primary structure of a biomolecule. In the case of proteins, a region is defined by a contiguous portion of the amino acid sequence of that protein. 
     The term “domain” as used herein refers to a structure of a biomolecule that contributes to a known or suspected function of the biomolecule. Domains may be co-extensive with regions or portions thereof; domains may also include distinct, non-contiguous regions of a biomolecule. Examples of protein domains include, but are not limited to, an Ig domain, an extracellular domain, a transmembrane domain, and a cytoplasmic domain. 
     As used herein, the term “molecule” means any compound, including, but not limited to, a small molecule, peptide, protein, sugar, nucleotide, nucleic acid, lipid, etc., and such a compound can be natural or synthetic. 
     “Carbon-based Products of Interest” include alcohols such as ethanol, propanol, isopropanol, butanol, fatty alcohols, fatty acid esters, wax esters; hydrocarbons and alkanes such as propane, octane, diesel, Jet Propellant 8 (JP8); polymers such as terephthalate, 1,3-propanediol, 1,4-butanediol, polyols, Polyhydroxyalkanoates (PHA), poly-beta-hydroxybutyrate (PHB), acrylate, adipic acid, ε-caprolactone, isoprene, caprolactam, rubber; commodity chemicals such as lactate, Docosahexaenoic acid (DHA), 3-hydroxypropionate, γ-valerolactone, lysine, serine, aspartate, aspartic acid, sorbitol, ascorbate, ascorbic acid, isopentenol, lanosterol, omega-3 DHA, lycopene, itaconate, 1,3-butadiene, ethylene, propylene, succinate, citrate, citric acid, glutamate, malate, 3-hydroxypropionic acid (HPA), lactic acid, THF, gamma butyrolactone, pyrrolidones, hydroxybutyrate, glutamic acid, levulinic acid, acrylic acid, malonic acid; specialty chemicals such as carotenoids, isoprenoids, itaconic acid; pharmaceuticals and pharmaceutical intermediates such as 7-aminodeacetoxycephalosporanic acid (7-ADCA)/cephalosporin, erythromycin, polyketides, statins, paclitaxel, docetaxel, terpenes, peptides, steroids, omega fatty acids and other such suitable products of interest. Such products are useful in the context of biofuels, industrial and specialty chemicals, as intermediates used to make additional products, such as nutritional supplements, neutraceuticals, polymers, paraffin replacements, personal care products and pharmaceuticals. 
     Biofuel: A biofuel refers to any fuel that derives from a biological source. Biofuel can refer to one or more hydrocarbons, one or more alcohols, one or more fatty esters or a mixture thereof. 
     The term “hydrocarbon” generally refers to a chemical compound that consists of the elements carbon (C), hydrogen (H) and optionally oxygen (O). There are essentially three types of hydrocarbons, e.g., aromatic hydrocarbons, saturated hydrocarbons and unsaturated hydrocarbons such as alkenes, alkynes, and dienes. The term also includes fuels, biofuels, plastics, waxes, solvents and oils. Hydrocarbons encompass biofuels, as well as plastics, waxes, solvents and oils. A “fatty acid” is a carboxylic acid with a long unbranched aliphatic tail (chain), which is either saturated or unsaturated. Most naturally occurring fatty acids have a chain of four to 28 carbons. 
     Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this present invention pertains. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice of the present invention and will be apparent to those of skill in the art. All publications and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. The materials, methods, and examples are illustrative only and not intended to be limiting. 
     Throughout this specification and claims, the word “comprise” or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. 
     Nucleic Acid Sequences 
     Esters are chemical compounds with the basic formula: 
     
       
         
         
             
             
         
       
     
     where R and R′ denote any alkyl or aryl group. In one embodiment, the invention provides one or more isolated or recombinant nucleic acids encoding one or more genes which, when recombinantly expressed in a photosynthetic microorganism, catalyze the synthesis of esters by the microorganism. The first gene is a thioesterase, which catalyzes the synthesis of fatty acids from an acyl-Acyl Carrier Protein (“acyl-ACP”) molecule. The second gene is an acyl-CoA synthetase, which synthesizes fatty acyl-CoA from a fatty acid. The third gene is a wax synthase, which synthesizes esters from a fatty acyl-CoA molecule and an alcohol (e.g., methanol, ethanol, proponal, butanol, etc.). In certain related embodiments, additional genes expressing a recombinant resistance nodulation cell division type (“RND-type”) transporter such as TolC/AcrAB are also recombinantly expressed to facilitate the transport of ethyl esters outside of the engineered photosynthetic cell and into the culture medium. 
     Accordingly, the present invention provides isolated nucleic acid molecules for genes encoding thioesterase, acyl-CoA synthetases and wax synthase enzymes, and variants thereof. An exemplary full-length expression optimized nucleic acid sequence for a gene encoding a thioesterase is presented as SEQ ID NO: 4. The corresponding amino acid sequences is presented as SEQ ID NO: 1. Additional genes encoding thioesterases are presented in Table 3A. An exemplary full-length expression-optimized nucleic acid sequence for a gene encoding an acyl-CoA synthetase is presented as SEQ ID NO: 5, and the corresponding amino acid sequence is presented as SEQ ID NOs: 2. Additional genes encoding acyl-CoA synthetases are presented in Table 3B. An exemplary full-length expression-optimized nucleic acid sequence for a gene encoding an acyl-CoA synthetase is presented as SEQ ID NO: 6, and the corresponding amino acid sequence is presented as SEQ ID NOs: 3. Additional genes encoding acyl-CoA synthetases are presented in Table 3C. 
     One skilled in the art will recognize that the redundancy of the genetic code will allow many other nucleic acid sequences to encode the identical enzymes. The sequences of the nucleic acids disclosed herein can be optimized as needed to yield the desired expression levels in a particular photosynthetic microorganism. Such a nucleic acid sequence can have 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.9% or even higher identity to the native gene sequence. 
     In another embodiment, the nucleic acid molecule of the present invention encodes a polypeptide having the amino acid sequence of SEQ ID NO:1, 2, 3, 7, 8, or 9. Preferably, the nucleic acid molecule of the present invention encodes a polypeptide sequence of at least 50%, 60, 70%, 80%, 85%, 90% or 95% identity to SEQ ID NO:1, 2, 3, 7, 8 or 9 and the identity can even more preferably be 96%, 97%, 98%, 99%, 99.9% or even higher. 
     The present invention also provides nucleic acid molecules that hybridize under stringent conditions to the above-described nucleic acid molecules. As defined above, and as is well known in the art, stringent hybridizations are performed at about 25° C. below the thermal melting point (T m ) for the specific DNA hybrid under a particular set of conditions, where the T m  is the temperature at which 50% of the target sequence hybridizes to a perfectly matched probe. Stringent washing is performed at temperatures about 5° C. lower than the T m  for the specific DNA hybrid under a particular set of conditions. 
     Nucleic acid molecules comprising a fragment of any one of the above-described nucleic acid sequences are also provided. These fragments preferably contain at least 20 contiguous nucleotides. More preferably the fragments of the nucleic acid sequences contain at least 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 or even more contiguous nucleotides. 
     The nucleic acid sequence fragments of the present invention display utility in a variety of systems and methods. For example, the fragments may be used as probes in various hybridization techniques. Depending on the method, the target nucleic acid sequences may be either DNA or RNA. The target nucleic acid sequences may be fractionated (e.g., by gel electrophoresis) prior to the hybridization, or the hybridization may be performed on samples in situ. One of skill in the art will appreciate that nucleic acid probes of known sequence find utility in determining chromosomal structure (e.g., by Southern blotting) and in measuring gene expression (e.g., by Northern blotting). In such experiments, the sequence fragments are preferably detectably labeled, so that their specific hydridization to target sequences can be detected and optionally quantified. One of skill in the art will appreciate that the nucleic acid fragments of the present invention may be used in a wide variety of blotting techniques not specifically described herein. 
     It should also be appreciated that the nucleic acid sequence fragments disclosed herein also find utility as probes when immobilized on microarrays. Methods for creating microarrays by deposition and fixation of nucleic acids onto support substrates are well known in the art. Reviewed in  DNA Microarrays: A Practical Approach  (Practical Approach Series), Schena (ed.), Oxford University Press (1999) (ISBN: 0199637768); Nature Genet. 21(1)(suppl):1-60 (1999);  Microarray Biochip: Tools and Technology , Schena (ed.), Eaton Publishing Company/BioTechniques Books Division (2000) (ISBN: 1881299376), the disclosures of which are incorporated herein by reference in their entireties. Analysis of, for example, gene expression using microarrays comprising nucleic acid sequence fragments, such as the nucleic acid sequence fragments disclosed herein, is a well-established utility for sequence fragments in the field of cell and molecular biology. Other uses for sequence fragments immobilized on microarrays are described in Gerhold et al.,  Trends Biochem. Sci.  24:168-173 (1999) and Zweiger,  Trends Biotechnol.  17:429-436 (1999);  DNA Microarrays: A Practical Approach  (Practical Approach Series), Schena (ed.), Oxford University Press (1999) (ISBN: 0199637768); Nature Genet. 21(1)(suppl):1-60 (1999);  Microarray Biochip: Tools and Technology , Schena (ed.), Eaton Publishing Company/BioTechniques Books Division (2000) (ISBN: 1881299376), the disclosure of each of which is incorporated herein by reference in its entirety. 
     As is well known in the art, enzyme activities can be measured in various ways. For example, the pyrophosphorolysis of OMP may be followed spectroscopically (Grubmeyer et al., (1993)  J. Biol. Chem.  268:20299-20304). Alternatively, the activity of the enzyme can be followed using chromatographic techniques, such as by high performance liquid chromatography (Chung and Sloan, (1986)  J. Chromatogr.  371:71-81). As another alternative the activity can be indirectly measured by determining the levels of product made from the enzyme activity. These levels can be measured with techniques including aqueous chloroform/methanol extraction as known and described in the art (Cf M. Kates (1986)  Techniques of Lipidology; Isolation, analysis and identification of Lipids . Elsevier Science Publishers, New York (ISBN: 0444807322)). More modern techniques include using gas chromatography linked to mass spectrometry (Niessen, W. M. A. (2001).  Current practice of gas chromatography—mass spectrometry . New York, N.Y.: Marcel Dekker. (ISBN: 0824704738)). Additional modern techniques for identification of recombinant protein activity and products including liquid chromatography-mass spectrometry (LCMS), high performance liquid chromatography (HPLC), capillary electrophoresis, Matrix-Assisted Laser Desorption Ionization time of flight-mass spectrometry (MALDI-TOF MS), nuclear magnetic resonance (NMR), near-infrared (NIR) spectroscopy, viscometry (Knothe, G (1997)  Am. Chem. Soc. Symp. Series,  666: 172-208), titration for determining free fatty acids (Komers (1997)  Fett/Lipid,  99(2): 52-54), enzymatic methods (Bailer (1991)  Fresenius J. Anal. Chem.  340(3): 186), physical property-based methods, wet chemical methods, etc. can be used to analyze the levels and the identity of the product produced by the organisms of the present invention. Other methods and techniques may also be suitable for the measurement of enzyme activity, as would be known by one of skill in the art. 
     Vectors 
     Also provided are vectors, including expression vectors, which comprise the above nucleic acid molecules of the present invention, as described further herein. In a first embodiment, the vectors include the isolated nucleic acid molecules described above. In an alternative embodiment, the vectors of the present invention include the above-described nucleic acid molecules operably linked to one or more expression control sequences. The vectors of the instant invention may thus be used to express a thioesterase, an acyl-CoA synthease, and/or a wax synthase, contributing to the synthesis of esters by the cell. 
     In a related embodiment, vectors may include nucleic acid molecules encoding an RND-type transporter such as TolC/AcrAB to facilitate the extracellular transport of esters. Exemplary vectors of the invention include any of the vectors expressing a thioesterase, an acyl-CoA synthease, wax synthase, and/or TolC/AcrAB transporter disclosed here, e.g., pJB532, pJB634, pJB578 and pJB1074. The invention also provides other vectors such as pJB161 which are capable of receiving nucleic acid sequences of the invention. Vectors such as pJB161 comprise sequences which are homologous with sequences that are present in plasmids which are endogenous to certain photosynthetic microorganisms (e.g., plasmids pAQ7 or pAQ1 of certain  Synechococcus  species). Recombination between pJB161 and the endogenous plasmids in vivo yield engineered microbes expressing the genes of interest from their endogenous plasmids. Alternatively, vectors can be engineered to recombine with the host cell chromosome, or the vector can be engineered to replicate and express genes of interest independent of the host cell chromosome or any of the host cell&#39;s endogenous plasmids. 
     Vectors useful for expression of nucleic acids in prokaryotes are well known in the art. 
     Isolated Polypeptides 
     According to another aspect of the present invention, isolated polypeptides (including muteins, allelic variants, fragments, derivatives, and analogs) encoded by the nucleic acid molecules of the present invention are provided. In one embodiment, the isolated polypeptide comprises the polypeptide sequence corresponding to SEQ ID NO:1, 2, 3, 7, 8, or 9. In an alternative embodiment of the present invention, the isolated polypeptide comprises a polypeptide sequence at least 85% identical to SEQ ID NO:1, 2, 3, 7, 8, or 9. Preferably the isolated polypeptide of the present invention has at least 50%, 60, 70%, 80%, 85%, 90%, 95%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or even higher identity to SEQ ID NO:1, 2, 3, 7, 8 or 9. 
     According to other embodiments of the present invention, isolated polypeptides comprising a fragment of the above-described polypeptide sequences are provided. These fragments preferably include at least 20 contiguous amino acids, more preferably at least 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 or even more contiguous amino acids. 
     The polypeptides of the present invention also include fusions between the above-described polypeptide sequences and heterologous polypeptides. The heterologous sequences can, for example, include sequences designed to facilitate purification, e.g. histidine tags, and/or visualization of recombinantly-expressed proteins. Other non-limiting examples of protein fusions include those that permit display of the encoded protein on the surface of a phage or a cell, fusions to intrinsically fluorescent proteins, such as green fluorescent protein (GFP), and fusions to the IgG Fc region. 
     Host Cell Transformants 
     In another aspect of the present invention, host cells transformed with the nucleic acid molecules or vectors of the present invention, and descendants thereof, are provided. In some embodiments of the present invention, these cells carry the nucleic acid sequences of the present invention on vectors, which may but need not be freely replicating vectors. In other embodiments of the present invention, the nucleic acids have been integrated into the genome of the host cells and/or into an endogenous plasmid of the host cells. 
     In a preferred embodiment, the host cell comprises one or more recombinant thioesterase-, acyl-CoA synthase-, wax synthase-, or TolC/AcrAB-encoding nucleic acids which express thioesterase-, acyl-CoA synthase, wax synthase or TolC/AcrAB respectively in the host cell. 
     In an alternative embodiment, the host cells of the present invention can be mutated by recombination with a disruption, deletion or mutation of the isolated nucleic acid of the present invention so that the activity of a native thioesterase, acyl-CoA synthase, wax synthase, and/or TolC/AcrAB protein in the host cell is reduced or eliminated compared to a host cell lacking the mutation. 
     Selected or Engineered Microorganisms for the Production of Fatty Acids, Esters, and Other Carbon-Based Products of Interest 
     Microorganism: Includes prokaryotic and eukaryotic microbial species from the Domains Archaea, Bacteria and Eucarya, the latter including yeast and filamentous fungi, protozoa, algae, or higher Protista. The terms “microbial cells” and “microbes” are used interchangeably with the term microorganism. 
     A variety of host organisms can be transformed to produce a product of interest. Photoautotrophic organisms include eukaryotic plants and algae, as well as prokaryotic cyanobacteria, green-sulfur bacteria, green non-sulfur bacteria, purple sulfur bacteria, and purple non-sulfur bacteria. 
     Extremophiles are also contemplated as suitable organisms. Such organisms withstand various environmental parameters such as temperature, radiation, pressure, gravity, vacuum, desiccation, salinity, pH, oxygen tension, and chemicals. They include hyperthermophiles, which grow at or above 80° C. such as  Pyrolobus fumarii ; thermophiles, which grow between 60-80° C. such as  Synechococcus lividis ; mesophiles, which grow between 15-60° C. and psychrophiles, which grow at or below 15° C. such as  Psychrobacter  and some insects. Radiation tolerant organisms include  Deinococcus radiodurans . Pressure-tolerant organisms include piezophiles, which tolerate pressure of 130 MPa. Weight-tolerant organisms include barophiles. Hypergravity (e.g., &gt;1 g) hypogravity (e.g., &lt;1 g) tolerant organisms are also contemplated. Vacuum tolerant organisms include tardigrades, insects, microbes and seeds. Dessicant tolerant and anhydrobiotic organisms include xerophiles such as  Artemia salina ; nematodes, microbes, fungi and lichens. Salt-tolerant organisms include halophiles (e.g., 2-5 M NaCl)  Halobacteriacea  and  Dunaliella salina . pH-tolerant organisms include alkaliphiles such as  Natronobacterium, Bacillus firmus  OF4 , Spirulina  spp. (e.g., pH&gt;9) and acidophiles such as  Cyanidium caldarium, Ferroplasma  sp. (e.g., low pH). Anaerobes, which cannot tolerate O 2  such as  Methanococcus jannaschii ; microaerophils, which tolerate some O 2  such as  Clostridium  and aerobes, which require O 2  are also contemplated. Gas-tolerant organisms, which tolerate pure CO 2  include  Cyanidium caldarium  and metal tolerant organisms include metalotolerants such as  Ferroplasma acidarmanus  (e.g., Cu, As, Cd, Zn),  Ralstonia  sp. CH34 (e.g., Zn, Co, Cd, Hg, Pb). Gross, Michael.  Life on the Edge: Amazing Creatures Thriving in Extreme Environments . New YorK: Plenum (1998) and Seckbach, J. “Search for Life in the Universe with Terrestrial Microbes Which Thrive Under Extreme Conditions.” In Cristiano Batalli Cosmovici, Stuart Bowyer, and Dan Wertheimer, eds.,  Astronomical and Biochemical Origins and the Search for Life in the Universe , p. 511. Milan: Editrice Compositori (1997). 
     Plants include but are not limited to the following genera:  Arabidopsis, Beta, Glycine, Jatropha, Miscanthus, Panicum, Phalaris, Populus, Saccharum, Salix, Simmondsia  and  Zea.    
     Algae and cyanobacteria include but are not limited to the following genera:  Acanthoceras, Acanthococcus, Acaryochloris, Achnanthes, Achnanthidium, Actinastrum, Actinochloris, Actinocyclus, Actinotaenium, Amphichrysis, Amphidinium, Amphikrikos, Amphipleura, Amphiprora, Amphithrix, Amphora, Anabaena, Anabaenopsis, Aneumastus, Ankistrodesmus, Ankyra, Anomoeoneis, Apatococcus, Aphanizomenon, Aphanocapsa, Aphanochaete, Aphanothece, Apiocystis, Apistonema, Arthrodesmus, Artherospira, Ascochloris, Asterionella, Asterococcus, Audouinella, Aulacoseira, Bacillaria, Balbiania, Bambusina, Bangia, Basichlamys, Batrachospermum, Binuclearia, Bitrichia, Blidingia, Botrdiopsis, Botrydium, Botryococcus, Botryosphaerella, Brachiomonas, Brachysira, Brachytrichia, Brebissonia, Bulbochaete, Bumilleria, Bumilleriopsis, Caloneis, Calothrix, Campylodiscus, Capsosiphon, Carteria, Catena, Cavinula, Centritractus, Centronella, Ceratium, Chaetoceros, Chaetochloris, Chaetomorpha, Chaetonella, Chaetonema, Chaetopeltis, Chaetophora, Chaetosphaeridium, Chamaesiphon, Chara, Characiochloris, Characiopsis, Characium, Charales, Chilomonas, Chlainomonas, Chlamydoblepharis, Chlamydocapsa, Chlamydomonas, Chlamydomonopsis, Chlamydomyxa, Chlamydonephris, Chlorangiella, Chlorangiopsis, Chlorella, Chlorobotrys, Chlorobrachis, Chlorochytrium, Chlorococcum, Chlorogloea, Chlorogloeopsis, Chlorogonium, Chlorolobion, Chloromonas, Chlorophysema, Chlorophyta, Chlorosaccus, Chlorosarcina, Choricystis, Chromophyton, Chromulina, Chroococcidiopsis, Chroococcus, Chroodactylon, Chroomonas, Chroothece, Chrysamoeba, Chrysapsis, Chrysidiastrum, Chrysocapsa, Chrysocapsella, Chrysochaete, Chrysochromulina, Chrysococcus, Chrysocrinus, Chrysolepidomonas, Chrysolykos, Chrysonebula, Chrysophyta, Chrysopyxis, Chrysosaccus, Chrysophaerella, Chrysostephanosphaera, Clodophora, Clastidium, Closteriopsis, Closterium, Coccomyxa, Cocconeis, Coelastrella, Coelastrum, Coelosphaerium, Coenochloris, Coenococcus, Coenocystis, Colacium, Coleochaete, Collodictyon, Compsogonopsis, Compsopogon, Conjugatophyta, Conochaete, Coronastrum, Cosmarium, Cosmioneis, Cosmocladium, Crateriportula, Craticula, Crinalium, Crucigenia, Crucigeniella, Cryptoaulax, Cryptomonas, Cryptophyta, Ctenophora, Cyanodictyon, Cyanonephron, Cyanophora, Cyanophyta, Cyanothece, Cyanothomonas, Cyclonexis, Cyclostephanos, Cyclotella, Cylindrocapsa, Cylindrocystis, Cylindrospermum, Cylindrotheca, Cymatopleura, Cymbella, Cymbellonitzschia, Cystodinium Dactylococcopsis, Debarya, Denticula, Dermatochrysis, Dermocarpa, Dermocarpella, Desmatractum, Desmidium, Desmococcus, Desmonema, Desmosiphon, Diacanthos, Diacronema, Diadesmis, Diatoma, Diatomella, Dicellula, Dichothrix, Dichotomococcus, Dicranochaete, Dictyochloris, Dictyococcus, Dictyosphaerium, Didymocystis, Didymogenes, Didymosphenia, Dilabifilum, Dimorphococcus, Dinobryon, Dinococcus, Diplochloris, Diploneis, Diplostauron, Distrionella, Docidium, Draparnaldia, Dunaliella, Dysmorphococcus, Ecballocystis, Elakatothrix, Ellerbeckia, Encyonema, Enteromorpha, Entocladia, Entomoneis, Entophysalis, Epichrysis, Epipyxis, Epithemia, Eremosphaera, Euastropsis, Euastrum, Eucapsis, Eucocconeis, Eudorina, Euglena, Euglenophyta, Eunotia, Eustigmatophyta, Eutreptia, Fallacia, Fischerella, Fragilaria, Fragilariforma, Franceia, Frustulia, Curcilla, Geminella, Genicularia, Glaucocystis, Glaucophyta, Glenodiniopsis, Glenodinium, Gloeocapsa, Gloeochaete, Gloeochrysis, Gloeococcus, Gloeocystis, Gloeodendron, Gloeomonas, Gloeoplax, Gloeothece, Gloeotila, Gloeotrichia, Gloiodictyon, Golenkinia, Golenkiniopsis, Gomontia, Gomphocymbella, Gomphonema, Gomphosphaeria, Gonatozygon, Gongrosia, Gongrosira, Goniochloris, Gonium, Gonyostomum, Granulochloris, Granulocystopsis, Groenbladia, Gymnodinium, Gymnozyga, Gyrosigma, Haematococcus, Hafniomonas, Hallassia, Hammatoidea, Hannaea, Hantzschia, Hapalosiphon, Haplotaenium, Haptophyta, Haslea, Hemidinium, Hemitoma, Heribaudiella, Heteromastix, Heterothrix, Hibberdia, Hildenbrandia, Hillea, Holopedium, Homoeothrix, Hormanthonema, Hormotila, Hyalobrachion, Hyalocardium, Hyalodiscus, Hyalogonium, Hyalotheca, Hydrianum, Hydrococcus, Hydrocoleum, Hydrocoryne, Hydrodictyon, Hydrosera, Hydrurus, Hyella, Hymenomonas, Isthmochloron, Johannesbaptistia, Juranyiella, Karayevia, Kathablepharis, Katodinium, Kephyrion, Keratococcus, Kirchneriella, Klebsormidium, Kolbesia, Koliella, Komarekia, Korshikoviella, Kraskella, Lagerheimia, Lagynion, Lamprothamnium, Lemanea, Lepocinclis, Leptosira, Lobococcus, Lobocystis, Lobomonas, Luticola, Lyngbya, Malleochloris, Mallomonas, Mantoniella, Marssoniella, Martyana, Mastigocoleus, Gastogloia, Melosira, Merismopedia, Mesostigma, Mesotaenium, Micractinium, Micrasterias, Microchaete, Microcoleus, Microcystis, Microglena, Micromonas, Microspora, Microthamnion, Mischococcus, Monochrysis, Monodus, Monomastix, Monoraphidium, Monostroma, Mougeotia, Mougeotiopsis, Myochloris, Myromecia, Myxosarcina, Naegeliella, Nannochloris, Nautococcus, Navicula, Neglectella, Neidium, Nephroclamys, Nephrocytium, Nephrodiella, Nephroselmis, Netrium, Nitella, Nitellopsis, Nitzschia, Nodularia, Nostoc, Ochromonas, Oedogonium, Oligochaetophora, Onychonema, Oocardium, Oocystis, Opephora, Ophiocytium, Orthoseira, Oscillatoria, Oxyneis, Pachycladella, Palmella, Palmodictyon, Pnadorina, Pannus, Paralia, Pascherina, Paulschulzia, Pediastrum, Pedinella, Pedinomonas, Pedinopera, Pelagodictyon, Penium, Peranema, Peridiniopsis, Peridinium, Peronia, Petroneis, Phacotus, Phacus, Phaeaster, Phaeodermatium, Phaeophyta, Phaeosphaera, Phaeothamnion, Phormidium, Phycopeltis, Phyllariochloris, Phyllocardium, Phyllomitas, Pinnularia, Pitophora, Placoneis, Planctonema, Planktosphaeria, Planothidium, Plectonema, Pleodorina, Pleurastrum, Pleurocapsa, Pleurocladia, Pleurodiscus, Pleurosigma, Pleurosira, Pleurotaenium, Pocillomonas, Podohedra, Polyblepharides, Polychaetophora, Polyedriella, Polyedriopsis, Polygoniochloris, Polyepidomonas, Polytaenia, Polytoma, Polytomella, Porphyridium, Posteriochromonas, Prasinochloris, Prasinocladus, Prasinophyta, Prasiola, Prochlorphyta, Prochlorothrix, Protoderma, Protosiphon, Provasoliella, Prymnesium, Psammodictyon, Psammothidium, Pseudanabaena, Pseudenoclonium, Psuedocarteria, Pseudochate, Pseudocharacium, Pseudococcomyxa, Pseudodictyosphaerium, Pseudokephyrion, Pseudoncobyrsa, Pseudoquadrigula, Pseudosphaerocystis, Pseudostaurastrum, Pseudostaurosira, Pseudotetrastrum, Pteromonas, Punctastruata, Pyramichlamys, Pyramimonas, Pyrrophyta, Quadrichloris, Quadricoccus, Quadrigula, Radiococcus, Radiofilum, Raphidiopsis, Raphidocelis, Raphidonema, Raphidophyta, Peimeria, Rhabdoderma, Rhabdomonas, Rhizoclonium, Rhodomonas, Rhodophyta, Rhoicosphenia, Rhopalodia, Rivularia, Rosenvingiella, Rossithidium, Roya, Scenedesmus, Scherffelia, Schizochlamydella, Schizochlamys, Schizomeris, Schizothrix, Schroederia, Scolioneis, Scotiella, Scotiellopsis, Scourfieldia, Scytonema, Selenastrum, Selenochloris, Sellaphora, Semiorbis, Siderocelis, Diderocystopsis, Dimonsenia, Siphononema, Sirocladium, Sirogonium, Skeletonema, Sorastrum, Spermatozopsis, Sphaerellocystis, Sphaerellopsis, Sphaerodinium, Sphaeroplea, Sphaerozosma, Spiniferomonas, Spirogyra, Spirotaenia, Spirulina, Spondylomorum, Spondylosium, Sporotetras, Spumella, Staurastrum, Stauerodesmus, Stauroneis, Staurosira, Staurosirella, Stenopterobia, Stephanocostis, Stephanodiscus, Stephanoporos, Stephanosphaera, Stichococcus, Stichogloea, Stigeoclonium, Stigonema, Stipitococcus, Stokesiella, Strombomonas, Stylochrysalis, Stylodinium, Styloyxis, Stylosphaeridium, Surirella, Sykidion, Symploca, Synechococcus, Synechocystis, Synedra, Synochromonas, Synura, Tabellaria, Tabularia, Teilingia, Temnogametum, Tetmemorus, Tetrachlorella, Tetracyclus, Tetradesmus, Tetraedriella, Tetraedron, Tetraselmis, Tetraspora, Tetrastrum, Thalassiosira, Thamniochaete, Thorakochloris, Thorea, Tolypella, Tolypothrix, Trachelomonas, Trachydiscus, Trebouxia, Trentepholia, Treubaria, Tribonema, Trichodesmium, Trichodiscus, Trochiscia, Tryblionella, Ulothrix, Uroglena, Uronema, Urosolenia, Urospora, Uva, Vacuolaria, Vaucheria, Volvox, Volvulina, Westella, Woloszynskia, Xanthidium, Xanthophyta, Xenococcus, Zygnema, Zygnemopsis , and  Zygonium.    
     Additional cyanobacteria include members of the genus Chamaesiphon,  Chroococcus, Cyanobacterium, Cyanobium, Cyanothece, Dactylococcopsis, Gloeobacter, Gloeocapsa, Gloeothece, Microcystis, Prochlorococcus, Prochloron, Synechococcus, Synechocystis, Cyanocystis, Dermocarpella, Stanieria, Xenococcus, Chroococcidiopsis, Myxosarcina, Arthrospira, Borzia, Crinalium, Geitlerinemia, Leptolyngbya, Limnothrix, Lyngbya, Microcoleus, Oscillatoria, Planktothrix, Prochiorothrix, Pseudanabaena, Spirulina, Starria, Symploca, Trichodesmium, Tychonema, Anabaena, Anabaenopsis, Aphanizomenon, Cyanospira, Cylindrospermopsis, Cylindrospermum, Nodularia, Nostoc, Scylonema, Calothrix, Rivularia, Tolypothrix, Chlorogloeopsis, Fischerella, Geitieria, Iyengariella, Nostochopsis, Stigonema  and  Thermosynechococcus.    
     Green non-sulfur bacteria include but are not limited to the following genera:  Chloroflexus, Chloronema, Oscillochloris, Heliothrix, Herpetosiphon, Roseiflexus , and  Thermomicrobium.    
     Green sulfur bacteria include but are not limited to the following genera: 
       Chlorobium, Clathrochloris , and  Prosthecochloris.    
     Purple sulfur bacteria include but are not limited to the following genera:  Allochromatium, Chromatium, Halochromatium, Isochromatium, Marichromatium, Rhodovulum, Thermochromatium, Thiocapsa, Thiorhodococcus , and  Thiocystis,    
     Purple non-sulfur bacteria include but are not limited to the following genera:  Phaeospirillum, Rhodobaca, Rhodobacter, Rhodomicrobium, Rhodopila, Rhodopseudomonas, Rhodothalassium, Rhodospirillum, Rodovibrio , and  Roseospira.    
     Aerobic chemolithotrophic bacteria include but are not limited to nitrifying bacteria such as  Nitrobacteraceae  sp.,  Nitrobacter  sp.,  Nitrospina  sp.,  Nitrococcus  sp.,  Nitrospira  sp.,  Nitrosomonas  sp.,  Nitrosococcus  sp.,  Nitrosospira  sp.,  Nitrosolobus  sp.,  Nitrosovibrio  sp.; colorless sulfur bacteria such as,  Thiovulum  sp.,  Thiobacillus  sp.,  Thiomicrospira  sp.,  Thiosphaera  sp.,  Thermothrix  sp.; obligately chemolithotrophic hydrogen bacteria such as  Hydrogenobacter  sp., iron and manganese-oxidizing and/or depositing bacteria such as  Siderococcus  sp., and magnetotactic bacteria such as  Aquaspirillum  sp. 
     Archaeobacteria include but are not limited to methanogenic archaeobacteria such as  Methanobacterium  sp.,  Methanobrevibacter  sp.,  Methanothermus  sp.,  Methanococcus  sp.,  Methanomicrobium  sp.,  Methanospirillum  sp.,  Methanogenium  sp.,  Methanosarcina  sp.,  Methanolobus  sp.,  Methanothrix  sp.,  Methanococcoides  sp.,  Methanoplanus  sp.; extremely thermophilic S-Metabolizers such as  Thermoproteus  sp.,  Pyrodictium  sp.,  Sulfolobus  sp.,  Acidianus  sp. and other microorganisms such as,  Bacillus subtilis, Saccharomyces cerevisiae, Streptomyces  sp.,  Ralstonia  sp.,  Rhodococcus  sp.,  Corynebacteria  sp.,  Brevibacteria  sp.,  Mycobacteria  sp., and oleaginous yeast. 
     Preferred organisms for the manufacture of esters according to the methods disclosed herein include:  Arabidopsis thaliana, Panicum virgatum, Miscanthus giganteus , and  Zea mays  (plants);  Botryococcus braunii, Chlamydomonas reinhardtii  and  Dunaliela salina  (algae);  Synechococcus  sp PCC 7002 , Synechococcus  sp. PCC 7942,  Synechocystis  sp. PCC 6803 , Thermosynechococcus elongatus  BP-1 (cyanobacteria);  Chlorobium tepidum  (green sulfur bacteria),  Chloroflexus auranticus  (green non-sulfur bacteria);  Chromatium tepidum  and  Chromatium vinosum  (purple sulfur bacteria);  Rhodospirillum rubrum, Rhodobacter capsulatus , and  Rhodopseudomonas palusris  (purple non-sulfur bacteria). 
     Yet other suitable organisms include synthetic cells or cells produced by synthetic genomes as described in Venter et al. US Pat. Pub. No. 2007/0264688, and cell-like systems or synthetic cells as described in Glass et al. US Pat. Pub. No. 2007/0269862. 
     Still, other suitable organisms include microorganisms that can be engineered to fix carbon dioxide, such as  Escherichia coli, Acetobacter aceti, Bacillus subtilis , yeast and fungi such as  Clostridium ljungdahlii, Clostridium thermocellum, Penicillium chrysogenum, Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pseudomonas fluorescens , or  Zymomonas mobilis.    
     The capability to use carbon dioxide as the sole source of cell carbon (autotrophy) is found in almost all major groups of prokaryotes. The CO 2  fixation pathways differ between groups, and there is no clear distribution pattern of the four presently-known autotrophic pathways. See, e.g., Fuchs, G. 1989 . Alternative pathways of autotrophic CO   2    fixation,  p. 365-382, in H. G. Schlegel, and B. Bowien (ed.), Autotrophic bacteria. Springer-Verlag, Berlin, Germany. The reductive pentose phosphate cycle (Calvin-Bassham-Benson cycle) represents the CO 2  fixation pathway in almost all aerobic autotrophic bacteria, for example, the cyanobacteria. 
     For producing esters via the recombinant expression of thioesterase, acyl-CoA synthetase and/or wax synthase enzymes, an engineered cyanobacteria, e.g., a  Synechococcus  or  Thermosynechococcus  species, is especially preferred. Other preferred organisms include  Synechocystis, Klebsiella oxytoca, Escherichia coli  or  Saccharomyces cerevisiae . Other prokaryotic, archaea and eukaryotic host cells are also encompassed within the scope of the present invention. Engineered ester-producing organisms expressing thioesterase, acyl-CoA synthetase and/or wax synthase enzymes can be further engineered to express recombinant TolC/AcrAB to enhance the extracellular transport of esters. 
     Carbon-Based Products of Interest: Esters 
     In various embodiments of the invention, desired esters or a mixture thereof can be produced. For example, by including a particular alcohol or mixture of alcohols in the culture media, methyl esters, ethyl esters, propyl esters, butyl esters, and esters of higher chain length alcohols (or mixtures thereof, depending on the substrate alcohols available to the photosynthetic microbe) can be synthesized. The carbon chain lengths of the esters can vary from C 10  to C 20 , e.g., using ethanol as a substrate, diverse esters including, e.g., ethyl myristate, ethyl palmitate, ethyl oleate, and/or ethyl stearate and/or mixtures thereof can be produced by a single engineered photosynthetic microorganism of the invention. Accordingly, the invention provides methods and compositions for the production of various chain lengths of esters, each of which is suitable for use as a fuel or any other chemical use. 
     In preferred aspects, the methods provide culturing host cells for direct product secretion for easy recovery without the need to extract biomass. These carbon-based products of interest are secreted directly into the medium. Since the invention enables production of various defined chain length of hydrocarbons and alcohols, the secreted products are easily recovered or separated. The products of the invention, therefore, can be used directly or used with minimal processing. 
     Media and Culture Conditions 
     One skilled in the art will recognize that a variety of media and culture conditions can be used in conjunction with the methods and engineered cyanobacteria disclosed herein for the bioproduction of fatty acid esters (see, e.g., Rogers and Gallon,  Biochemistry of the Algae and Cyanobacteria , Clarendon Press Oxford (1988); Burlwe,  Algal Culture: From Laboratory to Pilot Plant , Carnegie Institution of Washington Publication 600 Washington, D.C., (1961); and Round, F. E. The Biology of the Algae. St Martin&#39;s Press, New York, 1965; Golden S S et al. (1987)  Methods Enzymol  153:215-231; Golden and Sherman,  J. Bacteriology  158:36 (1984), each of which is incorporated herein by reference). Exemplary culture conditions and media are also described in, e.g., WO/2010/068288, filed May 21, 2009, published Jun. 17, 2010, and incorporated by reference herein. Typical culture conditions for the methods of the present invention include the use of JB 2.1 culture media or A+ media. A recipe for one liter of JB 2.1 appears in Table A, below. 
     
       
         
           
               
             
               
                 TABLE A 
               
             
            
               
                   
               
               
                 JB 2.1 media (1 L) 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 mg/L 
                   
                   
                   
                   
               
               
                 Chemical 
                 added 
                 FW 
                 Molarity 
                 Units 
                 Source 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 NaCl 
                 18000 
                 58.44 
                 308 
                 mM 
                 Fisher 
               
               
                 KCl 
                 600 
                 74.55 
                 8.05 
                 mM 
                 Fisher 
               
               
                 NaNO 3   
                 4000 
                 84.99 
                 47.06 
                 mM 
                 Sigma 
               
               
                   
                   
                   
                   
                   
                 Aldrich 
               
               
                 MgSO 4 —7H 2 O 
                 5000 
                 246.47 
                 20.29 
                 mM 
                 Sigma 
               
               
                   
                   
                   
                   
                   
                 Aldrich 
               
               
                 KH 2 PO 4   
                 200 
                 136.09 
                 1.47 
                 mM 
                 Fisher 
               
               
                 CaCl 2   
                 266 
                 110.99 
                 2.40 
                 mM 
                 Sigma 
               
               
                 NaEDTA tetra   
                 30 
                 372.24 
                 80.59 
                 μM 
                 Fisher 
               
               
                 Ferric Citrate 
                 14.1 
                 244.95 
                 57.48 
                 μM 
                 Acros 
               
               
                   
                   
                   
                   
                   
                 Organics 
               
               
                 Tris 
                 1000 
                 121.14 
                 8.25 
                 mM 
                 Fisher 
               
               
                 Vitamin B 12   
                 0.004 
                 1355.37 
                 2.95E−03 
                 μM 
                 Sigma 
               
               
                 (Cyanoco- 
                   
                   
                   
                   
                 Aldrich 
               
               
                 balamin) 
               
               
                 H 3 BO 3   
                 34 
                 61.83 
                 554 
                 μM 
                 Acros 
               
               
                   
                   
                   
                   
                   
                 Organics 
               
               
                 MnCl 2 —4H 2 O 
                 4.3 
                 197.91 
                 21.83 
                 μM 
                 Sigma 
               
               
                 ZnCl 
                 0.32 
                 136.28 
                 2.31 
                 μM 
                 Sigma 
               
               
                 MoO 3   
                 0.030 
                 143.94 
                 0.21 
                 μM 
                 Sigma 
               
               
                   
                   
                   
                   
                   
                 Aldrich 
               
               
                 CuSO 4 —5H 2 O 
                 0.0030 
                 249.69 
                 0.012 
                 μM 
                 Sigma 
               
               
                   
                   
                   
                   
                   
                 Aldrich 
               
               
                 CoCl 2 —6H 2 O 
                 0.012 
                 237.93 
                 0.051 
                 μM 
                 Sigma 
               
               
                   
               
            
           
         
       
     
     As described in more detail in the Examples, below, in certain embodiments one or more alcohols (e.g., methanol, ethanol, propanol, butanol, etc.) may be added during culturing to produce the desired fatty acid ester(s) of interest (e.g., a fatty acid methyl ester, a fatty acid ethyl ester, etc., and mixtures thereof). For organisms that require or metabolize most efficiently in the presence of light and carbon dioxide, either carbon dioxide or bicarbonate can be used during culturing. 
     Fuel Compositions 
     In various embodiments, compositions produced by the methods of the invention are used as fuels. Such fuels comply with ASTM standards, for instance, standard specifications for diesel fuel oils D 975-09b, and Jet A, Jet A-1 and Jet B as specified in ASTM Specification D. 1655-68. Fuel compositions may require blending of several products to produce a uniform product. The blending process is relatively straightforward, but the determination of the amount of each component to include in a blend is much more difficult. Fuel compositions may, therefore, include aromatic and/or branched hydrocarbons, for instance, 75% saturated and 25% aromatic, wherein some of the saturated hydrocarbons are branched and some are cyclic. Preferably, the methods of the invention produce an array of hydrocarbons, such as C 13 -C 17  or C 10 -C 15  to alter cloud point. Furthermore, the compositions may comprise fuel additives, which are used to enhance the performance of a fuel or engine. For example, fuel additives can be used to alter the freezing/gelling point, cloud point, lubricity, viscosity, oxidative stability, ignition quality, octane level, and flash point. Fuels compositions may also comprise, among others, antioxidants, static dissipater, corrosion inhibitor, icing inhibitor, biocide, metal deactivator and thermal stability improver. 
     In addition to many environmental advantages of the invention such as CO 2  conversion and renewable source, other advantages of the fuel compositions disclosed herein include low sulfur content, low emissions, being free or substantially free of alcohol and having high cetane number. 
     Carbon Fingerprinting 
     Biologically-produced carbon-based products, e.g., ethanol, fatty acids, alkanes, isoprenoids, represent a new commodity for fuels, such as alcohols, diesel and gasoline. Such biofuels have not been produced using biomass but use CO2 as its carbon source. These new fuels may be distinguishable from fuels derived form petrochemical carbon on the basis of dual carbon-isotopic fingerprinting. Such products, derivatives, and mixtures thereof may be completely distinguished from their petrochemical derived counterparts on the basis of  14 C (fM) and dual carbon-isotopic fingerprinting, indicating new compositions of matter. 
     There are three naturally occurring isotopes of carbon:  12 C,  13 C, and  14 C. These isotopes occur in above-ground total carbon at fractions of 0.989, 0.011, and 10 −12 , respectively. The isotopes  12 C and  13 C are stable, while  14 C decays naturally to  14 N, a beta particle, and an anti-neutrino in a process with a half-life of 5730 years. The isotope  14 C originates in the atmosphere, due primarily to neutron bombardment of  14 N caused ultimately by cosmic radiation. Because of its relatively short half-life (in geologic terms),  14 C occurs at extremely low levels in fossil carbon. Over the course of 1 million years without exposure to the atmosphere, just 1 part in 10 50  will remain  14 C. 
     The  13 C: 12 C ratio varies slightly but measurably among natural carbon sources. Generally these differences are expressed as deviations from the  13 C: 12 C ratio in a standard material. The international standard for carbon is Pee Dee Belemnite, a form of limestone found in South Carolina, with a  13 C fraction of 0.0112372. For a carbon source a, the deviation of the  13 C: 12 C ratio from that of Pee Dee Belemnite is expressed as: δ a =(R a /R s )−1, where R a = 13 C: 12 C ratio in the natural source, and R s = 13 C: 12 C ratio in Pee Dee Belemnite, the standard. For convenience, δ a  is expressed in parts per thousand, or ‰. A negative value of δ a  shows a bias toward  12 C over  13 C as compared to Pee Dee Belemnite. Table 1 shows δ a  and  14 C fraction for several natural sources of carbon. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 13C:12C variations in natural carbon sources 
               
            
           
           
               
               
               
            
               
                 Source 
                 −δ a  (‰) 
                 References 
               
               
                   
               
            
           
           
               
               
               
            
               
                 Underground coal 
                 32.5 
                 Farquhar et al. (1989)  Plant Mol. Biol. , 40: 503-37 
               
               
                 Fossil fuels 
                 26 
                 Farquhar et al. (1989)  Plant Mol. Biol. , 40: 503-37 
               
               
                 Ocean DIC* 
                   0-1.5 
                 Goericke et al. (1994) Chapter 9 in 
               
               
                   
                   
                   Stable Isotopes in Ecology and Environmental Science , 
               
               
                   
                   
                 by K. Lajtha and R. H. Michener, Blackwell Publishing; 
               
               
                   
                   
                 Ivlev (2010)  Separation Sci. Technol . 36: 1819-1914 
               
               
                 Atmospheric 
                 6-8 
                 Ivlev (2010)  Separation Sci. Technol.  36: 1819-1914; 
               
               
                 CO2 
                   
                 Farquhar et al. (1989)  Plant Mol. Biol. , 40: 503-37 
               
               
                 Freshwater DIC* 
                  6-14 
                 Dettman et al. (1999)  Geochim. Cosmochim. Acta   
               
               
                   
                   
                 63: 1049-1057 
               
               
                 Pee Dee Belemnite 
                 0 
                 Ivlev (2010)  Separation Sci. Technol.  36: 1819-1914 
               
               
                   
               
               
                 *DIC = dissolved inorganic carbon 
               
            
           
         
       
     
     Biological processes often discriminate among carbon isotopes. The natural abundance of  14 C is very small, and hence discrimination for or against  14 C is difficult to measure. Biological discrimination between  13 C and  12 C, however, is well-documented. For a biological product p, we can define similar quantities to those above: δ p =(R p /R s )−1, where R p = 13 C: 12 C ratio in the biological product, and R s = 13 C: 12 C ratio in Pee Dee Belemnite, the standard. Table 2 shows measured deviations in the  13 C: 12 C ratio for some biological products. 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                   13 C: 12 C variations in selected biological products 
               
            
           
           
               
               
               
               
            
               
                 Product 
                 −δ p (‰) 
                 −D(‰)* 
                 References 
               
               
                   
               
               
                 Plant sugar/starch from 
                 18-28 
                     10-20 
                 Ivlev (2010)  Separation Sci. Technol.  36: 1819-1914 
               
               
                 atmospheric CO 2   
               
               
                 Cyanobacterial biomass from 
                 18-31 
                 16.5-31 
                 Goericke et al. (1994) Chapter 9 in 
               
               
                 marine DIC 
                   
                   
                   Stable Isotopes in Ecology and Environmental Science , 
               
               
                   
                   
                   
                 by K. Lajtha and R. H. Michener, Blackwell Publishing; 
               
               
                   
                   
                   
                 Sakata et al. (1997)  Geochim. Cosmochim. Acta , 
               
               
                   
                   
                   
                 61: 5379-89 
               
               
                 Cyanobacterial lipid from 
                 39-40 
                 37.5-40 
                 Sakata et al. (1997)  Geochim. Cosmochim. Acta , 
               
               
                 marine DIC 
                   
                   
                 61: 5379-89 
               
               
                 Algal lipid from marine DIC 
                 17-28 
                 15.5-28 
                 Goericke et al. (1994) Chapter 9 in 
               
               
                   
                   
                   
                   Stable Isotopes in Ecology and Environmental Science , 
               
               
                   
                   
                   
                 by K. Lajtha and R. H. Michener, Blackwell Publishing; 
               
               
                   
                   
                   
                 Abelseon et al. (1961)  Proc. Natl. Acad. Sci. , 47: 623-32 
               
               
                 Algal biomass from 
                 17-36 
                   3-30 
                 Marty et al. (2008)  Limnol. Oceanogr.: Methods  6: 51-63 
               
               
                 freshwater DIC 
               
               
                   E. coli  lipid from plant sugar 
                 15-27 
                 near 0 
                 Monson et al. (1980)  J. Biol. Chem. , 255: 11435-41 
               
               
                 Cyanobacterial lipid from fossil 
                 63.5-66     
                 37.5-40 
                 — 
               
               
                 carbon 
               
               
                 Cyanobacterial biomass from 
                 42.5-57     
                 16.5-31 
                 — 
               
               
                 fossil carbon 
               
               
                   
               
               
                 *D = discrimination by a biological process in its utilization of  12 C vs.  13 C (see text) 
               
            
           
         
       
     
     Table 2 introduces a new quantity, D. This is the discrimination by a biological process in its utilization of  12 C vs.  13 C. We define D as follows: D=(R p /R a )−1. This quantity is very similar to δ a  and δ p , except we now compare the biological product directly to the carbon source rather than to a standard. Using D, we can combine the bias effects of a carbon source and a biological process to obtain the bias of the biological product as compared to the standard. Solving for δ p , we obtain: δ p =(D)(δ a )+D+δ a , and, because (D)(δ a ) is generally very small compared to the other terms, δ p ≈δ a +D. 
     For a biological product having a production process with a known D, we may therefore estimate δ p  by summing δ a  and D. We assume that D operates irrespective of the carbon source. This has been done in Table 1 for cyanobacterial lipid and biomass produced from fossil carbon. As shown in the Table 1 and Table 2, above, cyanobacterial products made from fossil carbon (in the form of, for example, flue gas or other emissions) will have a higher δ p  than those of comparable biological products made from other sources, distinguishing them on the basis of composition of matter from these other biological products. In addition, any product derived solely from fossil carbon will have a negligible fraction of  14 C, while products made from above-ground carbon will have a  14 C fraction of approximately 10 −12 . 
     Accordingly, in certain aspects, the invention provides various carbon-based products of interest characterized as −δ p (‰) of about 63.5 to about 66 and −D(‰) of about 37.5 to about 40. 
     The following examples are for illustrative purposes and are not intended to limit the scope of the present invention. 
     Example 1 
     Recombinant Genes for the Biosynthesis of Biodiesel and Biodiesel-Like Compounds 
     In one embodiment of the invention, a  cyanobacterium  strain is transformed or engineered to express one or more enzymes selected from the following list: a wax synthase (EC: 2.3.175), a thioesterase (EC: 3.1.2.-, 3.1.2.14), and an acyl-CoA synthase (EC: 6.2.1.3). For example, a typical embodiment utilizes a thioesterase gene from  E. coli  (tesA; SEQ ID NO:1), an acyl-CoA synthetase gene from  E. coli  (fadD; SEQ ID NO:2), and a wax synthase gene from  A. baylyi  (wax; SEQ ID NO:3). Thioesterase generates fatty acid from acyl-ACP. Acyl-CoA synthetase (also referred to as acyl-CoA ligase) generates fatty acyl-CoA from fatty acid. Wax synthase (EC 2.3.1.75) generates fatty acid esters using acyl-CoA and acyl alcohol as substrates (e.g., methanol, ethanol, butanol, etc). 
     Additional thioesterase, acyl-CoA synthetase and wax synthases genes that can be recombinantly expressed in cyanobacteria are set forth in Table 3A, Table 3B, and Table 3C, respectively. 
     
       
         
           
               
             
               
                 TABLE 3A 
               
             
            
               
                   
               
               
                 Exemplary Thioesterases* 
               
            
           
           
               
               
               
               
            
               
                   
                   
                 GenBank: 
                 Genbank: 
               
               
                   
                   
                 gene 
                 protein 
               
               
                   
                   
                 accession 
                 accession 
               
               
                 Source 
                 Enzyme 
                 number 
                 number 
               
               
                   
               
               
                 
                   E. coli 
                 
                 C-18:1 
                 NC_000913 
                 NP_415027 
               
               
                   
                 thioesterase 
               
               
                 
                   Cuphea 
                 
                 C-8:0 to C-10:0 
                 U39834.1 
                 AAC49269 
               
               
                 
                   hookeriana 
                 
                 thioesterase 
               
               
                 
                   Umbellularia 
                 
                 C-12:0 
                 M94159.1 
                 Q41635 
               
               
                 
                   california 
                 
                 thioesterase 
               
               
                 
                   Cinnamonum 
                 
                 C-14:0 
                 U17076.1 
                 Q39473 
               
               
                 
                   camphorum 
                 
                 thioesterase 
               
               
                 
                   Arabidopsis 
                 
                 C-18:1 
                 822102 
                 NP_189147.1 
               
               
                 
                   thaliana 
                 
                 thioesterase 
               
               
                   
               
               
                 *where leader sequences are present in the native protein, as in the case of  E. coli  tesA, the leader sequences are typically removed before the activity is recombinantly expressed 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 3B 
               
             
            
               
                   
               
               
                 Exemplary Acyl-CoA Synthetases 
               
            
           
           
               
               
               
               
            
               
                   
                   
                 GenBank: 
                 Genbank: 
               
               
                   
                   
                 gene 
                 protein 
               
               
                   
                   
                 accession 
                 accession 
               
               
                 Source 
                 Gene name 
                 number 
                 number 
               
               
                   
               
               
                 
                   E. coli 
                 
                 Acyl-CoA 
                 NC_000913 
                 NP_416319.1 
               
               
                   
                 synthetase 
               
               
                 
                   Geobacillus 
                 
                 Acyl-CoA 
                 CP000557.1 
                 ABO66726.1 
               
               
                 
                   thermodenitrificans 
                 
                 synthetase 
               
               
                 NG80-2 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 3C 
               
             
            
               
                   
               
               
                 Exemplary Wax Synthases 
               
            
           
           
               
               
               
               
            
               
                   
                   
                 GenBank: 
                 Genbank: 
               
               
                   
                   
                 gene 
                 protein 
               
               
                   
                 Gene or 
                 accession 
                 accession 
               
               
                 Source 
                 protein name 
                 number 
                 number 
               
               
                   
               
               
                 
                   Acinetobacter baylyi 
                 
                 wxs 
                 AF529086.1 
                 AAO17391.1 
               
               
                 
                   Mycobacterium 
                 
                 acyltransferase, 
                   
                 NP_218257.1 
               
               
                   tuberculosis  H37Rv 
                 WS/DGAT/MGAT 
               
               
                 
                   Saccharomyces 
                 
                 Eeb1 
                   
                 NP_015230 
               
               
                 
                   cerevisiae 
                 
               
               
                 
                   Saccharomyces 
                 
                 YMR210w 
                   
                 NP_013937 
               
               
                 
                   cerevisiae 
                 
               
               
                 
                   Rattus 
                 
                 FAEE synthase 
                   
                 P16303 
               
               
                   norvegicus  (rat) 
               
               
                 
                   Fundibacter 
                 
                 wst9 
               
               
                 
                   jadensis 
                 
               
               
                 DSM 12178 
               
               
                   Acinetobacter  sp. 
                 Wshn 
               
               
                 H01-N 
               
               
                 
                   H. sapiens 
                 
                 mWS 
               
               
                 
                   Fragaria xananassa 
                 
                 SAAT 
               
               
                 
                   Malus xdomestica 
                 
                 mpAAT 
               
               
                 
                   Simmondsia 
                 
                 JjWs 
                   
                 Q9XGY6 
               
               
                 
                   chinensis 
                 
               
               
                 
                   Mus musculus 
                 
                 mWS 
                   
                 Q6E1M8 
               
               
                   
               
            
           
         
       
     
     The engineered  cyanobacterium  expressing one or more of the thioesterase, acyl-CoA synthetase, and wax synthase genes set forth above is grown in suitable media, under appropriate conditions (e.g., temperature, shaking, light, etc.). After a certain optical density is reached, the cells are separated from the spent medium by centrifugation. The cell pellet is re-suspended and the cell suspension and the spent medium are then extracted with a suitable solvent, e.g., ethyl acetate. The resulting ethyl acetate phases from the cell suspension and the supernatant are subjected to GC-MS analysis. The fatty acid esters in the ethyl acetate phases can be quantified, e.g., using commercial palmitic acid ethyl ester as a reference standard. 
     Fatty acid esters can be made according to this method by adding an alcohol (e.g., methanol, propanol, isopropanol, butanol, etc.) to the fermentation media, whereby fatty acid esters of the added alcohols are produced by the engineered  cyanobacterium . Alternatively, one or more alcohols can be synthesized by the engineered  cyanobacterium , natively or recombinantly, and used as substrates for fatty acid ester synthesis by a recombinantly expressed wax synthase. As detailed in the Examples below, the engineered  cyanobacterium  can also be modified to recombinantly expresses a TolC/AcrAB transporter to facilitate secretion of the fatty acid esters into the culture medium. 
     Example 2 
     Synthesis of Ethyl and Methyl Fatty Acid Esters by an Engineered  Cyanobacterium    
     Genes and Plasmids: 
     The pJB5 base vector was designed as an empty expression vector for recombination into  Synechococcus  sp. PCC 7002. Two regions of homology, the Upstream Homology Region (UHR) and the Downstream Homology Region (DHR), are designed to flank the construct of interest. These 500 bp regions of homology correspond to positions 3301-3800 and 3801-4300 (Genbank Accession NC — 005025) for UHR and DHR respectively. The aadA promoter, gene sequence, and terminator were designed to confer spectinomycin and streptomycin resistance to the integrated construct. For expression, pJB5 was designed with the aphII kanamycin resistance cassette promoter and ribosome binding site (RBS). Downstream of this promoter and RBS, the restriction endonuclease recognition site for NdeI, EcoRI, SpeI and PacI were inserted. Following the EcoRI site, the natural terminator from the alcohol dehydrogenase gene from  Zymomonas mobilis  (adhII) terminator was included. Convenient XbaI restriction sites flank the UHR and the DHR allowing cleavage of the DNA intended for recombination from the rest of the vector. 
     The  E. coli  thioesterase tesA gene with the leader sequence removed (SEQ ID NO:4; Genbank # NC — 000913; Chot and Cronan, 1993), the  E. coli  acyl-CoA synthetase fadD (SEQ ID NO:5; Genbank # NC — 000913; Kameda and Nunn, 1981) and the wax synthase gene (wax) from  Acinetobacter  baylyi strain ADPI (SEQ ID NO:6; Genbank # AF529086.1; Stöveken et al. 2005) were purchased from DNA 2.0, following codon optimization, checking for secondary structure effects, and removal of any unwanted restriction sites (NdeI, XhoI, BamHI, NgoMIV, NcoI, SacI, BsrGI, AvrII, BmtI, MluI, EcoRI, SbfI, NotI, SpeI, XbaI, Pad, AscI, FseI). These genes were received on a pJ201 vector and assembled into a three-gene operon (tesA-fadD-wax, SEQ ID NO: 10) with flanking NdeI-EcoRI sites on the recombination vector pJB5 under the control of the PaphII kanamycin resistance cassette promoter. A second plasmid (pJB532; SEQ ID NO:11) was constructed which is identical to pJB494 except the PaphII promoter was replaced with SEQ ID NO:12, a Ptrc promoter and a lacIq repressor. As a control, a third plasmid (pJB413) was prepared with only tesA under the control of the PaphII promoter. These plasmid constructs were named pJB494, pJB532, and pJB413, respectively. 
     Strain Construction: 
     The constructs described above were integrated onto the plasmid pAQ1 in  Synechococcus  sp. PCC 7002 according to the following protocol.  Synechococcus  7002 was grown for 48 h from colonies in an incubated shaker flask at 37° C. at 2% CO 2  to an OD 730  of 1 in A +  medium described in Frigaard et al.,  Methods Mol. Biol.,  274:325-340 (2004). 450 μL of culture was added to a epi-tube with 50 μL of 5 μg of plasmid DNA digested with XbaI ((New England Biolabs; Ipswitch, Mass.)) that was not purified following restriction digest. Cells were incubated in the dark for four hours at 37° C. The entire volume of cells was plated on A +  medium plates with 1.5% agarose and grown at 37° C. in a lighted incubator (40-60 μE/m2/s PAR, measured with a LI-250A light meter (LI-COR)) for about 24 hours. 25 μg/mL of spectinomycin was underlayed on the plates. Resistant colonies were visible in 7-10 days after further incubation, and recombinant strains were confirmed by PCR using internal and external primers to check insertion and confirm location of the genes on pAQ1 in the strains (Table 4). 
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 Joule Culture Collection (JCC) numbers of  Synechococcus   
               
               
                 sp. PCC 7002 recombinant strains with gene insertions 
               
               
                 on the native plasmid pAQ1 
               
            
           
           
               
               
               
               
               
            
               
                   
                 JCC # 
                 Promoter 
                 Genes 
                 Marker 
               
               
                   
                   
               
               
                   
                 JCC879 
                 PaphII 
                 — 
                 aadA 
               
               
                   
                 JCC750 
                 PaphII 
                 tesA 
                 aadA 
               
               
                   
                 JCC723 
                 PaphII 
                 tesA-fadD-wax 
                 aadA 
               
               
                   
                 JCC803 
                 lacIq Ptrc 
                 tesA-fadD-wax 
                 aadA 
               
               
                   
                   
               
            
           
         
       
     
     Ethyl Ester Production Culturing Conditions: 
     One colony of each of the four strains listed in Table 4 was inoculated into 10 ml of A+ media containing 50 μg/ml spectinomycin and 1% ethanol (v/v). These cultures were incubated for about 4 days in a bubble tube at 37° C. sparged at approximately 1-2 bubbles of 1% CO 2 /air every 2 seconds in light (40-50 μE/m2/s PAR, measured with a LI-250A light meter (LI-COR)). The cultures were then diluted so that the following day they would have OD 730  of 2-6. The cells were washed with 2×10 ml JB 2.1/spec200, and inoculated into duplicate 28 ml cultures in JB 2.1/spec200+1% ethanol (v/v) media to an OD 730 =0.07. IPTG was added to the JCC803 cultures to a final concentration of 0.5 mM. These cultures were incubated in a shaking incubator at 150 rpm at 37° C. under 2% CO 2 /air and continuous light (70-130 μE m2/s PAR, measured with a LI-250A light meter (LI-COR)) for ten days. Water loss through evaporation was replaced with the addition of sterile Milli-Q water. 0.5% (v/v) ethanol was added to the cultures to replace loss due to evaporation every 48 hours. At 68 and 236 hours, 5 ml and 3 ml of culture were removed from each flask for ethyl ester analysis, respectively. The OD 730  values reached by the cultures are given in Table 5. 
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 OD 730 s reached by recombinant  Synechococcus  sp. PCC 7002 strains 
               
               
                 at timepoints 68 and 236 h 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                   
                 JCC879 
                 JCC879 
                 JCC750 
                 JCC750 
                 JCC723 
                 JCC723 
                 JCC803 
                 JCC803 
               
               
                 Time point 
                 #1 
                 #2 
                 #1 
                 #2 
                 #1 
                 #2 
                 #1 
                 #2 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                  68 h 
                 3.6 
                 4.0 
                 4.6 
                 5.0 
                 6.6 
                 6.0 
                 5.4 
                 5.8 
               
               
                 236 h 
                 21.2 
                 18.5 
                 19.4 
                 20.9 
                 22.2 
                 21.4 
                 17.2 
                 17.7 
               
               
                   
               
            
           
         
       
     
     The culture aliquots were pelleted using a Sorvall RC6 Plus superspeed centrifuge (Thermo Electron Corp) and a F13S-14X50CY rotor (5000 rpm for 10 min). The spent media supernatant was removed and the cells were resuspended in 1 ml of Milli-Q water. The cells were pelleted again using a benchtop centrifuge, the supernatant discarded and the cell pellet was stored at −80° C. until analyzed for the presence of ethyl esters. 
     Detection and Quantification of Ethyl Esters in Strains: 
     Cell pellets were thawed and 1 ml aliquots of acetone (Acros Organics 326570010) containing 100 mg/L butylated hydroxytoluene (Sigma-Aldrich B1378) and 50 mg/L ethyl valerate (Fluka 30784) were added. The cell pellets were mixed with the acetone using a Pasteur pipettes and vortexed twice for 10 seconds (total extraction time of 1-2 min). The suspensions were centrifuged for 5 min to pellet debris, and the supernatants were removed with Pasteur pipettes and subjected to analysis with a gas chromatograph using flame ionization detection (GC/FID). 
     An Agilent 7890A GC/FID equipped with a 7683 series autosampler was used to detect the ethyl esters. One μL of each sample was injected into the GC inlet (split 5:1, pressure: 20 psi, pulse time: 0.3 min, purge time: 0.2 min, purge flow: 15 mL/min) and an inlet temperature of 280° C. The column was a HP-5MS (Agilent, 30 m×0.25 mm×0.25 μm) and the carrier gas was helium at a flow of 1.0 mL/min. The GC oven temperature program was 50° C., hold one minute; 10°/min increase to 280° C.; hold ten minutes. The GC/MS interface was 290° C., and the MS range monitored was 25 to 600 amu. Ethyl myristate [C14:0; retention time (rt): 17.8 min], ethyl palmitate (C16:0; rt: 19.8 min) and ethyl stearate (C18:0; rt: 21.6 min) were identified based on comparison to a standard mix of C4-C24 even carbon saturated fatty acid ethyl esters (Supelco 49454-U). Ethyl oleate (C18:1; rt: 21.4 min) was identified by comparison with an ethyl oleate standard (Sigma Aldrich 268011). These identifications were confirmed by GC/MS (see following Methyl Ester Production description for details). Calibration curves were constructed for these ethyl esters using the commercially available standards, and the concentrations of ethyl esters present in the extracts were determined and normalized to the concentration of ethyl valerate (internal standard). 
     Four different ethyl esters were found in the extracts of JCC723 and JCC803 (Table 6 and Table 7). In general, JCC803 produced 2-10× the amount of each ethyl ester than JCC723, but ethyl myristate (C14:0) was only produced in low quantities of 1 mg/L or less for all these cultures. Both JCC723 and JCC803 produced ethyl esters with the relative amounts C16:0&gt;C18:0&gt;C18:1 (cis-9)&gt;C14:0. No ethyl esters were found in the extracts of JCC879 or JCC750, indicating that the strain cannot make ethyl esters naturally and that expression of only the tesA gene is not sufficient to confer production of ethyl esters. 
     
       
         
           
               
             
               
                 TABLE 6 
               
             
            
               
                   
               
               
                 Amounts of respective ethyl esters found in the cell 
               
               
                 pellet extracts of JCC723 given as mg/L of culture 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                   
                   
                 C18:1 
                   
                   
               
               
                   
                 C14:0 
                 C16:0 
                 (cis-9) 
                 C18:0 
                 % 
               
               
                 Sample 
                 myristate 
                 palmitate 
                 oleate 
                 stearate 
                 Yield* 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 JCC723 #1 68 h 
                 0.08 
                 0.34 
                 0.22 
                 0.21 
                 0.04 
               
               
                 JCC723 #2 68 h 
                 0.12 
                 1.0 
                 0.43 
                 0.40 
                 0.1 
               
               
                 JCC803 #1 68 h 
                 0.45 
                 6.6 
                 1.4 
                 0.74 
                 0.6 
               
               
                 JCC803 #2 68 h 
                 0.63 
                 8.6 
                 2.0 
                 0.94 
                 0.7 
               
               
                 JCC723 #1 236 h 
                 1.04 
                 15.3 
                 2.1 
                 4.5 
                 0.3 
               
               
                 JCC723 #2 236 h 
                 0.59 
                 9.0 
                 1.3 
                 3.7 
                 0.2 
               
               
                 JCC803 #1 236 h 
                 0.28 
                 35.3 
                 13.4 
                 19.2 
                 1.3 
               
               
                 JCC803 #2 236 h 
                 0.49 
                 49.4 
                 14.9 
                 21.2 
                 1.6 
               
               
                   
               
               
                 *Yield (%) = ((sum of EEs)/dry cell weight)*100 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 7 
               
             
            
               
                   
               
               
                 % of total ethyl esters by mass 
               
            
           
           
               
               
               
               
               
            
               
                   
                 C14:0 
                 C16:0 
                 C18:1 
                 C18:0 
               
               
                 Sample 
                 myristate 
                 palmitate 
                 oleate 
                 stearate 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 JCC723 #1 68 h 
                 9.4 
                 40.0 
                 25.9 
                 24.7 
               
               
                 JCC723 #2 68 h 
                 6.2 
                 51.3 
                 22.1 
                 20.5 
               
               
                 JCC803 #1 68 h 
                 4.9 
                 71.8 
                 15.2 
                 8.1 
               
               
                 JCC803 #2 68 h 
                 5.2 
                 70.7 
                 16.4 
                 7.7 
               
               
                 JCC723 #1 236 h 
                 4.5 
                 66.7 
                 9.2 
                 19.6 
               
               
                 JCC723 #2 236 h 
                 4.0 
                 61.7 
                 8.9 
                 25.4 
               
               
                 JCC803 #1 236 h 
                 0.4 
                 51.8 
                 19.7 
                 28.2 
               
               
                 JCC803 #2 236 h 
                 0.6 
                 57.4 
                 17.3 
                 24.7 
               
               
                   
               
            
           
         
       
     
     Methyl Ester Production Culturing Conditions: 
     One colony of JCC803 (Table 1) was inoculated into 10 mL of A+ media containing 50 μg/ml spectinomycin and 1% ethanol (v/v). This culture was incubated for 3 days in a bubble tube at 37° C. sparged at approximately 1-2 bubbles of 1% CO 2 /air every 2 seconds in light (40-50 μE/m2/s PAR, measured with a LI-250A light meter (LI-COR)). The culture was innoculated into two flasks to a final volume of 20.5 ml and OD 730 =0.08 in A+ media containing 200 μg/ml spectinomycin and 0.5 mM IPTG with either 0.5% methanol or 0.5% ethanol (v/v). These cultures were incubated in a shaking incubator at 150 rpm at 37° C. under 2% CO 2 /air and continuous light (70-130 μE m2/s PAR, measured with a LI-250A light meter (LI-COR)) for three days. Water loss through evaporation was replaced with the addition of sterile Milli-Q water. Samples of 5 ml of these cultures (OD 730 =5-6) were analyzed for the presence of ethyl or methyl esters. 
     Detection of Methyl Esters and Comparison with Ethyl Ester Production in the Same Strain: 
     Cell pellets were thawed and 1 ml aliquots of acetone (Acros Organics 326570010) containing 100 mg/L butylated hydroxytoluene (Sigma-Aldrich B1378) and 50 mg/L ethyl valerate (Fluka 30784) were added. The cell pellets were mixed with the acetone using a Pasteur pipette and vortexed twice for 10 seconds (total extraction time of 1-2 min). The suspensions were centrifuged for 5 min to pellet debris, and the supernatants were removed with Pasteur pipettes and subjected to analysis with a gas chromatograph using mass spectral detection (GC/MS). 
     An Agilent 7890A GC/5975C EI-MS equipped with a 7683 series autosampler was used to measure the ethyl esters. One μL of each sample was injected into the GC inlet using pulsed splitless injection (pressure: 20 psi, pulse time: 0.3 min, purge time: 0.2 min, purge flow: 15 mL/min) and an inlet temperature of 280° C. The column was a HP-5MS (Agilent, 30 m×0.25 mm×0.25 μm) and the carrier gas was helium at a flow of 1.0 mL/min. The GC oven temperature program was 50° C., hold one minute; 10°/min increase to 280° C.; hold ten minutes. The GC/MS interface was 290° C., and the MS range monitored was 25 to 600 amu. Compounds indicated by peaks present in total ion chromatograms were identified by matching experimentally determined mass spectra associated with the peaks with mass spectral matches found by searching in a NIST 08 MS database. 
     The culture of JCC803 incubated with ethanol contained ethyl palmitate [C16:0; retention time (rt): 18.5 min], ethyl heptadecanoate (C17rt: 19.4 min), ethyl oleate (C18:1; rt: 20.1 min) and ethyl stearate (C18:0; rt: 20.3 min) ( FIG. 1 ). The relative amounts produced were C16:0&gt;C18:0&gt;C18:1&gt;C17:0. The production of low levels of C17:0 and the absence of measured levels of C14:0/myristate in this experiment is likely a result of the use of A+ medium (JB 2.1 was used to generate the date in Table 7, above). 
     No ethyl esters were detected in the strain incubated with methanol. Instead, methyl palmitate (C16:0; retention time (“rt”): 17.8 min), methyl heptadecanoate (C17:0; rt: 18.8 min) and methyl stearate (C18:0) were found ( FIG. 1 ; methyl palmitate: 0.1 mg/L; methyl heptadecanoate: 0.062 mg/L; methyl stearate: 0.058 mg/L; total FAMEs: 0.22 mg/L; % of DCW: 0.01). 
     The data presented herein shows that JCC803 and other cyanobacterial strains engineered with tesA-fadD-wax genes can utilize methanol, ethanol, butanol, and other alcohols, including exogenously added alcohols, to produce a variety of fatty acid esters. In certain embodiments, multiple types of exogenous or endogenous alcohols (e.g., methanol and ethanol; butanol or ethanol; methanol and butanol; etc.) could be added to the culture medium and utilized as substrates. 
     Example 3 
     Production of Fatty-Acid Esters Through Heterologous Expression of an Acyl-CoA Synthetase and a Wax Synthase 
     In order to compare the yields of fatty-acid esters produced by recombinant strains expressing tesA-fadD or fadD-wax (i.e., two of the three genes in the tesA-fadD-wax synthetic operon), fadD-wax and tesA-fadD and were assembled as two-gene operons and inserted into pJB5 to yield pJB634 and pJB578, respectively. These recombination plasmids were transformed into  Synechococcus  sp. PCC 7002 as described in Example 1, above to generate the strains listed in Table 8. Table 8 also lists JCC723, described above. 
     
       
         
           
               
             
               
                 TABLE 8 
               
             
            
               
                   
               
               
                 Joule Culture Collection (JCC) numbers of the  
               
               
                   Synechococcus  sp. PCC 7002 
               
               
                 recombinant strains with gene insertions on the  
               
               
                 native plasmid pAQ1. 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                   
                   
                 Promoter- 
                   
                   
                 %  
               
               
                   
                   
                   
                 operon  
                   
                   
                 DCW 
               
               
                 Strain # 
                 Promoter 
                 Genes 
                 sequences 
                 Marker 
                 OD 730   
                 FAEE 
               
               
                   
               
               
                 JCC723 
                 PaphII 
                 tesA-fadD- 
                 SEQ ID  
                 aadA 
                 15.35 
                 0.20 
               
               
                   
                   
                 wax 
                 NO: 10 
                   
                   
                   
               
               
                 JCC1215 
                 PaphII 
                 fadD-wax 
                 SEQ ID  
                 aadA 
                 10.10 
                 0.04 
               
               
                   
                   
                   
                 NO: 13 
                   
                   
                   
               
               
                 JCC1216 
                 PaphII 
                 tesA-fadD 
                 SEQ ID  
                 aadA 
                 10.00 
                 0.00 
               
               
                   
                   
                   
                 NO: 14 
               
               
                   
               
            
           
         
       
     
     One 30-ml culture of each strain listed in Table 1 was prepared in JB 2.1 medium containing 200 mg/L spectinomycin and 1% ethanol (vol/vol) at an OD 730 =0.1 in 125 ml flasks equipped with foam plugs (inocula were from five ml A+ cultures containing 200 mg/L spectinomyin started from colonies incubated for 3 days in a Multitron II Infors shaking photoincubator under continuous light of ˜100 μE m −2 s −1  photosynthetically active radiation (PAR) at 37° C. at 150 rpm in 2% CO 2 -enriched air). The cultures were incubated for seven days in the Infors incubators under continuous light of ˜100 μE m −2 s −1  photosynthetically active radiation (PAR) at 37° C. at 150 rpm in 2% CO 2 -enriched air. Fifty percent of the starting volume of ethanol was added approximately at day 5 based on experimentally determined stripping rates of ethanol under these conditions. Water loss was compensated by adding back milli-Q water (based on weight loss of flasks). Optical density measurements at 730 nm (OD 730 ) were taken (Table 8), and esters were extracted from cell pellets using the acetone procedure detailed in Example 2, above. Ethyl arachidate (Sigma A9010) at 100 mg/L was used as an internal standard instead of ethyl valerate. The dry cell weights (DCWs) were estimated based on the OD measurement using an experimentally determined average of 300 mg L −1  OD 730   −1 . 
     The acetone extracts were analyzed by GC/FID (for instrument conditions, see Example 2). In order to quantify the various esters, response factors (RF) were estimated from RFs measured for authentic ethyl ester standards and these RFs were used to determine the titres in the acetone extracts. The % DCW of the fatty-acid esters and the sum of the esters as % DCW is given in Table 8. Expression of fadD-wax was sufficient to allow production of fatty-acid ethyl esters (FAEEs), while expression of tesA-fadD did not result in any FAEEs ( FIG. 2 ). The overall yield was lower than JCC723, indicating that the co-expression of tesA is beneficial for increasing yields of FAEEs in this strain. 
     Example 4 
     Production of Longer-Chain Fatty-Acid Esters by Addition of Respective Alcohols to tesA-fadD-Wax Cultures 
     Seven 30-ml cultures of JCC803 (prepared from a single JCC803 culture that was diluted into 250 ml of JB 2.1 media containing 200 mg/L spectinomycin at an OD 730 =0.1) in 125-ml flasks were used to evaluate the ability of JCC803 to esterify different alcohols with fatty acids. Seven different alcohols were added at concentrations previously determined to allow growth of JCC803 (Table 9). The cultures were incubated for seven days in a Multitron II Infors shaking photoincubator under continuous light of ˜100 μE m −2 s −1  photosynthetically active radiation (PAR) at 37° C. at 150 rpm in 2% CO 2 -enriched air. Water loss was compensated by adding back milli-Q water (based on weight loss of flasks). Optical density measurements at 730 nm (OD 730 ) were taken (Table 3), and esters were extracted from cell pellets using the acetone procedure detailed in Example 2, above. Ethyl arachidate (Sigma A9010) at 100 mg/L was used as an internal standard instead of ethyl valerate. The dry cell weights (DCWs) were also determined for each culture so that the % DCW of the esters could be reported. 
     
       
         
           
               
               
               
               
             
               
                 TABLE 9 
               
               
                   
               
               
                   
                   
                 Concentration % 
                 Final 
               
               
                 Alcohol 
                 Catalog # 
                 (vol/vol) 
                 OD 730   
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
            
               
                 Propanol 
                 256404 (Sigma) 
                 0.25 
                 12.6 
               
               
                 Isopropanol 
                 BP2632 (Fisher) 
                 0.25 
                 12.6 
               
               
                 Butanol 
                 34867 (Sigma) 
                 0.1 
                 12.5 
               
               
                 Hexanol 
                 H13303 (Sigma) 
                 0.01 
                 8.6 
               
               
                 Cyclohexanol 
                 105899 (Sigma) 
                 0.01 
                 13.6 
               
               
                 Isoamyl alcohol 
                 A393 (Fisher) 
                 0.05 
                 13.6 
               
               
                 Ethanol 
                 2716 (Decon Labs Inc.) 
                 1.0 
                 14.0 
               
               
                   
               
            
           
         
       
     
     The acetone extracts were analyzed by GC/MS and GC/FID, as described above. The compounds indicated by peaks present in the total ion chromatograms were identified by matching the mass spectra associated with the peaks with mass spectral matches found by searching the NIST 08 MS database or by interpretation of the mass spectra when a respective mass spectrum of an authentic standard was not available in the database. In all cases, the corresponding alcohol esters of fatty acids were produced by JCC803 ( FIG. 3 ). Six fatty-acid esters were detected and quantified in the cell pellet extracts: myristate (C14:0), palmitoleate (C16:1Δ9), palmitate (C16:0), margarate (C17:0), oleate (C18:1Δ9) and stearate (C18:0). Magnified chromatograms for JCC803 incubated with ethanol and butanol are shown in  FIG. 4  and  FIG. 5 , respectively, so that the lower-yielding palmitoleate and margarate esters could be indicated on the chromatograms. In order to quantify the various esters, response factors (RF) were estimated from RFs measured for authentic ethyl ester and these RFs were used to determine the titres in the acetone extracts. The % DCW of the different esters and the sum of the esters as % DCW is given in Table 10. The % of the individual esters by weight and the total ester yield in mg/L is given in Table 11. 
     In general, the provision of longer-chain alcohols increased the yields of fatty-acid esters. The addition of butanol resulted in the highest yields of fatty-acid esters. Because butanol can be made biosynthetically (Nielsen et al. 2009, and references therein), exogenous butanol biosynthetic pathways could be expressed by one skilled in the art to generate a photosynthetic strain which can produce butyl esters without the addition of butanol. The use of butanol and butanol-producing pathways in other microbes containing the tesA-fadD-wax pathway would also be expected to increase yields of fatty-acid esters. 
     
       
         
           
               
             
               
                 TABLE 10 
               
             
            
               
                   
               
               
                 The yield of the fatty acid-esters individually and total as % dry cell weight 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                   
                   
                   
                 Total 
               
               
                   
                 Myristate 
                 Palmitoleate 
                 Palmitate 
                 Margarate  
                 Oleate 
                 Stearate  
                 Ester 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 Ethyl 
                 0.05 
                 0.02 
                 0.94 
                 0.01 
                 0.11 
                 0.15 
                 1.3 
               
               
                 Propyl 
                 0.26 
                 0.06 
                 3.22 
                 0.03 
                 0.21 
                 0.48 
                 4.3 
               
               
                 Isopropyl 
                 0.20 
                 0.04 
                 2.42 
                 0.02 
                 0.08 
                 0.42 
                 3.2 
               
               
                 Butyl 
                 0.59 
                 0.06 
                 3.67 
                 0.03 
                 0.19 
                 0.56 
                 5.1 
               
               
                 Hexyl 
                 0.11 
                 0.04 
                 1.33 
                 0.02 
                 0.17 
                 0.19 
                 1.8 
               
               
                 Cyclohexyl 
                 0.09 
                 0.03 
                 1.88 
                 0.01 
                 0.09 
                 0.31 
                 2.4 
               
               
                 Isoamyl 
                 0.31 
                 0.05 
                 2.84 
                 0.02 
                 0.15 
                 0.46 
                 3.8 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 11 
               
             
            
               
                   
               
               
                 The % of the individual esters by weight and total ester yield in mg/L. 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                   
                   
                   
                 Total 
               
               
                   
                 Myristate 
                 Palmitoleate 
                 Palmitate 
                 Margarate 
                 Oleate  
                 Stearate 
                 Ester 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 Ethyl 
                 4.2 
                 1.2 
                 73.4 
                 0.7 
                 8.6 
                 12.0 
                 77.6 
               
               
                 Propyl 
                 6.0 
                 1.3 
                 76.0 
                 0.7 
                 4.9 
                 11.1 
                 251.7 
               
               
                 Isopropyl 
                 6.2 
                 1.2 
                 76.4 
                 0.8 
                 2.4 
                 13.0 
                 188.5 
               
               
                 Butyl 
                 11.4 
                 1.1 
                 72.6 
                 0.5 
                 3.7 
                 10.8 
                 308.9 
               
               
                 Hexyl 
                 6.0 
                 2.1 
                 71.9 
                 1.1 
                 8.9 
                 10.0 
                 65.3 
               
               
                 Cyclohexyl 
                 3.6 
                 1.1 
                 78.5 
                 0.6 
                 3.6 
                 12.7 
                 139.6 
               
               
                 Isoamyl 
                 8.1 
                 1.2 
                 74.6 
                 0.5 
                 3.9 
                 11.8 
                 226.8 
               
               
                   
               
            
           
         
       
     
     Example 5 
     Reproducibility of Butanol Yields in tesA-fadD-Wax Cultures 
     Six 30-ml cultures of JCC803 (prepared from a single JCC803 culture that was diluted into 200 ml of JB 2.1 media/spec200 at an OD 730 =0.1) in 125 ml flasks were used to evaluate the ability of JCC803 cultures to produce butyl esters when containing different concentrations of butanol. Six different concentrations were tested (Table 12). The cultures were incubated for 21 days in a Multitron II Infors shaking photoincubator under continuous light at ˜100 μE m −2 s −1  PAR at 37° C. at 150 rpm in 2% CO 2 -enriched air. Fifty percent of the starting volume of butanol was added approximately every 3.5 days based on experimentally determined stripping rates of butanol under these conditions. Water loss was compensated by adding back milli-Q water (based on weight loss of flasks). OD 730 s were taken and esters were extracted from cell pellets using the acetone procedure detailed above. 100 mg/L ethyl arachidate (Sigma A9010) was used as an internal standard instead of ethyl valerate. The dry cell weights (DCWs) were also determined for each culture so that the % DCW of the esters could be reported. 
     An Agilent 7890A GC/FID equipped with a 7683 series autosampler was used to quantify the butyl esters. One microliter of each sample was injected into the GC inlet (split 5:1, pressure: 20 psi, pulse time: 0.3 min, purge time: 0.2 min, purge flow: 15 mL/min), which was at a temperature of 280° C. The column was an HP-5MS (Agilent, 30 m×0.25 mm×0.25 μm), and the carrier gas was helium at a flow of 1.0 mL/min. The GC oven temperature program was: 50° C., hold one minute; 10°/min increase to 280° C.; hold ten minutes. Butyl myristate, butyl palmitate, butyl margarate, butyl oleate and butyl stearate were quantified by determining appropriate response factors for the number of carbons present in the butyl esters from commercially available fatty-acid ethyl esters (“FAEEs”) and fatty acid butyl esters (“FABEs”). The calibration curves were prepared for ethyl laurate (Sigma 61630), ethyl myristate (Sigma E39600), ethyl palmitate (Sigma P9009), ethyl oleate (Sigma 268011), ethyl stearate (Fluka 85690), butyl laurate (Sigma W220604) and butyl stearate (Sigma S5001). The concentrations of the butyl esters present in the extracts were determined and normalized to the concentration of ethyl arachidate (internal standard). 
     The yields of the JCC803 cultures as given by the % DCW of the fatty acid butyl esters is given in Table 12. The highest yield of 14.7% resulted from the culture incubated with 0.05% butanol (vol/vol) although the 0.075% butanol-containing culture was approximately the same. 
     
       
         
           
               
             
               
                 TABLE 12 
               
             
            
               
                   
               
               
                 Yield of total FABES as % DCW for the JCC803 
               
               
                 cultures containing different concentrations of butanol 
               
               
                 and final OD 730  of the cultures. 
               
            
           
           
               
               
               
            
               
                 Concentration 
                   
                   
               
               
                 of butanol 
               
               
                 % (vol/vol) 
                 OD 730   
                 % DCW 
               
               
                   
               
            
           
           
               
               
               
            
               
                 0.2 
                 10.6 
                 11.75 
               
               
                 0.1 
                 9.0 
                 12.43 
               
               
                 0.075 
                 12.8 
                 14.53 
               
               
                 0.05 
                 12.0 
                 14.71 
               
               
                 0.025 
                 13.4 
                 10.43 
               
               
                 0.01 
                 16.0 
                 6.12 
               
               
                   
               
            
           
         
       
     
     Example 6 
     Secretion of Esters Produced by an Engineered  Cyanobacterium    
     Plasmids. 
       Escherichia coli  exports alkanes and other hydrophobic molecules out of the cell via the TolC-AcrAB transporter complex (Tsukagoshi and Aono, 2000; Chollet et al. 2004). PCR primer sets were designed to amplify tolC (Genbank # NC — 000913.2, locus b3035) and acrA-acrB as an operon (Genbank # NC — 000913.2, loci b0463, b0462) from  E. coli  MG1655 (ATCC #700926). The tolC and acrAB genes were amplified from MG1655 genomic DNA using the Phusion High-Fidelity PCR kit F-553 from New England BioLabs (Ipswich, Mass.) following the manufacturer&#39;s instructions. Buffer GC and 3% dimethyl sulfoxide (DMSO) were used for the PCR reactions. The amplicons were assembled into a three-gene, two-promoter construct (“transporter insert”; P psaA -tolC-P tsr2142 -acrAB) and placed in multiple cloning site of recombination vector pJB161 (SEQ ID #15) to yield pJB1074. pJB161 (and pJB161-derived plasmids, including pJB1074) contain an upstream homology region (UHR) and a downstream homology region (DHR) that allows recombination into the pAQ7 plasmid of  Synechococcus  sp. PCC7002 at the lactate dehydrogenase locus (for pAQ7 plasmid sequence, see Genbank # CP000957). The homology regions flank a multiple cloning site (mcs), the natural terminator from the alcohol dehydrogenase gene from  Zymomonas mobilis  (adhII) and a kanamycin cassette which provides resistance in both  E. coli  and  Synechococcus  sp. PCC 7002. The transporter insert with flanking homology regions is provided as SEQ ID 16. 
     Strain Construction. 
     As described above, JCC803 is a strain of  Synechococcus  sp. PCC 7002 that has been engineered to produce esters of fatty acids (such as those found in biodiesel) when incubated in the presence of alcohols. The strain contains a thioesterase (tesA), an acyl-CoA synthetase (fadD) and a wax synthase (wxs) inserted into plasmid pAQ1 by homologous recombination. 
     The genes present in pJB161 and pJB1074 were integrated into the plasmid pAQ7 in  Synechococcus  sp. PCC 7002 (specifically, strain JCC803) using the following procedure. A 5 ml culture of JCC803 in A+ medium containing 200 mg/L spectinomycin was incubated in an Infors shaking incubator at 150 rpm at 37° C. under 2% CO2/air and continuous light (70-130 μE m −2  s −1  PAR, measured with a LI-250A light meter (LI-COR)) until it reached an OD730 of 1.14. For each plasmid, 500 μl of culture and 5 μg of plasmid DNA were added into a microcentrifuge tube. The tubes were then incubated at 37° C. in the dark rotating on a Rotamix RKSVD (ATR, Inc.) on a setting of approximately 20. After 4 hours for pJB161 or 7 hours for pJB1074, the cells were pelleted using a microcentrifuge. All but ˜100 μl of the supernatants were removed and the cell pellets were resuspended using the remaining supernatant and plated on A+ agar plates. The plates were incubated overnight in a Percival lighted incubator under constant illumination (40-60 μE m −2  s −1  PAR, measured with a LI-250A light meter (LI-COR)) at 37° C. for about 24 hours. On the following day, spectinomycin and kanamycin solution was added underneath the agar of the plates to estimated concentration of 25 mg/L spectinomycin and 50 mg/L kanamycin (assuming 40 ml A+ agar in the plate). These plates were placed back into the incubator until tiny colonies became visible. The plates were moved to another Percival incubator under the same conditions except that 1% CO 2  was maintained in the air (allows for faster growth). Approximately 110 colonies formed for recombinant strains resulting from the pJB1074 transformation and 2800 colonies resulting from the pJB160 transformation. A colony from the pJB161 transformation plate was designated JCC1132. 
     Thirty colonies were picked from the tolC-acrAB transformation plate and streaked onto both an A+ plate with 100 mg/L spectinomycin and 0.05 mg/L erythromycin and an A+ plate with 100 mg/L spectinomycin and 0.1 mg/L erythromycin. Erythromycin is a substrate for the TolC-AcrAB transporter (Chollet et al. 2004) and served to verify function of the transporter in naturally erythromycin-sensitive  Synechococcus  sp. PCC 7002. The plates were incubated in Percival lighted incubator at 37° C. under constant illumination (40-60 μE m −2  s −1  PAR, measured with a LI-250A light meter (LI-COR)) at 37° C. After two days, slight growth was visible on both plates. Eight days after streaking, variable growth and survival was evident on most of the streaks on the 0.05 mg/L erythromycin plate. On the 0.1 mg/L erythromycin plate, all of the streaks except for two had become nonviable. The same source colonies that produced the two viable streaks on 0.1 mg/L erythromycin produced streaks that were healthy on the 0.05 mg/L erythromycin plate. One of these strains on the 0.1 mg/L erythromycin plate was designated JCC1585 (see Table 13 for a list of strains). 
     
       
         
           
               
             
               
                 TABLE 13 
               
             
            
               
                   
               
               
                 Strains and control strain investigated 
               
               
                 for the secretion of butyl esters. 
               
            
           
           
               
               
               
               
            
               
                   
                 Parent 
                 Recombinant genes/Promoters 
                   
               
               
                 JCC # 
                 strain 
                 with loci 
                 Marker 
               
               
                   
               
               
                 JCC1132 
                 JCC803 
                 pAQ1:: p trc -tesa-fadd-wxs-aada; 
                 spectinomycin 
               
               
                   
                   
                 pAQ7::kan r   
                 kanamycin 
               
               
                 JCC1585 
                 JCC803 
                 pAQ1:: p trc -tesa-fadd-wxs-aada; 
                 spectinomycin 
               
               
                   
                   
                 pAQ7:: p psaa -tolc-p tsr2142 - 
                 kanamycin 
               
               
                   
                   
                 acrab-kanr 
               
               
                   
               
            
           
         
       
     
     Erythromycin Tolerance in Liquid Culture. 
     To verify the improved tolerance of JCC1585 to erythromycin compared to JCC1132, a 5 ml A+ culture containing 200 mg/L spectinomycin and 0.5 mg/L erythromycin (JCC1585) or containing 200 mg/L spectinomycin and 50 mg/L kanamycin (JCC1132) were used to inoculate 30 ml of JB 2.1 containing 200 mg/L spectinomycin and 0.5, 0.6, 0.7, 0.8, 0.9, or 1 mg/L erythromycin in 125 ml culture flasks at an OD 730  of 0.1. These cultures were incubated in an Infors shaking incubator at 150 rpm at 37° C. under 2% CO 2 /air and continuous light (70-130 μE m −2  s −1  PAR, measured with a LI-250A light meter (LI-COR)). Timepoints were taken at 5 and 10 days of growth, during which water loss was replaced through addition of milli-Q water. Table 14 shows OD 730  values of JCC1132 and JCC1585 cultures at day 5 and 10 with different concentrations of erythromycin present in the medium. The JCC1585 cultures were tolerant of erythromycin concentrations of up to 1 mg/L (highest concentration tested) after 10 days while the JCC1132 cultures had bleached under all concentrations of erythromycin tested. 
     
       
         
           
               
               
               
               
               
             
               
                 TABLE 14 
               
               
                   
               
               
                   
                 Erythromycin 
                 OD 730   
                   
                   
               
               
                   
                 Concentration 
                 Start of 
                 OD 730   
                 OD 730   
               
               
                 Strain 
                 (mg/L) 
                 Experiment 
                 Day 5 
                 Day 10* 
               
               
                   
               
             
            
               
                 JCC1132 
                 0.5 
                 0.1 
                 5.72 
                 — 
               
               
                   
                 0.6 
                 0.1 
                 4.76 
                 — 
               
               
                   
                 0.7 
                 0.1 
                 4.98 
                 — 
               
               
                   
                 0.8 
                 0.1 
                 2.94 
                 — 
               
               
                   
                 0.9 
                 0.1 
                 2.50 
                 — 
               
               
                   
                 1.0 
                 0.1 
                 2.26 
                 — 
               
               
                 JCC1585 
                 0.5 
                 0.1 
                 6.60 
                 7.34 
               
               
                   
                 0.6 
                 0.1 
                 6.34 
                 6.20 
               
               
                   
                 0.7 
                 0.1 
                 5.82 
                 5.74 
               
               
                   
                 0.8 
                 0.1 
                 5.80 
                 4.84 
               
               
                   
                 0.9 
                 0.1 
                 5.34 
                 5.04 
               
               
                   
                 1.0 
                 0.1 
                 5.58 
                 5.12 
               
               
                   
               
               
                 *“—” indicates culture had bleached 
               
            
           
         
       
     
     To verify the improved tolerance of JCC1585 to erythromycin compared to JCC1132, a 5 ml A+ culture containing 200 mg/L spectinomycin and 0.5 mg/L erythromycin (JCC1585) or containing 200 mg/L spectinomycin and 50 mg/L kanamycin (JCC1132) were used to inoculate 30 ml of JB 2.1 media containing 200 mg/L spectinomycin and 0.5, 0.6, 0.7, 0.8, 0.9, or 1 mg/L erythromycin in 125 ml culture flasks at an OD730 of 0.1. These cultures were incubated in an Infors shaking incubator at 150 rpm at 37° C. under 2% CO2/air and continuous light (70-130 μE m2/s PAR, measured with a LI-250A light meter (LI-COR)). Timepoints were taken at 5 and 10 days of growth, during which water loss was replaced through addition of milli-Q water. The JCC1585 cultures were tolerant of erythromycin concentrations of up to 1 mg/L (highest concentration tested) after 10 days while the JCC1132 cultures had bleached under all concentrations of erythromycin tested (Table 14). 
     Culture Conditions. 
     To test for secretion of butyl esters, 5 ml A+ cultures with 200 mg/L spectinomycin and 50 mg/L kanamycin were inoculated from colonies for JCC1132 and JCC1585. These cultures were used to inoculate duplicate 30 ml cultures in JB2.1 medium containing 200 mg/L spectinomycin and 50 mg/L kanamycin. At the beginning of the experiment, 15 μl butanol (Sigma 34867) was added to each flask so that fatty acid butyl esters (FABEs) would be produced by the cultures. These cultures were incubated in an Infors shaking incubator at 150 rpm at 37° C. under 2% CO 2 /air and continuous light (70-130 μE m −2  s −1  PAR, measured with a LI-250A light meter (LI-COR)) for three days. At day 4 of the experiment, 7.5 μl butanol was added to the cultures to compensate for the experimentally determined stripping rate of butanol under these conditions. Water loss through evaporation was replaced with the addition of sterile Milli-Q water at day 7 and OD 730  readings were taken for each culture. 
     Detection of Butyl Esters. 
     An aliquot of 250 μl was removed from each culture and centrifuged at 1500 rpm in Microcentrifuge 5424 (Eppendorf) for ˜2 min. The supernatants were removed and the pellets were suspended in 500 μl milli-Q H 2 O. The samples were centrifuged and the supernatants discarded. An additional centrifugation step for 4 min was performed, and any remaining supernatant was removed. The weight of the tube and the cell pellet were measured. One milliliter of acetone (Acros Organics 326570010) containing 100 mg/L butylated hydroxytoluene (BHT, Sigma-Aldrich B1378) and 100 mg/L ethyl arachidate (Sigma A9010) were added to each pellet, and the mixture was pipetted up and down until none of the pellet remained on the wall of the tube. Each tube was then vortexed for 15 s, and the weight of the tube, acetone solution, and cells was taken. The tubes were then spun down and 500 μl of supernatant was submitted for GC analysis. From these samples, the percent dry cell weights of fatty acid butyl esters in the cell pellets were determined. 
     In order to quantify FABE&#39;s in the medium, 300 μL of a 20% (v/v) Span80 (Fluka 85548) solution was added to each flask and mixed by swirling for 30 seconds. These mixtures were then poured into 50 mL Falcon tubes. Five mL of isooctane containing 0.01% BHT and 0.005% ethyl arachidate was added to the flasks and swirled for several seconds. The solutions were then poured into the appropriate 50 mL Falcon tubes containing the culture from the flasks. The tube was then shaken for 10 seconds and centrifuged using a Sorvall RC6 Plus superspeed centrifuge (Thermo Electron Corp) and a F13S-14X50CY rotor (6000 rpm for 20 min). One milliliter of the organic phase (upper phase) was removed and submitted for GC analysis. 
     The butyl esters produced by JCC803 and JCC803-derived strains were identified by GC/MS employing an Agilent 7890A GC/5975C ELMS equipped with a 7683 series autosampler. One microliter of each sample was injected into the GC inlet using a pulsed splitless injection (pressure: 20 psi, pulse time: 0.3 min, purge time: 0.2 min, purge flow: 15 mL/min) and an inlet temperature of 280° C. The column was a HP-5MS (Agilent, 30 m×0.25 mm×0.25 μm) and the carrier gas was helium at a flow of 1.0 mL/min. The GC oven temperature program was 50° C., hold one minute; 10°/min increase to 280° C.; hold ten minutes. The GC/MS interface was 290° C., and the MS range monitored was 25 to 600 amu. Butyl myristate [retention time (rt): 19.72 min], butyl palmitate (rt: 21.58 min) butyl heptadecanoate (rt: 22.40 min), butyl oleate (rt: 23.04 min) and butyl stearate (rt: 23.24 min) were identified by matching experimentally determined mass spectra associated with the peaks with mass spectral matches found by searching in a NIST 08 MS database. 
     An Agilent 7890A GC/FID equipped with a 7683 series autosampler was used to quantify the butyl esters. One microliter of each sample was injected into the GC inlet (split 5:1, pressure: 20 psi, pulse time: 0.3 min, purge time: 0.2 min, purge flow: 15 mL/min), which was at a temperature of 280° C. The column was an HP-5MS (Agilent, 30 m×0.25 mm×0.25 μm), and the carrier gas was helium at a flow of 1.0 mL/min. The GC oven temperature program was 50° C., hold one minute; 10°/min increase to 280° C.; hold ten minutes. Butyl myristate (rt: 19.68 min], butyl palmitate (rt: 21.48 min), butyl heptadecanoate (rt: 22.32 min), butyl oleate (rt: 22.95 min) and butyl stearate (rt: 23.14 min) were quantified by determining appropriate response factors for the number of carbons present in the butyl esters from commercially-available fatty acid ethyl esters (FAEEs) and FABEs. The calibration curves were prepared for ethyl laurate (Sigma 61630), ethyl myristate (Sigma E39600), ethyl palmitate (Sigma P9009), ethyl oleate (Sigma 268011), ethyl stearate (Fluka 85690), butyl laurate (Sigma W220604) and butyl stearate (Sigma S5001). The concentrations of the butyl esters present in the extracts were determined and normalized to the concentration of ethyl arachidate (internal standard). 
     Peaks with areas greater than 0.05 could be integrated by the Chemstation™ software (Agilent®), and the concentrations of the butyl esters in both media and supernatant were determined from these values. The dry cell weight (DCW) of these strains was based on a measurement of OD 730  and calculated based on the observed average DCW/OD relationship of 0.29 g L −1  OD −1 . In the case of the JCC1585 culture supernatant, small peaks for butyl myristate (flask 1 area: 1.26, flask 2: 2.23) and butyl palmitate (flask 1 area: 5.16, flask 2: 5.62) were observed while no peak with an area greater than 0.05 at these retention times was found in the media extraction of the JCC1132 cultures. The OD 730  percent dry cell weights of the FABEs in the cell pellets and the media are given in Table 15. The total % DCW of FABE&#39;s found in the cell pellets is indicated, as is the % DCW of butyl myristate and butyl palmitate found in the pellets and the media. 
     
       
         
           
               
               
               
               
               
             
               
                 TABLE 15 
               
               
                   
               
               
                   
                   
                   
                 Pellet butyl 
                 Media butyl 
               
               
                   
                   
                   
                 myristate + 
                 myristate + 
               
               
                 Strain 
                   
                 FABEs 
                 butyl pal- 
                 butyl pal- 
               
               
                 (flask) 
                 OD730 
                 (% DCW) 
                 mitate (% DCW) 
                 mitate (% DCW) 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 JCC1585 (1) 
                 9.65 
                 7.76 
                 6.59 
                 0.013 
               
               
                 JCC1132 (1) 
                 5.44 
                 4.93 
                 4.20 
                 0 
               
               
                 JCC1585 (2) 
                 8.50 
                 7.79 
                 6.65 
                 0.018 
               
               
                 JCC1132 (2) 
                 4.48 
                 4.60 
                 3.85 
                 0 
               
               
                   
               
            
           
         
       
     
     Table 15 shows that the recombinant expression of to/C in an engineered  cyanobacterium  provides for the secretion of a detectable fraction of esters (in this case, butyl esters) synthesized by the engineered cell. The amount of secretion achieved can be modulated by increasing concentrations of erythromycin or other transporter substrates, and/or through optimization of expression levels (promoter strength and codon optimization strategies) and/or specifically targeting a cyanobacterial membrane by employing appropriate cyanobacterial N-terminal leader sequences. 
     Example 7 
     Secretion of Fatty Acids in  Thermosynechococcus elongatis  BP-1 (Δaas) 
     Strain Construction. 
       Thermosynechoccocus elongatus  BP-1 long-chain-fatty-acid CoA ligase gene (aas, GenBank accession number NP — 682091.1) was replaced with a thermostable kanamycin resistance marker (kan_HTK, GenBank accession number AB121443.1) as follows: 
     Regions of homology flanking the BP-1 aas gene (Accession Number: NP — 682091.1) were amplified directly from BP-1 genomic DNA using the primers in Table 16. PCR amplifications were performed with Phusion High Fidelity PCR Master Mix (New England BioLabs) and standard amplification conditions. 
     
       
         
           
               
               
               
               
             
               
                 TABLE 16 
               
               
                   
               
               
                   
                   
                 SEQ 
                   
               
               
                   
                   
                 ID 
                 Restriction 
               
               
                 Primer 
                 Sequence 
                 NO: 
                 site added 
               
               
                   
               
             
            
               
                 Upstream  
                 5′-GCTATGCCTGCAGGGGCCTTTTATGAGGAGCGGTA-3′ 
                 21 
                 SbfI 
               
               
                 forward 
                   
                   
                   
               
               
                   
               
               
                 Upstream 
                 5′-GCTATGGCGGCCGCTCTTCATGACAGACCCTATGGATACTA-3′ 
                 22 
                 NotI 
               
               
                 reverse 
                   
                   
                   
               
               
                   
               
               
                 Down- 
                 5′-GCTATGGGCGCGCCTTATCTGACTCCAGACGCAACA-3′ 
                 23 
                 AscI 
               
               
                 stream 
                   
                   
                   
               
               
                 forward 
                   
                   
                   
               
               
                   
               
               
                 Down- 
                 5′-GCTATGGGCCGGCCGATCCTTGGATCAACTCACCCT-3′ 
                 24 
                 FseI 
               
               
                 stream 
                   
                   
                   
               
               
                 reverse 
               
               
                   
               
            
           
         
       
     
     The amplified upstream homologous region (UHR) was cloned into the UHR of a pJB5 expression vector containing kan_HTK by digesting the insert and vector individually with SbfI and NotI restriction endonucleases (New England BioLabs) following well known laboratory techniques. Digestions were isolated on 1% TAE agarose gel, purified using a Gel Extraction Kit (Qiagen), and ligated with T4 DNA Ligase (New England BioLabs) incubated at room temperature for 1 hour. The ligated product was transformed into NEB 5-alpha chemically competent  E. coli  cells (New England BioLabs) using standard techniques and confirmed by PCR. The downstream homologous region (DHR) was cloned into the resulting plasmid following a similar protocol using AscI and FseI restriction endonucleases (New England BioLabs). The final plasmid (pJB1349) was purified using QIAprep Spin Miniprep kit (Qiagen) and the construct was confirmed by digestion with HindIII, AseI, and PstI restriction endonucleases (New England BioLabs). 
     BP-1 was grown in 5 ml B-HEPES liquid media in a glass test tube (45° C., 120 rpm, 2% CO 2 ) to OD 730 1.28. A 1 ml aliquot of culture was transferred to a fresh tube and combined with 1 ug of purified pJB1349. The culture was incubated in the dark (45° C., 120 rpm, 2% CO 2 ) for 4 hours. 4 ml of fresh B-HEPES liquid media were added and the culture was incubated with light (45° C., 120 rpm, 2% CO 2 ) overnight. 500 μl of the resulting culture were plated in 3 ml of B-HEPES soft agar on B-HEPES plates containing 60 μg/ml kanamycin and placed in an illuminated incubator (45° C., ambient CO 2 ) until colonies appeared (1 week), then moved into a 2% CO 2  illuminated incubator for an additional week. 
     Four randomly selected colonies (samples A-D) were independently grown in 5 ml B-HEPES liquid media with 60 μg/ml kanamycin in glass test tubes (45° C., 120 rpm, 2% CO 2 ) for one week. Replacement of aas gene was confirmed by PCR of whole cell genomic DNA by a culture PCR protocol as follows. Briefly, 100 μl of each culture was resuspended in 50 μl lysis buffer (96.8% diH 2 O, 1% Triton X-100, 2% 1M Tris pH 8.5, 0.2% 1M EDTA). 10 μl of each suspension were heated 10 min at 98° C. to lyse cells. 1 μl of lysate was used in 15 μl standard PCR reactions using Quick-Load Taq 2× Master Mix (New England BioLabs). The PCR product showed correct bands for an unsegregated knockout. 
     All cultures were maintained in fresh B-HEPES liquid media with 60 μg/ml kanamycin for an additional week. The PCR reaction described above was repeated, again showing correct bands for an unsegregated knockout. Cultures were maintained in liquid culture, and one representative culture was saved as JCC1862. 
     Detection and Quantification of Free Fatty Acids in Strains. 
     Each of the four independently inoculated cultures described above (samples A-D), as well as BP-1, was analyzed for secretion of free fatty acids. OD 730  was measured, and the volume in each culture tube was recorded. Fresh B-HEPES liquid media was added to each tube to bring the total volume to 5 ml and free fatty acids were extracted as follows: 
     Samples were acidified with 50 μl 1N HCl. 500 μl of 250 g/L methyl-β-cyclodextrin solution was added and samples were transferred to 15-ml conical tubes after pulse-vortexing. 1 ml of 50 mg/L butylated hydroxytoluene in isooctane was added to each tube. Samples were vortexed 20 s, then centrifuged 5 min at 6000 RCF to fractionate. 500 μl of the isooctane layer were placed into a new tube and submitted for GC analysis. 
     Concentrations of octanoic acid, decanoic acid, lauric acid, myristic acid, palmitoleic acid, palmitic acid, oleic acid, stearic acid, and 1-nonadecene extractants were quantitated by gas chromatography/flame ionization detection (GC/FID). Unknown peak areas in biological samples were converted to concentrations via linear calibration relationships determined between known authentic standard concentrations and their corresponding GC-FID peak areas. Standards were obtained from Sigma. GC-FID conditions were as follows. An Agilent 7890A GC/FID equipped with a 7683 series autosampler was used. 1 μl of each sample was injected into the GC inlet (split 5:1, pressure: 20 psi, pulse time: 0.3 min, purge time: 0.2 min, purge flow: 15 ml/min) and an inlet temperature of 280° C. The column was a HP-5MS (Agilent, 30 m×0.25 mm×0.25 μm) and the carrier gas was helium at a flow of 1.0 ml/min. The GC oven temperature program was 50° C., hold one minute; 10° C./min increase to 280° C.; hold ten minutes. 
     GC results showed that the unsegregated aas knockout increased fatty acid production relative to BP-1 (Table 17), with myristic and oleic acid making up the majority of the increase (Table 18). 
     
       
         
           
               
             
               
                 TABLE 17 
               
             
            
               
                   
               
               
                 Fatty Acid Production by Sample 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                   
                 Fatty acids 
                 Fatty acids 
               
               
                   
                 Sample 
                 OD 730   
                 (% DCW in media) 
                 (mg/L) 
               
               
                   
                   
               
               
                   
                 A 
                 6.25 
                 0.20 
                 3.66 
               
               
                   
                 B 
                 5.20 
                 0.11 
                 1.71 
               
               
                   
                 C 
                 5.60 
                 0.24 
                 3.85 
               
               
                   
                 D 
                 5.80 
                 0.23 
                 3.83 
               
               
                   
                 BP-1 
                 6.90 
                 0.04 
                 0.88 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 18 
               
             
            
               
                   
               
               
                 Fatty Acid Production by Type 
               
            
           
           
               
               
               
               
            
               
                 Sample 
                 Myristic (mg/L) 
                 Palmitic (mg/L) 
                 Oleic (mg/L) 
               
               
                   
               
               
                 A 
                 0.119 
                 0.051 
                 0.032 
               
               
                 B 
                 0.000 
                 0.072 
                 0.042 
               
               
                 C 
                 0.134 
                 0.063 
                 0.040 
               
               
                 D 
                 0.130 
                 0.060 
                 0.038 
               
               
                 BP-1 
                 0.000 
                 0.044 
                 0.000 
               
               
                   
               
            
           
         
       
     
     Example 8 
     Increased Production of Fatty Acids and Fatty Esters in  Thermosynechococcus elongatis  BP-1 (Δaas) 
     Transformation of BP-1. 
     As disclosed in PCT/US2010/042667, filed Jul. 20, 2010 , Thermosynechococcus elongatus  BP-1 is transformed with integration or expression plasmids using the following protocol. 400 ml  Thermosynechococcus elongatus  BP-1 in B-HEPES medium is grown in a 2.8 l Fernbach flask to an OD 730  of 1.0 in an Infors Multritron II shaking photoincubator (55° C.; 3.5% CO 2 ; 150 rpm). For each transformation, 50 ml cell culture is pelleted by centrifugation for 20 min (22° C.; 6000 rpm). After removing the supernatant, the cell pellet is resuspended in 500 μl B-HEPES and transferred to a 15 ml Falcon tube. To each 500 μl BP-1 cell suspension (OD 730  of ˜100), 25 μg undigested plasmid (or no DNA) is added. The cell-DNA suspension is incubated in a New Brunswick shaking incubator (45° C.; 250 rpm) in low light (˜3 μmol photons m −2  s 1 ). Following this incubation, the cell-DNA suspension is made up to 1 ml by addition of B-HEPES, mixed by gentle vortexing with 2.5 ml of molten B-HEPES 0.82% top agar solution equilibrated at 55° C., and spread out on the surface of a B-HEPES 1.5% agar plate (50 ml volume). Plates are left to sit at room temperature for 10 min to allow solidification of the top agar, after which time plates are placed in an inverted position in a Percival photoincubator and left to incubate for 24 hr (45° C.; 1% CO 2 ; 95% relative humidity) in low light (7-12 μmol photons m −2  s 1 ). After 24 hr, the plates are underlaid with 300 μl of 10 mg/ml kanamycin so as to obtain a final kanamycin concentration of 60 μg/ml following complete diffusion in the agar. Underlaid plates are placed back in the Percival incubator and left to incubate (45° C.; 1% CO 2 ; 95% relative humidity; 7-12 μmol photons m −2  s 1 ) for twelve days. 
     Increased Fatty Acids in BP-1. 
       Thermosynechococcus elongatus  BP-1 (Δaas) is first constructed as described in the above Example. BP-1(Δaas) is shown to have elevated levels of both intracellular and extracellular levels of free fatty acids relative to wild-type because mechanistic analysis suggests that cells lacking an acyl-ACP synthetase have the inability to recycle exogenous or extracellular fatty acids; the extracellular fatty acid chains are diverted away from transport into the inner cellular membrane while other transport systems are thought to continue to export fatty acids. Therefore, to up-regulate fatty acid production, BP-1(Δaas) is transformed with a plasmid (e.g., pJB1349) carrying a thioesterase gene (see Table 3A). Increased cellular level of fatty acid production may be attributed to the combination of the aas deletion decreasing extracellular import of fatty acids and the addition of the thioesterase gene and/or thioesterase gene homologues. 
     Fatty Acid Esters. 
     The thioesterase gene with or without the leader sequence removed (Genbank # NC 000913, ref: Chot and Cronan, 1993), the  E. coli  acyl-CoA synthetase fadD (Genbank # NC 000913, ref: Kameda and Nunn, 1981) and the wax synthase (wxs) from  Acinetobacter  baylyi strain ADPI (Genbank # AF529086.1, ref: Stóveken et al. 2005) genes are designed for codon optimization, checking for secondary structure effects, and removal of any unwanted restriction sites (NdeI, XhoI, BamHI, NgoMIV, NcoI, SacI, BsrGI, AvrII, BmtI, MiuI, EcoRI, SbfI, NotI, SpeI, XbaI, Pad, AscI, FseI). These genes are engineered into plasmid or integration vectors (e.g., pJB1349) and assembled into a two gene operon (fadD-wxs) or a three gene operon (tesA-fadD-wxs) with flanking sites on the integration vector corresponding to integration sites for transformation into  Thermosynechococcus elongatus  BP-1. Integration sites include TS1, TS2, TS3 and TS4. A preferred integration site is the site of the aas gene. Host cells are cultured in the presence of small amounts of ethanol (1-10%) in the growth media under an appropriate promoter such as Pnir for the production of fatty acid esters. 
     In another embodiment,  Thermosynechococcus elongatus  BP-1 host cell with a two gene operon (fadD-wxs) or a three gene operon (tesA-fadD-wxs) is engineered to have ethanol producing genes (PCT/US2009/035937, filed Mar. 3, 2009; PCT/US2009/055949, filed Sep. 3, 2009; PCT/US2009/057694, filed Sep. 21, 2009) conferring the ability to produce fatty acid esters. In one plasmid construct, genes for ethanol production, including pyruvate decarboxylase from  Zymomonas mobilis  (pdc Zm ) and alcohol dehydrogenase from  Moorella  sp. HUC22-1 (adhA M ), are engineered into a plasmid and transformed into BP-1. In an alternate plasmid construct, the pyruvate decarboxylase from  Zymobacter palmae  (pdc Zp ) and alcohol dehydrogenase from  Moorella  sp. HUC22-1 (adhA M ), are engineered into a plasmid and transformed into BP-1. These genes are engineered into plasmid or integration vectors (e.g., pJB1349) with flanking sites on the integration vector corresponding to integration sites for transformation into  Thermosynechococcus elongatus  BP-1. Integration sites include TS1, TS2, TS3 and TS4. A preferred integration site is the site of the aas gene. In one configuration, expression of pdcZm and adhAM are driven by λ phage cI (“PcI”) and pEM7 and in another expression strain driven by PcI and PtRNA Glu . In one embodiment, a single promoter is used to control the expression of both genes. In another embodiment each gene expression is controlled by separate promoters with PaphII or Pcpcb controlling one and PcI controlling the other. 
     Example 9 
       Synechococcus  Sp. PCC 7002 (Δaas) with Various Thioesterases 
     Strain Construction. 
     DNA sequences for thioesterase genes tesA, fatB, fatB1, and fatB2 were obtained from Genbank and were purchased from DNA 2.0 following codon optimization, checking for secondary structure effects, and removal of any unwanted restriction sites. Thioesterase gene fatB_mat is a modified form of fatB with its leader sequence removed. 
     
       
         
           
               
             
               
                 TABLE 19 
               
             
            
               
                   
               
               
                 Thioesterase sources 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 GenBank 
               
               
                   
                 Gene name 
                 Organism origin 
                 protein seq 
               
               
                   
                   
               
               
                   
                 tesA 
                 
                   Escherichia coli 
                 
                 AAC73596 
               
               
                   
                 fatB 
                 
                   Umbellularia californica 
                 
                 Q41635 
               
               
                   
                   
                 (California bay) 
               
               
                   
                 fatB1 
                 
                   Cinnamomum camphora 
                 
                 Q39473 
               
               
                   
                   
                 (camphor tree) 
               
               
                   
                 fatB2 
                 
                   Cuphea hookeriana 
                 
                 AAC49269 
               
               
                   
                   
               
            
           
         
       
     
     The thioesterase genes were cloned into a pJB5 expression vector containing upstream and downstream regions of homology to aquI (SYNPCC7002_A1189), pAQ3, and pAQ4 by digesting the inserts and vectors individually with AscI and NotI restriction endonucleases (New England BioLabs) following known laboratory techniques. Digestions were isolated on 1% TAE agarose gel, purified using a Gel Extraction Kit (Qiagen), and ligated with T4 DNA Ligase (New England BioLabs) incubated at room temperature for one hour. The ligated product was transformed into NEB 5-alpha chemically competent  E. coli  cells (New England BioLabs) using standard techniques. Purified plasmid was extracted using the QIAprep Spin Miniprep kit (Qiagen) and constructs were confirmed by PCR. 
       Synechococcus  sp. PCC 7002 (Δaas) was grown in 5 ml A+ liquid media with 25 μg/ml gentamicin in a glass test tube (37° C., 120 rpm, 2% CO 2 ) to OD 730  of 0.98-1.1. 500 μl of culture was combined with 1 μg purified plasmid in 1.5 ml microcentrifuge tubes and incubated in darkness 3-4 hours. Samples were then plated on A+ agar plates with 3 or 6 mM urea and incubated overnight 37° C. in the light. Selective antibiotics were introduced to the plates by placing stock solution spectinomycin under the agar at a final concentration of 10 μg/mL, and incubating to allow diffusion of the antibiotic. Plates were incubated at 37° C. with light until plates cleared and individual colonies formed. Plates were then moved to an illuminated incubator at 2% CO 2 . Cultures were maintained on liquid or agar A+ media containing 3-6 mM urea with 25 μg/ml gentamicin, 100-200 μg/ml spectomycin, to promote plasmid segregation. 
     Thioesterase integration and attenuation was confirmed by PCR of whole-cell genomic DNA by a “culture PCR” protocol. Briefly, 100 μl of each culture was resuspended in 50 μl water or lysis buffer (96.8% diH 2 O, 1% Triton X-100, 2% 1M tris pH 8.5, 0.2% 1M EDTA). 10 μl of each suspension were heated 10 min at 98° C. to lyse cells. 1 μl of lysate was used in 10 μl standard PCR reactions using Quick-Load Taq 2× Master Mix (New England BioLabs) or Platinum PCR Supermix HiFi (Invitrogen). PCR products showed correct bands for segregated aquI, pAQ4 and unsegregated (pAQ3) integrants. 
     Detection and Quantification of Free Fatty Acids in Strains. 
     Individual colonies were grown in A+ liquid media with 3 mM urea, 50 μg/ml gentamicin, 200 μg/ml spectomycin in glass test tubes (see Table 20). Cultures were maintained in liquid culture to promote segregation (37° C., 120 rpm, 2% CO 2 ). Liquid cultures were diluted to OD 730 =0.2 in 5 ml A+ liquid media with 3 mM urea and no antibiotics in glass test tubes and incubated for seven days (37° C., 120 rpm, 2% CO 2 ). After one week, OD 730  was recorded and free fatty acids were extracted as follows: 
     Samples were acidified with 50 μl 1N HCl. 500 μl of 250 g/L methyl-β-cyclodextrin solution was added, and samples were transferred to 15-ml conical tubes after pulse-vortexing. 1 ml of 50 mg/L butylated hydroxytoluene in isooctane was added to each tube. Samples were vortexed 20 s and immediately centrifuged 5 min at 6000 RCF to fractionate. 500 μl of the isooctane layer were sub-sampled into a new tube and submitted for GC analysis. 
     Concentrations of octanoic acid, decanoic acid, lauric acid, myristic acid, palmitoleic acid, palmitic acid, oleic acid, stearic acid, and 1-nonadecene extractants were quantitated by gas chromatography/flame ionization detection (GC/FID). Unknown peak areas in biological samples were converted to concentrations via linear calibration relationships determined between known authentic standard concentrations and their corresponding GC-FID peak areas. Standards were obtained from Sigma. GC-FID conditions were as follows. An Agilent 7890A GC/FID equipped with a 7683 series autosampler was used. 1 μl of each sample was injected into the GC inlet (split 5:1, pressure: 20 psi, pulse time: 0.3 min, purge time: 0.2 min, purge flow: 15 ml/min) and an inlet temperature of 280° C. The column was a HP-5MS (Agilent, 30 m×0.25 mm×0.25 μm) and the carrier gas was helium at a flow of 1.0 ml/min. The GC oven temperature program was 50° C., hold one minute; 10° C./min increase to 280° C.; hold ten minutes. 
     GC results showed increased fatty acid secretion in the thioesterase strains relative to  Synechococcus  sp. PCC 7002 JCC138 (Table 20). The specific enrichment profile of each culture was thioesterase dependent (Table 21). 
     
       
         
           
               
             
               
                 TABLE 20 
               
             
            
               
                   
               
               
                 Fatty acid secretion in tesA, fatB_mat strains 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                   
                   
                 Fatty Acids 
                   
               
               
                   
                   
                   
                   
                   
                   
                 (% DCW 
                 Fatty acids 
               
               
                 Sample 
                 Location 
                 Promoter 
                 Thioesterase 
                 Δaas 
                 OD 730   
                 in media)  
                 (mg/ml) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 JCC 138 
                 — 
                 — 
                 — 
                 — 
                 11.80 
                 0.11 
                 3.81 
               
               
                 JCC 
                 pAQ4 
                 P(nir07) 
                 tesA 
                 yes 
                 5.56 
                 2.76 
                 44.45 
               
               
                 1648 
                   
                   
                   
                   
                   
                   
                   
               
               
                 JCC 
                 pAQ3 
                 P(nir07) 
                 tesA 
                 yes 
                 7.68 
                 2.29 
                 51.10 
               
               
                 1751 
                   
                   
                   
                   
                   
                   
                   
               
               
                 JCC 
                 pAQ3 
                 P(nir07) 
                 fatB_mat 
                 yes 
                 3.92 
                 1.79 
                 20.38 
               
               
                 1755 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 21 
               
             
            
               
                   
               
               
                 Fatty acids by type 
               
            
           
           
               
               
            
               
                   
                 % DCW of compounds 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Sample 
                 Lauric  
                 Myristic 
                 Palmitoleic 
                 Palmitic 
                 Oleic 
                 Stearic 
               
               
                   
               
               
                 JCC 138 
                 0.000 
                 0.061 
                 0.000 
                 0.000 
                 0.000 
                 0.050 
               
               
                 JCC1648 
                 0.342 
                 1.557 
                 0.238 
                 0.000 
                 0.260 
                 0.360 
               
               
                 JCC 1751  
                 0.146 
                 0.539 
                 0.165 
                 1.145 
                 0.158 
                 0.143 
               
               
                 JCC1755 
                 0.940 
                 0.224 
                 0.289 
                 0.143 
                 0.197 
                 0.000 
               
               
                   
               
            
           
         
       
     
     Individual colonies of JCC1704, JCC1705, and JCC1706 were grown for three days in A+ liquid media with 3 mM urea, 25 μg/ml gentamicin, 100 μg/ml spectomycin in glass test tubes (37° C., 120 rpm, 2% CO 2 ). Cultures were diluted to OD 730 =0.2 in 5 ml A+ liquid media with 3 mM urea and no antibiotics in glass test tubes and incubated at 37° C., 120 rpm, 2% CO 2 . After 11 days, OD 730  was recorded and free fatty acids were extracted as follows: 
     Samples were acidified with 50 μl 1N HCl. 500 μl of 250 g/L methyl-β-cyclodextrin solution was added and samples were transferred to 15-ml conical tubes after pulse-vortexing. 1 ml of 50 mg/L butylated hydroxytoluene in isooctane was added to each tube. Samples were vortexed 20 s and immediately centrifuged 5 min at 6000 RCF to fractionate. 500 μl of the isooctane layer were sub-sampled into a new tube and submitted for GC analysis. 
     Concentrations of octanoic acid, decanoic acid, lauric acid, myristic acid, palmitoleic acid, palmitic acid, oleic acid, stearic acid, and 1-nonadecene extractants were quantitated by gas chromatograph/flange ionization detection (GC/FID), Unknown peak areas in biological samples were converted to concentrations via linear calibration relationships determined between known authentic standard concentrations and their corresponding GC-FID peak areas. Standards were obtained. from Sigma. GC-FID conditions were as follows. An Agilent 7890A GC/FID equipped with a 7683 series autosampler was used 1 μl of each sample was injected into the GC inlet (split 5:1, pressure: 20 psi, pulse time: 0.3 min, purge time: 0.2 min, purge flow: 15 ml/min) and an inlet temperature of 280° C. The column was a HP-5MS (Agilent, 30 m×0.25 mm×0.25 μm) and the carrier gas was helium at a flow of 1.0 ml/min. The GC oven temperature program was 50° C., hold one minute; 10° C./min increase to 280° C.; hold ten minutes. 
     GC results showed increased fatty acid secretion relative to JCC138 but to a lesser degree than tesA or fatB_mat (Table 22). The specific enrichment profile of each culture was thioesterase dependent (Table 23). 
     
       
         
           
               
             
               
                 TABLE 22 
               
             
            
               
                   
               
               
                 Fatty acid secretion in fatB, fatB1, fatB2 strains 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                   
                   
                 Fatty Acids 
                 Fatty 
               
               
                   
                   
                   
                   
                   
                   
                 (% DCW in 
                 acids 
               
               
                 Sample 
                 Location 
                 Promoter  
                 Thioesterase 
                 Δaas 
                 OD 730   
                 media) 
                 (mg/ml) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 JCC 1648 
                 pAQ4 
                 P(nir07) 
                 tesA 
                 yes 
                 11.2 
                 6.66 
                 216.283 
               
               
                 JCC 1648 
                 pAQ4 
                 P(nir07) 
                 tesA 
                 yes 
                 11.6 
                 5.74 
                 193.236 
               
               
                 JCC 1704 
                 aquI 
                 P(nir07) 
                 fatB 
                 yes 
                 15.80 
                 0.39 
                 17.72 
               
               
                 JCC 1704 
                 aquI 
                 P(nir07) 
                 fatB 
                 yes 
                 16.80 
                 0.40 
                 19.56 
               
               
                 JCC 1705 
                 aquI 
                 P(nir07) 
                 fatB1 
                 yes 
                 15.6 
                 0.42 
                 19.19 
               
               
                 JCC 1705 
                 aquI 
                 P(nir07) 
                 fatB1 
                 yes 
                 16.3 
                 0.43 
                 20.44 
               
               
                 JCC 1706 
                 aquI 
                 P(nir07) 
                 fatB2 
                 yes 
                 17.5 
                 0.40 
                 20.25 
               
               
                 JCC 1706 
                 aquI 
                 P(nir07) 
                 fatB2 
                 yes 
                 16.5 
                 0.41 
                 19.86 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 23 
               
             
            
               
                   
               
               
                 Fatty acids by type 
               
            
           
           
               
               
            
               
                   
                 % DCW of compounds 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Sample 
                 Lauric 
                 Myristic 
                 Palmitoleic 
                 Palmitic 
                 Oleic 
                 Stearic 
               
               
                   
               
               
                 JCC 1648 
                 0.233 
                 1.408 
                 0.264 
                 3.919 
                 0.223 
                 0.611 
               
               
                 JCC 1648 
                 0.201 
                 1.196 
                 0.183 
                 3.564 
                 0.131 
                 0.470 
               
               
                 JCC 1704 
                 0.000 
                 0.057 
                 0.107 
                 0.073 
                 0.087 
                 0.063 
               
               
                 JCC 1704  
                 0.000 
                 0.062 
                 0.113 
                 0.073 
                 0.094 
                 0.060 
               
               
                 JCC 1705  
                 0.000 
                 0.058 
                 0.110 
                 0.089 
                 0.099 
                 0.068 
               
               
                 JCC 1705 
                 0.000 
                 0.058 
                 0.107 
                 0.092 
                 0.101 
                 0.074 
               
               
                 JCC 1706  
                 0.000 
                 0.054 
                 0.098 
                 0.090 
                 0.085 
                 0.071 
               
               
                 JCC 1706  
                 0.000 
                 0.056 
                 0.106 
                 0.086 
                 0.100 
                 0.068 
               
               
                   
               
            
           
         
       
     
     Example 10 
     Fatty Acid Production Under Inducible or Repressible System 
     Construction of the Promoter-uidA Expression Plasmid. 
     The  E. coli  uidA gene (Genbank AAB30197) was synthesized by DNA 2.0 (Menlo Park, Calif.), and was subcloned into pJB5. The DNA sequences of the ammonia-repressible nitrate reductase promoters P(nirA) (SEQ ID NO:17), P(nir07) (SEQ ID NO:18), and P(nir09) (SEQ ID NO:19) were obtained from Genbank. The nickel-inducible P(nrsB) promoter (SEQ ID NO:20), nrsS and nrsR were amplified from  Synechocystis  sp. PCC 6803. The promoters were cloned between NotI and NdeI sites immediately upstream of uidA, which is flanked by NdeI and EcoRI. 
     In addition, plasmids containing two 750-bp regions of homology designed to remove the native aquI (A1189) or the ldh (G0164) gene from  Synechococcus  sp. PCC 7002 were obtained by contract synthesis from DNA 2.0 (Menlo Park, Calif.). Using these vectors, 4 constructs were engineered and tested for GUS activity. Final transformation constructs are listed in Table 24. All restriction and ligation enzymes were obtained from New England Biolabs (Ipswich, Mass.). Ligated constructs were transformed into NEB 5-α competent  E. coli  (High Efficiency) (New England Biolabs: Ipswich, Mass.). 
     
       
         
           
               
             
               
                 TABLE 24 
               
             
            
               
                   
               
               
                 Genotypes of JCC138 transformants 
               
            
           
           
               
               
               
               
            
               
                   
                 Insert location 
                 Promoter 
                 Marker 
               
               
                   
                   
               
               
                   
                 ldh 
                 P(nirA) 
                 kanamycin 
               
               
                   
                 aquI 
                 P(nir07) 
                 spectinomycin 
               
               
                   
                 aquI 
                 P(nir09) 
                 spectinomycin 
               
               
                   
                 ldh 
                 P(nrsB) 
                 kanamycin 
               
               
                   
                   
               
            
           
         
       
     
     Plasmid Transformation into JCC138. 
     The constructs as described above were integrated onto either the genome or pAQ7 of JCC138, both of which are maintained at approximately 7 copies per cell. The following protocol was used for integrating the DNA cassettes. JCC138 was grown in an incubated shaker flask at 37° C. at 1% CO 2  to an OD 730  of 0.8 in A +  medium. 500 μl of culture was added to a microcentrifuge tube with 1 μg of DNA. DNA was prepared using a Qiagen Qiaprep Spin Miniprep Kit (Valencia, Calif.) for each construct. Cells were incubated in the dark for one hour at 37° C. The entire volume of cells was plated on A +  plates with 1.5% agar supplemented with 3 mM urea when necessary and grown at 37° C. in an illuminated incubator (40-60 μE/m2/s PAR, measured with a LI-250A light meter (LI-COR)) for approximately 24 hours. 25 μg/mL of spectinomycin or 50 μg/mL of kanamycin was introduced to the plates by placing the stock solution of antibiotic under the agar, and allowing it to diffuse up through the agar. After further incubation, resistant colonies became visible in 6 days. One colony from each plate was restreaked onto A +  plates with 1.5% agar supplemented with 6 mM urea when necessary and 200 μg/mL spectinomycin or 50 μg/mL of kanamycin. 
     Measurement of GUS Activity. 
     The GUS (beta-glucuronidase) reporter system was used to test the inducibility or repressibility of several promoters. This system measures the activity of beta-glucuronidase, an enzyme from  E. coli  that transforms colorless or non-fluorescent substrates into colored or fluorescent products. In this case, MUG (4-methylumbelliferyl β-D-glucuronide) is the substrate, and is hydrolyzed by beta-glucuronidase to produce the florescent product MU (4-methylumbelliferone), which is subsequently detected and quantified with a fluorescent spectrophotometer. 
     Strains containing uidA constructs under urea repression were incubated to OD 730  between 1.8 and 4. These cells were subcultured to OD 730  0.2 in 5 mL A+ media supplemented with 0, 3, 6, or 12 mM urea plus either 100 μg/mL spectinomycin or 50 μg/ml kanamycin and incubated for 24 hours. JCC138 was cultured in 5 mL A+ media for 24 hours. The strain containing gus under nickel-inducible expression was cultured for 3 days, then subcultured to OD 730  0.2 in 5 mL A+ supplemented with 0, 2, 4, or 8 M NiSO 4 . These cells were incubated for 6 hours. To harvest cells, cultures were spun for 5 minute at 6000 rpm. Pellets were resuspended in 1 mL 1×GUS extraction buffer (1 mM EDTA, 5.6 mM 2-mercaptoethanol, 0.1 M sodium phosphate, pH 7) and lysed with microtip sonication pulsing 0.5 seconds on and 0.5 seconds off for 2 min. Total protein was analyzed with Bio-Rad (Hercules, Calif.) Quick Start Bradford assay, and extracts were subsequently analyzed for GUS activity using a Sigma (St Louis, Mo.) 0-Glucuronidase Fluorescent Activity Detection Kit. Relative activities of the 4 promoters are found in Table 25. 
     
       
         
           
               
             
               
                 TABLE 25 
               
             
            
               
                   
               
               
                 GUS activities of inducible/repressible promoters 
               
            
           
           
               
               
               
               
               
            
               
                   
                 promoter 
                 mM urea 
                 uM NiSO 4   
                 (ABS/mg × 10 6 ) 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 P(nirA) 
                 0 
                 — 
                 121.9 
               
               
                   
                   
                 3 
                 — 
                 8 
               
               
                   
                   
                 6 
                 — 
                 11.62 
               
               
                   
                   
                 12 
                 — 
                 7.81 
               
               
                   
                 P(nir07) 
                 0 
                 — 
                 396.39 
               
               
                   
                   
                 3 
                 — 
                 23.61 
               
               
                   
                   
                 6 
                 — 
                 30.89 
               
               
                   
                   
                 12 
                 — 
                 33.13 
               
               
                   
                 P(nir09) 
                 0 
                 — 
                 97.77 
               
               
                   
                   
                 3 
                 — 
                 12.47 
               
               
                   
                   
                 6 
                 — 
                 12.35 
               
               
                   
                   
                 12 
                 — 
                 12.1 
               
               
                   
                 P(nrsB) 
                 — 
                 0 
                 24.97 
               
               
                   
                   
                 — 
                 2 
                 286.96 
               
               
                   
                   
                 — 
                 4 
                 257.26 
               
               
                   
                   
                 — 
                 8 
                 423.77 
               
               
                   
                 no uidA gene 
                 — 
                 — 
                 6.4 
               
               
                   
                   
               
            
           
         
       
     
     A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. All publications, patents and other references mentioned herein are hereby incorporated by reference in their entirety. 
     REFERENCES 
     
         
         Cho, H. and Cronan, J. E. (1993)  The Journal of Biological Chemistry  268: 9238-9245. 
         Chollet, R et al. (2004) Antimicrobial Agents and Chemotherapy 48: 3621-3624. 
         Kalscheuer, R., et al. (2006a)  Microbiology  152: 2529-2536. 
         Kalscheuer, R. et al. (2006b) Applied and Environmental Microbiology 72: 1373-1379. 
         Kameda, K. and Nunn, W. D. (1981)  The Journal of Biological Chemistry  256: 5702-5707. 
         Lopez-Mauy et al.,  Cell  (2002) v. 43:247-256 
         Nielsen, D. R et al. (2009)  Metabolic Engineering  11: 262-273. 
         Qi et al.,  Applied and Environmental Microbiology  (2005) v. 71: 5678-5684 
         Stöveken, T. et al. (2005)  Journal of Bacteriology  187:1369-1376 
         Tsukagoshi, N. and Aono, R. (2000)  Journal of Bacteriology  182: 4803-4810 
       
    
     INFORMAL SEQUENCE LISTING 
       
     
       
         
           
               
               
            
               
                 SEQ ID NO: 1 
                   
               
               
                   E .  coli  TesA amino acid sequence 
                   
               
               
                 (leader sequence removed) 
               
               
                 MADTLLILGDSLSAGYRMSASAAWPALLNDKWQSKTSVVNASISGDTSQQGLARLPAL 
               
               
                   
               
               
                 LKQHQPRWVLVELGGNDGLRGFQPQQTEQTLRQILQDVKAANAEPLLMQIRLPANYGR 
               
               
                   
               
               
                 RYNEAFSAIYPKLAKEFDVPLLPFFMEEVYLKPQWMQDDGIHPNRDAQPFIADWMAKQ 
               
               
                   
               
               
                 LQPLVNHDS 
               
               
                   
               
               
                 SEQ ID NO: 2 
                   
               
               
                   E .  coli  FadD amino acid sequence 
                   
               
               
                 MKKVWLNRYPADVPTEINPDRYQSLVDMFEQSVARYADQPAFVNMGEVMTFRKLEER 
               
               
                   
               
               
                 SRAFAAYLQQGLGLKKGDRVALMMPNLLQYPVALFGILRAGMIVVNVNPLYTPRELEH 
               
               
                   
               
               
                 QLNDSGASAIVIVSNFAHTLEKVVDKTAVQHVILTRMGDQLSTAKGTVVNFVVKYIKRL 
               
               
                   
               
               
                 VPKYHLPDAISFRSALHNGYRMQYVKPELVPEDLAFLQYTGGTTGVAKGAMLTHRNM 
               
               
                   
               
               
                 LANLEQVNATYGPLLHPGKELVVTALPLYHIFALTINCLLFIELGGQNLLITNPRDIPGLV 
               
               
                   
               
               
                 KELAKYPFTAITGVNTLFNALLNNKEFQQLDFSSLHLSAGGGMPVQQVVAERWVKLTG 
               
               
                   
               
               
                 QYLLEGYGLTECAPLVSVNPYDIDYHSGSIGLPVPSTEAKLVDDDDNEVPPGQPGELCV 
               
               
                   
               
               
                 KGPQVMLGYWQRPDATDEIIKNGWLHTGDIAVMDEEGFLRIVDRKKDMILVSGFNVYP 
               
               
                   
               
               
                 NEIEDVVMQHPGVQEVAAVGVPSGSSGEAVKIFVVKKDPSLTEESLVTFCRRQLTGYKV 
               
               
                   
               
               
                 PKLVEFRDELPKSNVGKILRRELRDEARGKVDNKA 
               
               
                   
               
               
                 SEQ ID NO: 3 
                   
               
               
                   A .  baylyi  ADP1 wax synthase amino acids sequence 
                   
               
               
                 MRPLHPIDFIFLSLEKRQQPMHVGGLFLFQIPDNAPDTFIQDLVNDIRISKSIPVPPFNNKL 
               
               
                   
               
               
                 NGLFWDEDEEFDLDHHFRHIALPHPGRIRELLIYISQEHSTLLDRAKPLWTCNIIEGIEGNR 
               
               
                   
               
               
                 FAMYFKIHHAMVDGVAGMRLIEKSLSHDVTEKSIVPPWCVEGKRAKRLREPKTGKIKKI 
               
               
                   
               
               
                 MSGIKSQLQATPTVIQELSQTVFKDIGRNPDHVSSFQAPCSILNQRVSSSRRFAAQSFDLD 
               
               
                   
               
               
                 RFRNIAKSLNVTINDVVLAVCSGALRAYLMSHNSLPSKPLIAMVPASIRNDDSDVSNRIT 
               
               
                   
               
               
                 MILANLATHKDDPLQRLEIIRRSVQNSKQRFKRMTSDQILNYSAVVYGPAGLNIISGMMP 
               
               
                   
               
               
                 KRQAFNLVISNVPGPREPLYWNGAKLDALYPASIVLDGQALNITMTSYLDKLEVGLIAC 
               
               
                   
               
               
                 RNALPRMQNLLTHLEEEIQLFEGVIAKQEDIKTAN 
               
               
                   
               
               
                 SEQ ID NO: 4 
                   
               
               
                   E .  coli  tesA optimized nucleic acid sequence 
                   
               
               
                 ATGGCGGATACTCTGCTGATTCTGGGTGATTCTCTGTCTGCAGGCTACCGTATGTCCG 
               
               
                   
               
               
                 CCTCCGCGGCCTGGCCAGCTCTGCTGAATGATAAGTGGCAGTCTAAGACGTCCGTTG 
               
               
                   
               
               
                 TGAACGCATCCATCTCTGGCGACACGAGCCAGCAGGGCCTGGCCCGTCTGCCTGCAC 
               
               
                   
               
               
                 TGCTGAAACAGCACCAACCGCGCTGGGTCCTGGTGGAGCTGGGCGGTAACGACGGT 
               
               
                   
               
               
                 CTGCGCGGCTTCCAGCCGCAGCAGACCGAACAGACTCTGCGTCAGATTCTGCAGGA 
               
               
                   
               
               
                 CGTGAAAGCTGCTAACGCGGAACCGCTGCTGATGCAGATTCGTCTGCCAGCGAACT 
               
               
                   
               
               
                 ATGGCCGCCGTTACAACGAAGCGTTCTCTGCAATCTACCCAAAACTGGCGAAAGAG 
               
               
                   
               
               
                 TTTGACGTCCCGCTGCTGCCGTTCTTCATGGAGGAAGTATACCTGAAACCGCAGTGG 
               
               
                   
               
               
                 ATGCAAGATGACGGCATCCACCCGAACCGTGATGCGCAGCCGTTCATCGCTGACTG 
               
               
                   
               
               
                 GATGGCGAAGCAACTGCAGCCGCTGGTAAACCACGATTCCTAA 
               
               
                   
               
               
                 SEQ ID NO: 5 
                   
               
               
                   E .  coli  fadD optimized nucleic acid sequence 
                   
               
               
                 ATGAAGAAAGTTTGGCTGAACCGTTATCCGGCAGATGTACCGACTGAAATTAACCC 
               
               
                   
               
               
                 AGATCGTTACCAGTCCCTGGTTGACATGTTCGAACAGTCCGTGGCTCGCTACGCCGA 
               
               
                   
               
               
                 TCAGCCTGCTTTCGTCAACATGGGTGAGGTAATGACCTTTCGCAAACTGGAGGAGCG 
               
               
                   
               
               
                 TTCCCGTGCTTTCGCGGCATACCTGCAGCAGGGTCTGGGCCTGAAGAAAGGCGACC 
               
               
                   
               
               
                 GCGTGGCCCTGATGATGCCGAACCTGCTGCAATATCCTGTGGCGCTGTTCGGTATCC 
               
               
                   
               
               
                 TGCGTGCTGGTATGATCGTTGTCAATGTTAACCCTCTGTATACCCCTCGTGAACTGGA 
               
               
                   
               
               
                 GCACCAGCTGAATGACTCTGGTGCGTCTGCTATCGTTATCGTTTCCAATTTCGCACAT 
               
               
                   
               
               
                 ACGCTGGAGAAAGTGGTTGATAAAACCGCAGTGCAGCATGTCATTCTGACTCGCAT 
               
               
                   
               
               
                 GGGTGACCAGCTGTCCACCGCTAAAGGTACTGTAGTCAACTTCGTTGTGAAATACAT 
               
               
                   
               
               
                 TAAGCGCCTGGTTCCGAAATACCACCTGCCAGATGCAATTAGCTTTCGCTCTGCACT 
               
               
                   
               
               
                 GCATAACGGTTACCGTATGCAGTACGTAAAACCAGAGCTGGTGCCGGAAGACCTGG 
               
               
                   
               
               
                 CCTTTCTGCAGTATACCGGCGGCACCACCGGCGTGGCAAAGGGCGCGATGCTGACC 
               
               
                   
               
               
                 CATCGTAACATGCTGGCGAACCTGGAGCAGGTTAACGCAACGTACGGCCCGCTGCT 
               
               
                   
               
               
                 GCACCCGGGTAAAGAACTGGTAGTTACGGCACTGCCTCTGTATCACATCTTTGCACT 
               
               
                   
               
               
                 GACGATCAACTGTCTGCTGTTCATTGAACTGGGTGGTCAGAACCTGCTGATCACCAA 
               
               
                   
               
               
                 CCCGCGTGACATTCCGGGCCTGGTAAAAGAGCTGGCTAAGTACCCGTTCACCGCCAT 
               
               
                   
               
               
                 TACTGGCGTAAACACTCTGTTTAACGCGCTGCTGAACAACAAAGAGTTTCAGCAGCT 
               
               
                   
               
               
                 GGACTTCTCTAGCCTGCACCTGAGCGCTGGCGGTGGCATGCCGGTTCAGCAGGTTGT 
               
               
                   
               
               
                 GGCAGAGCGTTGGGTGAAACTGACCGGCCAGTATCTGCTGGAGGGTTATGGTCTGA 
               
               
                   
               
               
                 CCGAGTGTGCACCGCTGGTCAGCGTTAACCCGTATGATATTGATTACCACTCTGGTT 
               
               
                   
               
               
                 CTATTGGTCTGCCGGTTCCGTCCACGGAAGCCAAACTGGTGGACGATGACGACAAC 
               
               
                   
               
               
                 GAAGTACCTCCGGGCCAGCCGGGTGAGCTGTGTGTCAAGGGTCCGCAGGTTATGCT 
               
               
                   
               
               
                 GGGCTACTGGCAGCGCCCGGACGCCACCGACGAAATCATTAAAAACGGTTGGCTGC 
               
               
                   
               
               
                 ATACCGGTGATATCGCTGTAATGGACGAAGAAGGTTTCCTGCGTATCGTGGACCGTA 
               
               
                   
               
               
                 AGAAAGATATGATTCTGGTGAGCGGTTTCAACGTGTACCCGAACGAAATTGAGGAC 
               
               
                   
               
               
                 GTAGTTATGCAACACCCTGGCGTGCAGGAGGTGGCAGCCGTGGGCGTGCCGTCCGG 
               
               
                   
               
               
                 TTCTTCTGGTGAGGCTGTGAAAATCTTTGTCGTTAAAAAGGACCCGTCCCTGACCGA 
               
               
                   
               
               
                 AGAATCTCTGGTGACGTTTTGCCGCCGTCAACTGACTGGCTACAAAGTGCCGAAACT 
               
               
                   
               
               
                 GGTCGAGTTCCGCGATGAGCTGCCAAAATCTAACGTGGGTAAGATCCTGCGCCGCG 
               
               
                   
               
               
                 AGCTGCGTGACGAGGCACGTGGCAAAGTTGACAATAAAGCATAA 
               
               
                   
               
               
                 SEQ ID NO: 6 
                   
               
               
                   A .  baylyi  wsadpl optimized nucleic acid sequence 
                   
               
               
                 ATGCGCCCACTTCATCCGATCGATTTCATTTTCCTGTCCCTGGAGAAACGCCAGCAG 
               
               
                   
               
               
                 CCGATGCACGTAGGTGGTCTGTTCCTGTTCCAGATCCCGGATAACGCTCCGGACACC 
               
               
                   
               
               
                 TTTATTCAGGACCTGGTGAACGATATCCGTATCTCCAAGTCTATTCCGGTTCCGCCGT 
               
               
                   
               
               
                 TCAACAACAAGCTGAACGGTCTGTTCTGGGACGAAGACGAGGAGTTCGATCTGGAT 
               
               
                   
               
               
                 CACCATTTCCGTCATATTGCGCTGCCGCACCCGGGTCGCATCCGTGAGCTGCTGATT 
               
               
                   
               
               
                 TACATCTCTCAGGAACACAGCACTCTCCTCGATCGCGCTAAACCTCTGTGGACTTGC 
               
               
                   
               
               
                 AACATCATTGAAGGTATCGAGGGTAACCGTTTCGCCATGTACTTCAAGATTCATCAT 
               
               
                   
               
               
                 GCGATGGTGGATGGTGTGGCGGGTATGCGTCTGATTGAGAAAAGCCTGTCCCATGAT 
               
               
                   
               
               
                 GTTACTGAAAAGAGCATCGTACCGCCGTGGTGCGTTGAGGGCAAACGTGCTAAACG 
               
               
                   
               
               
                 CCTGCGTGAACCGAAGACCGGCAAAATTAAGAAAATCATGTCTGGTATTAAATCTC 
               
               
                   
               
               
                 AGCTCCAGGCCACCCCGACCGTTATTCAAGAACTGTCTCAGACGGTCTTCAAAGACA 
               
               
                   
               
               
                 TCGGCCGTAATCCGGACCACGTTTCCTCTTTCCAGGCGCCGTGCTCCATCCTCAACC 
               
               
                   
               
               
                 AGCGTGTGTCTTCTTCTCGTCGTTTCGCAGCACAGAGCTTTGACCTGGACCGTTTCCG 
               
               
                   
               
               
                 CAACATCGCCAAATCTCTGAACGTGACCATTAACGACGTTGTCCTGGCTGTGTGTAG 
               
               
                   
               
               
                 CGGTGCTCTGCGCGCTTATCTGATGTCTCATAACTCTCTGCCATCCAAACCGCTGATC 
               
               
                   
               
               
                 GCTATGGTCCCAGCAAGCATCCGCAACGATGATTCTGATGTGTCCAACCGTATTACT 
               
               
                   
               
               
                 ATGATTCTGGCCAACCTCGCTACTCACAAAGACGACCCTCTGCAGCGTCTGGAAATC 
               
               
                   
               
               
                 ATCCGCCGCTCCGTCCAGAACTCTAAACAGCGTTTTAAACGCATGACTTCCGACCAG 
               
               
                   
               
               
                 ATTCTGAACTATTCTGCGGTTGTATACGGCCCGGCTGGTCTGAACATTATCAGCGGT 
               
               
                   
               
               
                 ATGATGCCGAAACGTCAGGCTTTTAACCTGGTAATCAGCAACGTTCCTGGCCCGCGT 
               
               
                   
               
               
                 GAGCCGCTGTACTGGAACGGCGCAAAACTGGACGCACTGTACCCGGCTTCCATCGTT 
               
               
                   
               
               
                 CTGGATGGCCAGGCTCTGAACATCACTATGACCTCTTACCTGGACAAACTGGAAGTA 
               
               
                   
               
               
                 GGTCTGATCGCGTGTCGCAATGCACTGCCGCGCATGCAGAACCTGCTGACCCACCTG 
               
               
                   
               
               
                 GAGGAGGAAATCCAGCTGTTTGAGGGCGTTATCGCCAAACAGGAAGATATCAAAAC 
               
               
                   
               
               
                 GGCGAACTAA 
               
               
                   
               
               
                 SEQ ID NO: 7 
                   
               
               
                   E .  coli  TolC amino acid sequence 
                   
               
               
                 MKKLLPILIGLSLSGFSSLSQAENLMQVYQQARLSNPELRKSAADRDAAFEKINEARSPL 
               
               
                   
               
               
                 LPQLGLGADYTYSNGYRDANGINSNATSASLQLTQSIFDMSKWRALTLQEKAAGIQDVT 
               
               
                   
               
               
                 YQTDQQTLILNTATAYFNVLNAIDVLSYTQAQKEAIYRQLDQTTQRFNVGLVAITDVQN 
               
               
                   
               
               
                 ARAQYDTVLANEVTARNNLDNAVEQLRQITGNYYPELAALNVENFKTDKPQPVNALLK 
               
               
                   
               
               
                 EAEKRNLSLLQARLSQDLAREQIRQAQDGHLPTLDLTASTGISDTSYSGSKTRGAAGTQ 
               
               
                   
               
               
                 YDDSNMGQNKVGLSFSLPIYQGGMVNSQVKQAQYNFVGASEQLESAHRSVVQTVRSSF 
               
               
                   
               
               
                 NNINASISSINAYKQAVVSAQSSLDAMEAGYSVGTRTIVDVLDATTTLYNAKQELANAR 
               
               
                   
               
               
                 YNYLINQLNIKSALGTLNEQDLLALNNALSKPVSTNPENVAPQTPEQNAIADGYAPDSPA 
               
               
                   
               
               
                 PVVQQTSARTTTSNGHNPFRN 
               
               
                   
               
               
                 SEQ ID NO: 8 
                   
               
               
                   E .  coli  AcrA amino acid sequence 
                   
               
               
                 MNKNRGFTPLAVVLMLSGSLALTGCDDKQAQQGGQQMPAVGVVTVKTEPLQITTELP 
               
               
                   
               
               
                 GRTSAYRIAEVRPQVSGIILKRNFKEGSDIEAGVSLYQIDPATYQATYDSAKGDLAKAQA 
               
               
                   
               
               
                 AANIAQLTVNRYQKLLGTQYISKQEYDQALADAQQANAAVTAAKAAVETARINLAYT 
               
               
                   
               
               
                 KVTSPISGRIGKSNVTEGALVQNGQATALATVQQLDPIYVDVTQSSNDFLRLKQELANG 
               
               
                   
               
               
                 TLKQENGKAKVSLITSDGIKFPQDGTLEFSDVTVDQTTGSITLRAIFPNPDHTLLPGMFVR 
               
               
                   
               
               
                 ARLEEGLNPNAILVPQQGVTRTPRGDATVLVVGADDKVETRPIVASQAIGDKWLVTEGL 
               
               
                   
               
               
                 KAGDRVVISGLQKVRPGVQVKAQEVTADNNQQAASGAQPEQSKS 
               
               
                   
               
               
                 SEQ ID NO: 9 
                   
               
               
                   E .  coli  AcrB amino acid sequence 
                   
               
               
                 MPNFFIDRPIFAWVIAIIIMLAGGLAILKLPVAQYPTIAPPAVTISASYPGADAKTVQDTVT 
               
               
                   
               
               
                 QVIEQNMNGIDNLMYMSSNSDSTGTVQITLTFESGTDADIAQVQVQNKLQLAMPLLPQE 
               
               
                   
               
               
                 VQQQGVSVEKSSSSFLMVVGVINTDGTMTQEDISDYVAANMKDAISRTSGVGDVQLFG 
               
               
                   
               
               
                 SQYAMRIWMNPNELNKFQLTPVDVITAIKAQNAQVAAGQLGGTPPVKGQQLNASIIAQT 
               
               
                   
               
               
                 RLTSTEEFGKILLKVNQDGSRVLLRDVAKIELGGENYDIIAEFNGQPASGLGIKLATGAN 
               
               
                   
               
               
                 ALDTAAAIRAELAKMEPFFPSGLKIVYPYDTTPFVKISIHEVVKTLVEAIILVFLVMYLFL 
               
               
                   
               
               
                 QNFRATLIPTIAVPVVLLGTFAVLAAFGFSINTLTMFGMVLAIGLLVDDAIVVVENVERV 
               
               
                   
               
               
                 MAEEGLPPKEATRKSMGQIQGALVGIAMVLSAVFVPMAFFGGSTGAIYRQFSITIVSAM 
               
               
                   
               
               
                 ALSVLVALILTPALCATMLKPIAKGDHGEGKKGFFGWFNRMFEKSTHHYTDSVGGILRS 
               
               
                   
               
               
                 TGRYLVLYLIIVVGMAYLFVRLPSSFLPDEDQGVFMTMVQLPAGATQERTQKVLNEVT 
               
               
                   
               
               
                 HYYLTKEKNNVESVFAVNGFGFAGRGQNTGIAFVSLKDWADRPGEENKVEAITMRATR 
               
               
                   
               
               
                 AFSQIKDAMVFAFNLPAIVELGTATGFDFELIDQAGLGHEKLTQARNQLLAEAAKHPDM 
               
               
                   
               
               
                 LTSVRPNGLEDTPQFKIDIDQEKAQALGVSINDINTTLGAAWGGSYVNDFIDRGRVKKV 
               
               
                   
               
               
                 YVMSEAKYRMLPDDIGDWYVRAADGQMVPFSAFSSSRWEYGSPRLERYNGLPSMEILG 
               
               
                   
               
               
                 QAAPGKSTGEAMELMEQLASKLPTGVGYDWTGMSYQERLSGNQAPSLYAISLIVVFLC 
               
               
                   
               
               
                 LAALYESWSIPFSVMLVVPLGVIGALLAATFRGLTNDVYFQVGLLTTIGLSAKNAILIVEF 
               
               
                   
               
               
                 AKDLMDKEGKGLIEATLDAVRMRLRPILMTSLAFILGVMPLVISTGAGSGAQNAVGTGV 
               
               
                   
               
               
                 MGGMVTATVLAIFFVPVFFVVVRRRFSRKNEDIEHSHTVDHH 
               
               
                   
               
               
                 SEQ ID NO: 10 
                   
               
               
                 PaphII underlined; tesA, fadD and wsadpl are in bold and follow the promoter in order 
                   
               
               
                 GCGGCCGC GGGGGGGGGGGGGAAAGCCACGTTGTGTCTCAAAATCTCTGATGTTACATTGCACAAGAT   
               
               
                   
               
               
                   AAAAATATATCATCATGAACAATAAAACTGTCTGCTTACATAAACAGTAATACAAGGGGT CAT ATGG   
               
               
                   
               
               
                 
                   CGGATACTCTGCTGATTCTGGGTGATTCTCTGTCTGCAGGCTACCGTATGTCCGCCTCCGCGGC 
                 
               
               
                   
               
               
                 
                   CTGGCCAGCTCTGCTGAATGATAAGTGGCAGTCTAAGACGTCCGTTGTGAACGCATCCATCTCT 
                 
               
               
                   
               
               
                 
                   GGCGACACGAGCCAGCAGGGCCTGGCCCGTCTGCCTGCACTGCTGAAACAGCACCAACCGCGC 
                 
               
               
                   
               
               
                 
                   TGGGTCCTGGTGGAGCTGGGCGGTAACGACGGTCTGCGCGGCTTCCAGCCGCAGCAGACCGAA 
                 
               
               
                   
               
               
                 
                   CAGACTCTGCGTCAGATTCTGCAGGACGTGAAAGCTGCTAACGCGGAACCGCTGCTGATGCAGA 
                 
               
               
                   
               
               
                 
                   TTCGTCTGCCAGCGAACTATGGCCGCCGTTACAACGAAGCGTTCTCTGCAATCTACCCAAAACT 
                 
               
               
                   
               
               
                 
                   GGCGAAAGAGTTTGACGTCCCGCTGCTGCCGTTCTTCATGGAGGAAGTATACCTGAAACCGCAG 
                 
               
               
                   
               
               
                 
                   TGGATGCAAGATGACGGCATCCACCCGAACCGTGATGCGCAGCCGTTCATCGCTGACTGGATGG 
                 
               
               
                   
               
               
                   CGAAGCAACTGCAGCCGCTGGTAAACCACGATTCCTAA TTAAAGATCTGTAGTAGGATCCATGTAG 
               
               
                   
               
               
                 GGTGAGGTTATAGCT ATGAAGAAAGTTTGGCTGAACCGTTATCCGGCAGATGTACCGACTGAAAT   
               
               
                   
               
               
                 
                   TAACCCAGATCGTTACCAGTCCCTGGTTGACATGTTCGAACAGTCCGTGGCTCGCTACGCCGAT 
                 
               
               
                   
               
               
                 
                   CAGCCTGCTTTCGTCAACATGGGTGAGGTAATGACCTTTCGCAAACTGGAGGAGCGTTCCCGTG 
                 
               
               
                   
               
               
                 
                   CTTTCGCGGCATACCTGCAGCAGGGTCTGGGCCTGAAGAAAGGCGACCGCGTGGCCCTGATGAT 
                 
               
               
                   
               
               
                 
                   GCCGAACCTGCTGCAATATCCTGTGGCGCTGTTCGGTATCCTGCGTGCTGGTATGATCGTTGTC 
                 
               
               
                   
               
               
                 
                   AATGTTAACCCTCTGTATACCCCTCGTGAACTGGAGCACCAGCTGAATGACTCTGGTGCGTCTG 
                 
               
               
                   
               
               
                 
                   CTATCGTTATCGTTTCCAATTTCGCACATACGCTGGAGAAAGTGGTTGATAAAACCGCAGTGCAG 
                 
               
               
                   
               
               
                 
                   CATGTCATTCTGACTCGCATGGGTGACCAGCTGTCCACCGCTAAAGGTACTGTAGTCAACTTCGT 
                 
               
               
                   
               
               
                 
                   TGTGAAATACATTAAGCGCCTGGTTCCGAAATACCACCTGCCAGATGCAATTAGCTTTCGCTCTG 
                 
               
               
                   
               
               
                 
                   CACTGCATAACGGTTACCGTATGCAGTACGTAAAACCAGAGCTGGTGCCGGAAGACCTGGCCTT 
                 
               
               
                   
               
               
                 
                   TCTGCAGTATACCGGCGGCACCACCGGCGTGGCAAAGGGCGCGATGCTGACCCATCGTAACATG 
                 
               
               
                   
               
               
                 
                   CTGGCGAACCTGGAGCAGGTTAACGCAACGTACGGCCCGCTGCTGCACCCGGGTAAAGAACTG 
                 
               
               
                   
               
               
                 
                   GTAGTTACGGCACTGCCTCTGTATCACATCTTTGCACTGACGATCAACTGTCTGCTGTTCATTGA 
                 
               
               
                   
               
               
                 
                   ACTGGGTGGTCAGAACCTGCTGATCACCAACCCGCGTGACATTCCGGGCCTGGTAAAAGAGCTG 
                 
               
               
                   
               
               
                 
                   GCTAAGTACCCGTTCACCGCCATTACTGGCGTAAACACTCTGTTTAACGCGCTGCTGAACAACAA 
                 
               
               
                   
               
               
                 
                   AGAGTTTCAGCAGCTGGACTTCTCTAGCCTGCACCTGAGCGCTGGCGGTGGCATGCCGGTTCAG 
                 
               
               
                   
               
               
                 
                   CAGGTTGTGGCAGAGCGTTGGGTGAAACTGACCGGCCAGTATCTGCTGGAGGGTTATGGTCTGA 
                 
               
               
                   
               
               
                 
                   CCGAGTGTGCACCGCTGGTCAGCGTTAACCCGTATGATATTGATTACCACTCTGGTTCTATTGGT 
                 
               
               
                   
               
               
                 
                   CTGCCGGTTCCGTCCACGGAAGCCAAACTGGTGGACGATGACGACAACGAAGTACCTCCGGGCC 
                 
               
               
                   
               
               
                 
                   AGCCGGGTGAGCTGTGTGTCAAGGGTCCGCAGGTTATGCTGGGCTACTGGCAGCGCCCGGACG 
                 
               
               
                   
               
               
                 
                   CCACCGACGAAATCATTAAAAACGGTTGGCTGCATACCGGTGATATCGCTGTAATGGACGAAGA 
                 
               
               
                   
               
               
                 
                   AGGTTTCCTGCGTATCGTGGACCGTAAGAAAGATATGATTCTGGTGAGCGGTTTCAACGTGTAC 
                 
               
               
                   
               
               
                 
                   CCGAACGAAATTGAGGACGTAGTTATGCAACACCCTGGCGTGCAGGAGGTGGCAGCCGTGGGC 
                 
               
               
                   
               
               
                 
                   GTGCCGTCCGGTTCTTCTGGTGAGGCTGTGAAAATCTTTGTCGTTAAAAAGGACCCGTCCCTGA 
                 
               
               
                   
               
               
                 
                   CCGAAGAATCTCTGGTGACGTTTTGCCGCCGTCAACTGACTGGCTACAAAGTGCCGAAACTGGT 
                 
               
               
                   
               
               
                 
                   CGAGTTCCGCGATGAGCTGCCAAAATCTAACGTGGGTAAGATCCTGCGCCGCGAGCTGCGTGAC 
                 
               
               
                   
               
               
                   GAGGCACGTGGCAAAGTTGACAATAAAGCATAA CCGCGTAGGAGGACAGCT ATGCGCCCACTTCA   
               
               
                   
               
               
                 
                   TCCGATCGATTTCATTTTCCTGTCCCTGGAGAAACGCCAGCAGCCGATGCACGTAGGTGGTCTG 
                 
               
               
                   
               
               
                 
                   TTCCTGTTCCAGATCCCGGATAACGCTCCGGACACCTTTATTCAGGACCTGGTGAACGATATCCG 
                 
               
               
                   
               
               
                 
                   TATCTCCAAGTCTATTCCGGTTCCGCCGTTCAACAACAAGCTGAACGGTCTGTTCTGGGACGAA 
                 
               
               
                   
               
               
                 
                   GACGAGGAGTTCGATCTGGATCACCATTTCCGTCATATTGCGCTGCCGCACCCGGGTCGCATCC 
                 
               
               
                   
               
               
                 
                   GTGAGCTGCTGATTTACATCTCTCAGGAACACAGCACTCTCCTCGATCGCGCTAAACCTCTGTGG 
                 
               
               
                   
               
               
                 
                   ACTTGCAACATCATTGAAGGTATCGAGGGTAACCGTTTCGCCATGTACTTCAAGATTCATCATGC 
                 
               
               
                   
               
               
                 
                   GATGGTGGATGGTGTGGCGGGTATGCGTCTGATTGAGAAAAGCCTGTCCCATGATGTTACTGAA 
                 
               
               
                   
               
               
                 
                   AAGAGCATCGTACCGCCGTGGTGCGTTGAGGGCAAACGTGCTAAACGCCTGCGTGAACCGAAG 
                 
               
               
                   
               
               
                 
                   ACCGGCAAAATTAAGAAAATCATGTCTGGTATTAAATCTCAGCTCCAGGCCACCCCGACCGTTAT 
                 
               
               
                   
               
               
                 
                   TCAAGAACTGTCTCAGACGGTCTTCAAAGACATCGGCCGTAATCCGGACCACGTTTCCTCTTTCC 
                 
               
               
                   
               
               
                 
                   AGGCGCCGTGCTCCATCCTCAACCAGCGTGTGTCTTCTTCTCGTCGTTTCGCAGCACAGAGCTTT 
                 
               
               
                   
               
               
                 
                   GACCTGGACCGTTTCCGCAACATCGCCAAATCTCTGAACGTGACCATTAACGACGTTGTCCTGG 
                 
               
               
                   
               
               
                 
                   CTGTGTGTAGCGGTGCTCTGCGCGCTTATCTGATGTCTCATAACTCTCTGCCATCCAAACCGCTG 
                 
               
               
                   
               
               
                 
                   ATCGCTATGGTCCCAGCAAGCATCCGCAACGATGATTCTGATGTGTCCAACCGTATTACTATGAT 
                 
               
               
                   
               
               
                 
                   TCTGGCCAACCTCGCTACTCACAAAGACGACCCTCTGCAGCGTCTGGAAATCATCCGCCGCTCC 
                 
               
               
                   
               
               
                 
                   GTCCAGAACTCTAAACAGCGTTTTAAACGCATGACTTCCGACCAGATTCTGAACTATTCTGCGGT 
                 
               
               
                   
               
               
                 
                   TGTATACGGCCCGGCTGGTCTGAACATTATCAGCGGTATGATGCCGAAACGTCAGGCTTTTAAC 
                 
               
               
                   
               
               
                 
                   CTGGTAATCAGCAACGTTCCTGGCCCGCGTGAGCCGCTGTACTGGAACGGCGCAAAACTGGACG 
                 
               
               
                   
               
               
                 
                   CACTGTACCCGGCTTCCATCGTTCTGGATGGCCAGGCTCTGAACATCACTATGACCTCTTACCTG 
                 
               
               
                   
               
               
                 
                   GACAAACTGGAAGTAGGTCTGATCGCGTGTCGCAATGCACTGCCGCGCATGCAGAACCTGCTGA 
                 
               
               
                   
               
               
                 
                   CCCACCTGGAGGAGGAAATCCAGCTGTTTGAGGGCGTTATCGCCAAACAGGAAGATATCAAAAC 
                 
               
               
                   
               
               
                   GGCGAACTAA CCATGGTTGAATTC 
               
               
                   
               
               
                 SEQ ID NO: 11 
                   
               
               
                 pJB532 (UHR and DHR are lowercase; lacIq with promoter and P trc  underlined; tesA, fadD and wsadpl 
                   
               
               
                 are in bold and underlined and follow the promoter in order; aadA marker is italicized 
               
               
                 and underlined) 
               
               
                 CCTGCAGG G tcagcaagctctggaatttcccgattctctgatgggagatccaaaaattctcgcagtccctcaatcacgatatcggtcttggatcgcc 
               
               
                   
               
               
                 ctgtagcttccgacaactgctcaattttttcgagcatctctaccgggcatcggaatgaaattaacggtgttttagccatgtgttatacagtgtttac 
               
               
                   
               
               
                 aacttgactaacaaatacctgctagtgtatacatattgtattgcaatgtatacgctattttcactgctgtctttaatggggattatcgcaagcaagt 
               
               
                   
               
               
                 aaaaaagcctgaaaaccccaataggtaagggattccgagcttactcgataattatcacctttgagcgcccctaggaggaggcgaaaagctatgtctg 
               
               
                   
               
               
                 acaaggggtttgacccctgaagtcgttgcgcgagcattaaggtctgcggatagcccataacatacttttgttgaacttgtgcgcttttatcaacccc 
               
               
                   
               
               
                 ttaagggcttgggagcgttttatGCG 
               
               
                   
               
               
                 GCCGC TCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGC   
               
               
                   
               
               
                 
                   CAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTCACCAGTG 
                 
               
               
                   
               
               
                 
                   AGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTC 
                 
               
               
                   
               
               
                 
                   CACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTGACGGCGGGATATAAC 
                 
               
               
                   
               
               
                 
                   ATGAGCTGTCTTCGGTATCGTCGTATCCCACTACCGAGATATCCGCACCAACGCGCAGCCCG 
                 
               
               
                   
               
               
                 
                   GACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCTGATCGTTGGCAACCAGCATCGCAGT 
                 
               
               
                   
               
               
                 
                   GGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAACCGGACATGGCACTCCAGT 
                 
               
               
                   
               
               
                 
                   CGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATGCCAGCCAGCCA 
                 
               
               
                   
               
               
                 
                   GACGCAGACGCGCCGAGACAGAACTTAATGGGCCCGCTAACAGCGCGATTTGCTGGTGACC 
                 
               
               
                   
               
               
                 
                   CAATGCGACCAGATGCTCCACGCCCAGTCGCGTACCGTCTTCATGGGAGAAAATAATACTGT 
                 
               
               
                   
               
               
                 
                   TGATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGGAACATTAGTGCAGGCAGCTTC 
                 
               
               
                   
               
               
                 
                   CACAGCAATGGCATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCACTGACGCGCTGCG 
                 
               
               
                   
               
               
                 
                   CGAGAAGATTGTGCACCGCCGCTTTACAGGCTTCGACGCCGCTTCGTTCTACCATCGACACC 
                 
               
               
                   
               
               
                 
                   ACCACGCTGGCACCCAGTTGATCGGCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCGC 
                 
               
               
                   
               
               
                 
                   GTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTT 
                 
               
               
                   
               
               
                 
                   GTGCCACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTT 
                 
               
               
                   
               
               
                 
                   TCGCAGAAACGTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCGGC 
                 
               
               
                   
               
               
                 
                   ATACTCTGCGACATCGTATAACGTTACTGGTTTCATATTCACCACCCTGAATTGACTCTCTTC 
                 
               
               
                   
               
               
                 
                   CGGGCGCTATCATGCCATACCGCGAAAGGTTTTGCACCATTCGATGGTGTCAACGTAAATGC 
                 
               
               
                   
               
               
                 
                   ATGCCGCTTCGCCTTCCAATTGGACTGCACGGTGCACCAATGCTTCTGGCGTCAGGCAGCCA 
                 
               
               
                   
               
               
                 
                   TCGGAAGCTGTGGTATGGCTGTGCAGGTCGTAAATCACTGCATAATTCGTGTCGCTCAAGGC 
                 
               
               
                   
               
               
                 
                   GCACTCCCGTTCTGGATAATGTTTTTTGCGCCGACATCATAACGGTTCTGGCAAATATTCTGA 
                 
               
               
                   
               
               
                 
                   AATGAGCTGTTGACAATTAATCATCCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACA 
                 
               
               
                   
               
               
                 ATTTCACACAGGAAACAGCATGGCCAAGGAGGCCCAT   ATGGCGGATACTCTGCTGATTCT     
               
               
                   
               
               
                 
                   
                     GGGTGATTCTCTGTCTGCAGGCTACCGTATGTCCGCCTCCGCGGCCTGGCCAGCTCTG 
                   
                 
               
               
                   
               
               
                 
                   
                     CTGAATGATAAGTGGCAGTCTAAGACGTCCGTTGTGAACGCATCCATCTCTGGCGACA 
                   
                 
               
               
                   
               
               
                 
                   
                     CGAGCCAGCAGGGCCTGGCCCGTCTGCCTGCACTGCTGAAACAGCACCAACCGCGCTG 
                   
                 
               
               
                   
               
               
                 
                   
                     GGTCCTGGTGGAGCTGGGCGGTAACGACGGTCTGCGCGGCTTCCAGCCGCAGCAGAC 
                   
                 
               
               
                   
               
               
                 
                   
                     CGAACAGACTCTGCGTCAGATTCTGCAGGACGTGAAAGCTGCTAACGCGGAACCGCTG 
                   
                 
               
               
                   
               
               
                 
                   
                     CTGATGCAGATTCGTCTGCCAGCGAACTATGGCCGCCGTTACAACGAAGCGTTCTCTG 
                   
                 
               
               
                   
               
               
                 
                   
                     CAATCTACCCAAAACTGGCGAAAGAGTTTGACGTCCCGCTGCTGCCGTTCTTCATGGA 
                   
                 
               
               
                   
               
               
                 
                   
                     GGAAGTATACCTGAAACCGCAGTGGATGCAAGATGACGGCATCCACCCGAACCGTGAT 
                   
                 
               
               
                   
               
               
                 
                   
                     GCGCAGCCGTTCATCGCTGACTGGATGGCGAAGCAACTGCAGCCGCTGGTAAACCACG 
                   
                 
               
               
                   
               
               
                     ATTCCTAA   TTAAAGATCTGTAGTAGGATCCATGTAGGGTGAGGTTATAGCT   ATGAAGAAAG     
               
               
                   
               
               
                 
                   
                     TTTGGCTGAACCGTTATCCGGCAGATGTACCGACTGAAATTAACCCAGATCGTTACCAG 
                   
                 
               
               
                   
               
               
                 
                   
                     TCCCTGGTTGACATGTTCGAACAGTCCGTGGCTCGCTACGCCGATCAGCCTGCTTTCGT 
                   
                 
               
               
                   
               
               
                 
                   
                     CAACATGGGTGAGGTAATGACCTTTCGCAAACTGGAGGAGCGTTCCCGTGCTTTCGCG 
                   
                 
               
               
                   
               
               
                 
                   
                     GCATACCTGCAGCAGGGTCTGGGCCTGAAGAAAGGCGACCGCGTGGCCCTGATGATG 
                   
                 
               
               
                   
               
               
                 
                   
                     CCGAACCTGCTGCAATATCCTGTGGCGCTGTTCGGTATCCTGCGTGCTGGTATGATCG 
                   
                 
               
               
                   
               
               
                 
                   
                     TTGTCAATGTTAACCCTCTGTATACCCCTCGTGAACTGGAGCACCAGCTGAATGACTCT 
                   
                 
               
               
                   
               
               
                 
                   
                     GGTGCGTCTGCTATCGTTATCGTTTCCAATTTCGCACATACGCTGGAGAAAGTGGTTGA 
                   
                 
               
               
                   
               
               
                 
                   
                     TAAAACCGCAGTGCAGCATGTCATTCTGACTCGCATGGGTGACCAGCTGTCCACCGCT 
                   
                 
               
               
                   
               
               
                 
                   
                     AAAGGTACTGTAGTCAACTTCGTTGTGAAATACATTAAGCGCCTGGTTCCGAAATACCA 
                   
                 
               
               
                   
               
               
                 
                   
                     CCTGCCAGATGCAATTAGCTTTCGCTCTGCACTGCATAACGGTTACCGTATGCAGTACG 
                   
                 
               
               
                   
               
               
                 
                   
                     TAAAACCAGAGCTGGTGCCGGAAGACCTGGCCTTTCTGCAGTATACCGGCGGCACCAC 
                   
                 
               
               
                   
               
               
                 
                   
                     CGGCGTGGCAAAGGGCGCGATGCTGACCCATCGTAACATGCTGGCGAACCTGGAGCA 
                   
                 
               
               
                   
               
               
                 
                   
                     GGTTAACGCAACGTACGGCCCGCTGCTGCACCCGGGTAAAGAACTGGTAGTTACGGCA 
                   
                 
               
               
                   
               
               
                 
                   
                     CTGCCTCTGTATCACATCTTTGCACTGACGATCAACTGTCTGCTGTTCATTGAACTGGG 
                   
                 
               
               
                   
               
               
                 
                   
                     TGGTCAGAACCTGCTGATCACCAACCCGCGTGACATTCCGGGCCTGGTAAAAGAGCTG 
                   
                 
               
               
                   
               
               
                 
                   
                     GCTAAGTACCCGTTCACCGCCATTACTGGCGTAAACACTCTGTTTAACGCGCTGCTGAA 
                   
                 
               
               
                   
               
               
                 
                   
                     CAACAAAGAGTTTCAGCAGCTGGACTTCTCTAGCCTGCACCTGAGCGCTGGCGGTGGC 
                   
                 
               
               
                   
               
               
                 
                   
                     ATGCCGGTTCAGCAGGTTGTGGCAGAGCGTTGGGTGAAACTGACCGGCCAGTATCTGC 
                   
                 
               
               
                   
               
               
                 
                   
                     TGGAGGGTTATGGTCTGACCGAGTGTGCACCGCTGGTCAGCGTTAACCCGTATGATAT 
                   
                 
               
               
                   
               
               
                 
                   
                     TGATTACCACTCTGGTTCTATTGGTCTGCCGGTTCCGTCCACGGAAGCCAAACTGGTG 
                   
                 
               
               
                   
               
               
                 
                   
                     GACGATGACGACAACGAAGTACCTCCGGGCCAGCCGGGTGAGCTGTGTGTCAAGGGT 
                   
                 
               
               
                   
               
               
                 
                   
                     CCGCAGGTTATGCTGGGCTACTGGCAGCGCCCGGACGCCACCGACGAAATCATTAAAA 
                   
                 
               
               
                   
               
               
                 
                   
                     ACGGTTGGCTGCATACCGGTGATATCGCTGTAATGGACGAAGAAGGTTTCCTGCGTAT 
                   
                 
               
               
                   
               
               
                 
                   
                     CGTGGACCGTAAGAAAGATATGATTCTGGTGAGCGGTTTCAACGTGTACCCGAACGAA 
                   
                 
               
               
                   
               
               
                 
                   
                     ATTGAGGACGTAGTTATGCAACACCCTGGCGTGCAGGAGGTGGCAGCCGTGGGCGTG 
                   
                 
               
               
                   
               
               
                 
                   
                     CCGTCCGGTTCTTCTGGTGAGGCTGTGAAAATCTTTGTCGTTAAAAAGGACCCGTCCCT 
                   
                 
               
               
                   
               
               
                 
                   
                     GACCGAAGAATCTCTGGTGACGTTTTGCCGCCGTCAACTGACTGGCTACAAAGTGCCG 
                   
                 
               
               
                   
               
               
                 
                   
                     AAACTGGTCGAGTTCCGCGATGAGCTGCCAAAATCTAACGTGGGTAAGATCCTGCGCC 
                   
                 
               
               
                   
               
               
                     GCGAGCTGCGTGACGAGGCACGTGGCAAAGTTGACAATAAAGCATAA   CCGCGTAGGAG 
               
               
                   
               
               
                 GACAGCT   ATGCGCCCACTTCATCCGATCGATTTCATTTTCCTGTCCCTGGAGAAACGCC     
               
               
                   
               
               
                 
                   
                     AGCAGCCGATGCACGTAGGTGGTCTGTTCCTGTTCCAGATCCCGGATAACGCTCCGGA 
                   
                 
               
               
                   
               
               
                 
                   
                     CACCTTTATTCAGGACCTGGTGAACGATATCCGTATCTCCAAGTCTATTCCGGTTCCGC 
                   
                 
               
               
                   
               
               
                 
                   
                     CGTTCAACAACAAGCTGAACGGTCTGTTCTGGGACGAAGACGAGGAGTTCGATCTGGA 
                   
                 
               
               
                   
               
               
                 
                   
                     TCACCATTTCCGTCATATTGCGCTGCCGCACCCGGGTCGCATCCGTGAGCTGCTGATTT 
                   
                 
               
               
                   
               
               
                 
                   
                     ACATCTCTCAGGAACACAGCACTCTCCTCGATCGCGCTAAACCTCTGTGGACTTGCAAC 
                   
                 
               
               
                   
               
               
                 
                   
                     ATCATTGAAGGTATCGAGGGTAACCGTTTCGCCATGTACTTCAAGATTCATCATGCGAT 
                   
                 
               
               
                   
               
               
                 
                   
                     GGTGGATGGTGTGGCGGGTATGCGTCTGATTGAGAAAAGCCTGTCCCATGATGTTACT 
                   
                 
               
               
                   
               
               
                 
                   
                     GAAAAGAGCATCGTACCGCCGTGGTGCGTTGAGGGCAAACGTGCTAAACGCCTGCGTG 
                   
                 
               
               
                   
               
               
                 
                   
                     AACCGAAGACCGGCAAAATTAAGAAAATCATGTCTGGTATTAAATCTCAGCTCCAGGC 
                   
                 
               
               
                   
               
               
                 
                   
                     CACCCCGACCGTTATTCAAGAACTGTCTCAGACGGTCTTCAAAGACATCGGCCGTAATC 
                   
                 
               
               
                   
               
               
                 
                   
                     CGGACCACGTTTCCTCTTTCCAGGCGCCGTGCTCCATCCTCAACCAGCGTGTGTCTTCT 
                   
                 
               
               
                   
               
               
                 
                   
                     TCTCGTCGTTTCGCAGCACAGAGCTTTGACCTGGACCGTTTCCGCAACATCGCCAAATC 
                   
                 
               
               
                   
               
               
                 
                   
                     TCTGAACGTGACCATTAACGACGTTGTCCTGGCTGTGTGTAGCGGTGCTCTGCGCGCT 
                   
                 
               
               
                   
               
               
                 
                   
                     TATCTGATGTCTCATAACTCTCTGCCATCCAAACCGCTGATCGCTATGGTCCCAGCAAG 
                   
                 
               
               
                   
               
               
                 
                   
                     CATCCGCAACGATGATTCTGATGTGTCCAACCGTATTACTATGATTCTGGCCAACCTCG 
                   
                 
               
               
                   
               
               
                 
                   
                     CTACTCACAAAGACGACCCTCTGCAGCGTCTGGAAATCATCCGCCGCTCCGTCCAGAA 
                   
                 
               
               
                   
               
               
                 
                   
                     CTCTAAACAGCGTTTTAAACGCATGACTTCCGACCAGATTCTGAACTATTCTGCGGTTG 
                   
                 
               
               
                   
               
               
                 
                   
                     TATACGGCCCGGCTGGTCTGAACATTATCAGCGGTATGATGCCGAAACGTCAGGCTTT 
                   
                 
               
               
                   
               
               
                 
                   
                     TAACCTGGTAATCAGCAACGTTCCTGGCCCGCGTGAGCCGCTGTACTGGAACGGCGCA 
                   
                 
               
               
                   
               
               
                 
                   
                     AAACTGGACGCACTGTACCCGGCTTCCATCGTTCTGGATGGCCAGGCTCTGAACATCA 
                   
                 
               
               
                   
               
               
                 
                   
                     CTATGACCTCTTACCTGGACAAACTGGAAGTAGGTCTGATCGCGTGTCGCAATGCACT 
                   
                 
               
               
                   
               
               
                 
                   
                     GCCGCGCATGCAGAACCTGCTGACCCACCTGGAGGAGGAAATCCAGCTGTTTGAGGGC 
                   
                 
               
               
                   
               
               
                     GTTATCGCCAAACAGGAAGATATCAAAACGGCGAACTAA   CCATGGTTGAATTCGGTTTTC 
               
               
                   
               
               
                 CGTCCTGTCTTGATTTTCAAGCAAACAATGCCTCCGATTTCTAATCGGAGGCATTTGTTTTTG 
               
               
                   
               
               
                 TTTATTGCAAAAACAAAAAATATTGTTACAAATTTTTACAGGCTATTAAGCCTACCGTCATA 
               
               
                   
               
               
                 AATAATTTGCCATTTACTAGTTTTTAATTAA   CCAGAACCTTGACCGAACGCAGCGGTGGTAACG     
               
               
                   
               
               
                 
                   
                     GCGCAGTGGCGGTTTTCATGGCTTGTTATGACTGTTTTTTTGGGGTACAGTCTATGCCTCGGGCAT 
                   
                 
               
               
                   
               
               
                 
                   
                     CCAAGCAGCAAGCGCGTTACGCCGTGGGTCGATGTTTGATGTTATGGAGCAGCAACGATGTTACG 
                   
                 
               
               
                   
               
               
                 
                   
                     CAGCAGGGCAGTCGCCCTAAAACAAAGTTAAACATCATGAGGGAAGCGGTGATCGCCGAAGTATC 
                   
                 
               
               
                   
               
               
                 
                   
                     GACTCAACTATCAGAGGTAGTTGGCGTCATCGAGCGCCATCTCGAACCGACGTTGCTGGCCGTAC 
                   
                 
               
               
                   
               
               
                 
                   
                     ATTTGTACGGCTCCGCAGTGGATGGCGGCCTGAAGCCACACAGTGATATTGATTTGCTGGTTACGG 
                   
                 
               
               
                   
               
               
                 
                   
                     TGACCGTAAGGCTTGATGAAACAACGCGGCGAGCTTTGATCAACGACCTTTTGGAAACTTCGGCTT 
                   
                 
               
               
                   
               
               
                 
                   
                     CCCCTGGAGAGAGCGAGATTCTCCGCGCTGTAGAAGTCACCATTGTTGTGCACGACGACATCATTC 
                   
                 
               
               
                   
               
               
                 
                   
                     CGTGGCGTTATCCAGCTAAGCGCGAACTGCAATTTGGAGAATGGCAGCGCAATGACATTCTTGCAG 
                   
                 
               
               
                   
               
               
                 
                   
                     GTATCTTCGAGCCAGCCACGATCGACATTGATCTGGCTATCTTGCTGACAAAAGCAAGAGAACATA 
                   
                 
               
               
                   
               
               
                 
                   
                     GCGTTGCCTTGGTAGGTCCAGCGGCGGAGGAACTCTTTGATCCGGTTCCTGAACAGGATCTATTTG 
                   
                 
               
               
                   
               
               
                 
                   
                     AGGCGCTAAATGAAACCTTAACGCTATGGAACTCGCCGCCCGACTGGGCTGGCGATGAGCGAAAT 
                   
                 
               
               
                   
               
               
                 
                   
                     GTAGTGCTTACGTTGTCCCGCATTTGGTACAGCGCAGTAACCGGCAAAATCGCGCCGAAGGATGT 
                   
                 
               
               
                   
               
               
                 
                   
                     CGCTGCCGACTGGGCAATGGAGCGCCTGCCGGCCCAGTATCAGCCCGTCATACTTGAAGCTAGAC 
                   
                 
               
               
                   
               
               
                 
                   
                     AGGCTTATCTTGGACAAGAAGAAGATCGCTTGGCCTCGCGCGCAGATCAGTTGGAAGAATTTGTCC 
                   
                 
               
               
                   
               
               
                 
                   
                     ACTACGTGAAAGGCGAGATCACCAAGGTAGTCGGCAAATAATGTCTAACAATTCGTTCAAGCCGAC 
                   
                 
               
               
                   
               
               
                 
                   
                     GCCGCTTCGCGGCGCGGCTTAACTCAAGCGTTAGATGCACTAAGCACATAATTGCTCACAGCCAAA 
                   
                 
               
               
                   
               
               
                 
                   
                     CTATCAGGTCAAGTCTGCTTTTATTATTTTTAAGCGTGCATAATAAGCCCTACACAAATTGGGAGATA 
                   
                 
               
               
                   
               
               
                     TATCATGA   GGCGCGCCacgagtgcggggaaatttcgggggcgatcgcccctatatcgcaaaaaggagttaccccatcagagctatagtcg 
               
               
                   
               
               
                 agaagaaaaccatcattcactcaacaaggctatgtcagaagagaaactagaccggatcgaagcagccctagagcaattggataaggatgtgcaaac 
               
               
                   
               
               
                 gctccaaacagagcttcagcaatcccaaaaatggcaggacaggacatgggatgttgtgaagtgggtaggcggaatctcagcgggcctagcggtgag 
               
               
                   
               
               
                 cgcttccattgccctgttcgggttggtctttagattttctgtttccctgccataaaagcacattcttataagtcatacttgtttacatcaaggaac 
               
               
                   
               
               
                 aaaaacggcattgtgccttgcaaggcacaatgtctttctcttatgcacagatggggactggaaaccacacgcacaattcccttaaaaagcaaccgc 
               
               
                   
               
               
                 aaaaaataaccatcaaaataaaactggacaaattctcatgtgGGCCGGCC 
               
               
                   
               
               
                 SEQ ID NO: 12 
                   
               
               
                 Ptrc promoter and lacIq repressor 
                   
               
               
                 TCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACG 
               
               
                   
               
               
                 CGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTCACCAGTGAGACG 
               
               
                   
               
               
                 GGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTCCACGCT 
               
               
                   
               
               
                 GGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTGACGGCGGGATATAACATGAGC 
               
               
                   
               
               
                 TGTCTTCGGTATCGTCGTATCCCACTACCGAGATATCCGCACCAACGCGCAGCCCGGACTCG 
               
               
                   
               
               
                 GTAATGGCGCGCATTGCGCCCAGCGCCATCTGATCGTTGGCAACCAGCATCGCAGTGGGAAC 
               
               
                   
               
               
                 GATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAACCGGACATGGCACTCCAGTCGCCTTC 
               
               
                   
               
               
                 CCGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATGCCAGCCAGCCAGACGCA 
               
               
                   
               
               
                 GACGCGCCGAGACAGAACTTAATGGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGC 
               
               
                   
               
               
                 GACCAGATGCTCCACGCCCAGTCGCGTACCGTCTTCATGGGAGAAAATAATACTGTTGATGG 
               
               
                   
               
               
                 GTGTCTGGTCAGAGACATCAAGAAATAACGCCGGAACATTAGTGCAGGCAGCTTCCACAGC 
               
               
                   
               
               
                 AATGGCATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCACTGACGCGCTGCGCGAGAA 
               
               
                   
               
               
                 GATTGTGCACCGCCGCTTTACAGGCTTCGACGCCGCTTCGTTCTACCATCGACACCACCACG 
               
               
                   
               
               
                 CTGGCACCCAGTTGATCGGCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCGCGTGCAG 
               
               
                   
               
               
                 GGCCAGACTGGAGGTGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCCA 
               
               
                   
               
               
                 CGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAG 
               
               
                   
               
               
                 AAACGTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCGGCATACTC 
               
               
                   
               
               
                 TGCGACATCGTATAACGTTACTGGTTTCATATTCACCACCCTGAATTGACTCTCTTCCGGGCG 
               
               
                   
               
               
                 CTATCATGCCATACCGCGAAAGGTTTTGCACCATTCGATGGTGTCAACGTAAATGCATGCCG 
               
               
                   
               
               
                 CTTCGCCTTCCAATTGGACTGCACGGTGCACCAATGCTTCTGGCGTCAGGCAGCCATCGGAA 
               
               
                   
               
               
                 GCTGTGGTATGGCTGTGCAGGTCGTAAATCACTGCATAATTCGTGTCGCTCAAGGCGCACTC 
               
               
                   
               
               
                 CCGTTCTGGATAATGTTTTTTGCGCCGACATCATAACGGTTCTGGCAAATATTCTGAAATGAG 
               
               
                   
               
               
                 CTGTTGACAATTAATCATCCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCA 
               
               
                   
               
               
                 CACAGGAAACAGCAT 
               
               
                   
               
               
                 SEQ ID NO: 13 
                   
               
               
                 (UHR and DHR in lowercase; P aphII  underlined; fadD and wsadpl are in bold and underlined and 
                   
               
               
                 follow the promoter in order;  aadA  marker is italicized and underlined) 
               
               
                 CCTGCAGGgtcagcaagctctggaatttcccgattctctgatgggagatccaaaaattctcgcagtccctcaatcacgatatcggtcttggatcgc 
               
               
                   
               
               
                 cctgtagcttccgacaactgctcaattttttcgagcatctctaccgggcatcggaatgaaattaacggtgttttagccatgtgttatacagtgttt 
               
               
                   
               
               
                 acaacttgactaacaaatacctgctagtgtatacatattgtattgcaatgtatacgctattttcactgctgtctttaatggggattatcgcaagca 
               
               
                   
               
               
                 agtaaaaaagcctgaaaaccccaataggtaagggattccgagcttactcgataattatcacctttgagcgcccctaggaggaggcgaaaagctatg 
               
               
                   
               
               
                 tctgacaaggggtttgacccctgaagtcgttgcgcgagcattaaggtctgcggatagcccataacatacttttgttgaacttgtgcgcttttatca 
               
               
                   
               
               
                 accccttaagggcttgggagcgttttatGCGGCCGC GGGGGGGGGGGGGAAAGCCACGTTGTGTCTCAAAATCTCTGATGTTACATTGCACA   
               
               
                   
               
               
                 
                   AGATAAAAATATATCATCATGAACAATAAAACTGTCTGCTTACATAAACAGTAATACAAGG 
                 
               
               
                   
               
               
                   GGT CATAGATCTGTAGTAGGATCCATGTAGGGTGAGGTTATAGCT   ATGAAGAAAGTTTGGC     
               
               
                   
               
               
                 
                   
                     TGAACCGTTATCCGGCAGATGTACCGACTGAAATTAACCCAGATCGTTACCAGTCCCTG 
                   
                 
               
               
                   
               
               
                 
                   
                     GTTGACATGTTCGAACAGTCCGTGGCTCGCTACGCCGATCAGCCTGCTTTCGTCAACAT 
                   
                 
               
               
                   
               
               
                 
                   
                     GGGTGAGGTAATGACCTTTCGCAAACTGGAGGAGCGTTCCCGTGCTTTCGCGGCATAC 
                   
                 
               
               
                   
               
               
                 
                   
                     CTGCAGCAGGGTCTGGGCCTGAAGAAAGGCGACCGCGTGGCCCTGATGATGCCGAAC 
                   
                 
               
               
                   
               
               
                 
                   
                     CTGCTGCAATATCCTGTGGCGCTGTTCGGTATCCTGCGTGCTGGTATGATCGTTGTCAA 
                   
                 
               
               
                   
               
               
                 
                   
                     TGTTAACCCTCTGTATACCCCTCGTGAACTGGAGCACCAGCTGAATGACTCTGGTGCGT 
                   
                 
               
               
                   
               
               
                 
                   
                     CTGCTATCGTTATCGTTTCCAATTTCGCACATACGCTGGAGAAAGTGGTTGATAAAACC 
                   
                 
               
               
                   
               
               
                 
                   
                     GCAGTGCAGCATGTCATTCTGACTCGCATGGGTGACCAGCTGTCCACCGCTAAAGGTA 
                   
                 
               
               
                   
               
               
                 
                   
                     CTGTAGTCAACTTCGTTGTGAAATACATTAAGCGCCTGGTTCCGAAATACCACCTGCCA 
                   
                 
               
               
                   
               
               
                 
                   
                     GATGCAATTAGCTTTCGCTCTGCACTGCATAACGGTTACCGTATGCAGTACGTAAAACC 
                   
                 
               
               
                   
               
               
                 
                   
                     AGAGCTGGTGCCGGAAGACCTGGCCTTTCTGCAGTATACCGGCGGCACCACCGGCGTG 
                   
                 
               
               
                   
               
               
                 
                   
                     GCAAAGGGCGCGATGCTGACCCATCGTAACATGCTGGCGAACCTGGAGCAGGTTAACG 
                   
                 
               
               
                   
               
               
                 
                   
                     CAACGTACGGCCCGCTGCTGCACCCGGGTAAAGAACTGGTAGTTACGGCACTGCCTCT 
                   
                 
               
               
                   
               
               
                 
                   
                     GTATCACATCTTTGCACTGACGATCAACTGTCTGCTGTTCATTGAACTGGGTGGTCAGA 
                   
                 
               
               
                   
               
               
                 
                   
                     ACCTGCTGATCACCAACCCGCGTGACATTCCGGGCCTGGTAAAAGAGCTGGCTAAGTA 
                   
                 
               
               
                   
               
               
                 
                   
                     CCCGTTCACCGCCATTACTGGCGTAAACACTCTGTTTAACGCGCTGCTGAACAACAAAG 
                   
                 
               
               
                   
               
               
                 
                   
                     AGTTTCAGCAGCTGGACTTCTCTAGCCTGCACCTGAGCGCTGGCGGTGGCATGCCGGT 
                   
                 
               
               
                   
               
               
                 
                   
                     TCAGCAGGTTGTGGCAGAGCGTTGGGTGAAACTGACCGGCCAGTATCTGCTGGAGGGT 
                   
                 
               
               
                   
               
               
                 
                   
                     TATGGTCTGACCGAGTGTGCACCGCTGGTCAGCGTTAACCCGTATGATATTGATTACCA 
                   
                 
               
               
                   
               
               
                 
                   
                     CTCTGGTTCTATTGGTCTGCCGGTTCCGTCCACGGAAGCCAAACTGGTGGACGATGAC 
                   
                 
               
               
                   
               
               
                 
                   
                     GACAACGAAGTACCTCCGGGCCAGCCGGGTGAGCTGTGTGTCAAGGGTCCGCAGGTTA 
                   
                 
               
               
                   
               
               
                 
                   
                     TGCTGGGCTACTGGCAGCGCCCGGACGCCACCGACGAAATCATTAAAAACGGTTGGCT 
                   
                 
               
               
                   
               
               
                 
                   
                     GCATACCGGTGATATCGCTGTAATGGACGAAGAAGGTTTCCTGCGTATCGTGGACCGT 
                   
                 
               
               
                   
               
               
                 
                   
                     AAGAAAGATATGATTCTGGTGAGCGGTTTCAACGTGTACCCGAACGAAATTGAGGACG 
                   
                 
               
               
                   
               
               
                 
                   
                     TAGTTATGCAACACCCTGGCGTGCAGGAGGTGGCAGCCGTGGGCGTGCCGTCCGGTTC 
                   
                 
               
               
                   
               
               
                 
                   
                     TTCTGGTGAGGCTGTGAAAATCTTTGTCGTTAAAAAGGACCCGTCCCTGACCGAAGAA 
                   
                 
               
               
                   
               
               
                 
                   
                     TCTCTGGTGACGTTTTGCCGCCGTCAACTGACTGGCTACAAAGTGCCGAAACTGGTCG 
                   
                 
               
               
                   
               
               
                 
                   
                     AGTTCCGCGATGAGCTGCCAAAATCTAACGTGGGTAAGATCCTGCGCCGCGAGCTGCG 
                   
                 
               
               
                   
               
               
                     TGACGAGGCACGTGGCAAAGTTGACAATAAAGCATAA   CTCGACGCGTAGGAGGACAGCT 
               
               
                   
               
               
                 
                   
                     ATGCGCCCACTTCATCCGATCGATTTCATTTTCCTGTCCCTGGAGAAACGCCAGCAGCC 
                   
                 
               
               
                   
               
               
                 
                   
                     GATGCACGTAGGTGGTCTGTTCCTGTTCCAGATCCCGGATAACGCTCCGGACACCTTT 
                   
                 
               
               
                   
               
               
                 
                   
                     ATTCAGGACCTGGTGAACGATATCCGTATCTCCAAGTCTATTCCGGTTCCGCCGTTCAA 
                   
                 
               
               
                   
               
               
                 
                   
                     CAACAAGCTGAACGGTCTGTTCTGGGACGAAGACGAGGAGTTCGATCTGGATCACCAT 
                   
                 
               
               
                   
               
               
                 
                   
                     TTCCGTCATATTGCGCTGCCGCACCCGGGTCGCATCCGTGAGCTGCTGATTTACATCTC 
                   
                 
               
               
                   
               
               
                 
                   
                     TCAGGAACACAGCACTCTCCTCGATCGCGCTAAACCTCTGTGGACTTGCAACATCATTG 
                   
                 
               
               
                   
               
               
                 
                   
                     AAGGTATCGAGGGTAACCGTTTCGCCATGTACTTCAAGATTCATCATGCGATGGTGGA 
                   
                 
               
               
                   
               
               
                 
                   
                     TGGTGTGGCGGGTATGCGTCTGATTGAGAAAAGCCTGTCCCATGATGTTACTGAAAAG 
                   
                 
               
               
                   
               
               
                 
                   
                     AGCATCGTACCGCCGTGGTGCGTTGAGGGCAAACGTGCTAAACGCCTGCGTGAACCGA 
                   
                 
               
               
                   
               
               
                 
                   
                     AGACCGGCAAAATTAAGAAAATCATGTCTGGTATTAAATCTCAGCTCCAGGCCACCCC 
                   
                 
               
               
                   
               
               
                 
                   
                     GACCGTTATTCAAGAACTGTCTCAGACGGTCTTCAAAGACATCGGCCGTAATCCGGAC 
                   
                 
               
               
                   
               
               
                 
                   
                     CACGTTTCCTCTTTCCAGGCGCCGTGCTCCATCCTCAACCAGCGTGTGTCTTCTTCTCG 
                   
                 
               
               
                   
               
               
                 
                   
                     TCGTTTCGCAGCACAGAGCTTTGACCTGGACCGTTTCCGCAACATCGCCAAATCTCTGA 
                   
                 
               
               
                   
               
               
                 
                   
                     ACGTGACCATTAACGACGTTGTCCTGGCTGTGTGTAGCGGTGCTCTGCGCGCTTATCT 
                   
                 
               
               
                   
               
               
                 
                   
                     GATGTCTCATAACTCTCTGCCATCCAAACCGCTGATCGCTATGGTCCCAGCAAGCATCC 
                   
                 
               
               
                   
               
               
                 
                   
                     GCAACGATGATTCTGATGTGTCCAACCGTATTACTATGATTCTGGCCAACCTCGCTACT 
                   
                 
               
               
                   
               
               
                 
                   
                     CACAAAGACGACCCTCTGCAGCGTCTGGAAATCATCCGCCGCTCCGTCCAGAACTCTA 
                   
                 
               
               
                   
               
               
                 
                   
                     AACAGCGTTTTAAACGCATGACTTCCGACCAGATTCTGAACTATTCTGCGGTTGTATAC 
                   
                 
               
               
                   
               
               
                 
                   
                     GGCCCGGCTGGTCTGAACATTATCAGCGGTATGATGCCGAAACGTCAGGCTTTTAACC 
                   
                 
               
               
                   
               
               
                 
                   
                     TGGTAATCAGCAACGTTCCTGGCCCGCGTGAGCCGCTGTACTGGAACGGCGCAAAACT 
                   
                 
               
               
                   
               
               
                 
                   
                     GGACGCACTGTACCCGGCTTCCATCGTTCTGGATGGCCAGGCTCTGAACATCACTATG 
                   
                 
               
               
                   
               
               
                 
                   
                     ACCTCTTACCTGGACAAACTGGAAGTAGGTCTGATCGCGTGTCGCAATGCACTGCCGC 
                   
                 
               
               
                   
               
               
                 
                   
                     GCATGCAGAACCTGCTGACCCACCTGGAGGAGGAAATCCAGCTGTTTGAGGGCGTTAT 
                   
                 
               
               
                   
               
               
                     CGCCAAACAGGAAGATATCAAAACGGCGAACTAA   CCATGGTTGAATTCGGTTTTCCGTCC 
               
               
                   
               
               
                 TGTCTTGATTTTCAAGCAAACAATGCCTCCGATTTCTAATCGGAGGCATTTGTTTTTGTTTATT 
               
               
                   
               
               
                 GCAAAAACAAAAAATATTGTTACAAATTTTTACAGGCTATTAAGCCTACCGTCATAAATAAT 
               
               
                   
               
               
                 TTGCCATTTACTAGTTTTTAATTAA   CCAGAACCTTGACCGAACGCAGCGGTGGTAACGGCGCAG     
               
               
                   
               
               
                 
                   
                     TGGCGGTTTTCATGGCTTGTTATGACTGTTTTTTTGGGGTACAGTCTATGCCTCGGGCATCCAAGCA 
                   
                 
               
               
                   
               
               
                 
                   
                     GCAAGCGCGTTACGCCGTGGGTCGATGTTTGATGTTATGGAGCAGCAACGATGTTACGCAGCAGG 
                   
                 
               
               
                   
               
               
                 
                   
                     GCAGTCGCCCTAAAACAAAGTTAAACATCATGAGGGAAGCGGTGATCGCCGAAGTATCGACTCAAC 
                   
                 
               
               
                   
               
               
                 
                   
                     TATCAGAGGTAGTTGGCGTCATCGAGCGCCATCTCGAACCGACGTTGCTGGCCGTACATTTGTACG 
                   
                 
               
               
                   
               
               
                 
                   
                     GCTCCGCAGTGGATGGCGGCCTGAAGCCACACAGTGATATTGATTTGCTGGTTACGGTGACCGTA 
                   
                 
               
               
                   
               
               
                 
                   
                     AGGCTTGATGAAACAACGCGGCGAGCTTTGATCAACGACCTTTTGGAAACTTCGGCTTCCCCTGGA 
                   
                 
               
               
                   
               
               
                 
                   
                     GAGAGCGAGATTCTCCGCGCTGTAGAAGTCACCATTGTTGTGCACGACGACATCATTCCGTGGCGT 
                   
                 
               
               
                   
               
               
                 
                   
                     TATCCAGCTAAGCGCGAACTGCAATTTGGAGAATGGCAGCGCAATGACATTCTTGCAGGTATCTTC 
                   
                 
               
               
                   
               
               
                 
                   
                     GAGCCAGCCACGATCGACATTGATCTGGCTATCTTGCTGACAAAAGCAAGAGAACATAGCGTTGCC 
                   
                 
               
               
                   
               
               
                 
                   
                     TTGGTAGGTCCAGCGGCGGAGGAACTCTTTGATCCGGTTCCTGAACAGGATCTATTTGAGGCGCTA 
                   
                 
               
               
                   
               
               
                 
                   
                     AATGAAACCTTAACGCTATGGAACTCGCCGCCCGACTGGGCTGGCGATGAGCGAAATGTAGTGCT 
                   
                 
               
               
                   
               
               
                 
                   
                     TACGTTGTCCCGCATTTGGTACAGCGCAGTAACCGGCAAAATCGCGCCGAAGGATGTCGCTGCCG 
                   
                 
               
               
                   
               
               
                 
                   
                     ACTGGGCAATGGAGCGCCTGCCGGCCCAGTATCAGCCCGTCATACTTGAAGCTAGACAGGCTTAT 
                   
                 
               
               
                   
               
               
                 
                   
                     CTTGGACAAGAAGAAGATCGCTTGGCCTCGCGCGCAGATCAGTTGGAAGAATTTGTCCACTACGTG 
                   
                 
               
               
                   
               
               
                 
                   
                     AAAGGCGAGATCACCAAGGTAGTCGGCAAATAATGTCTAACAATTCGTTCAAGCCGACGCCGCTTC 
                   
                 
               
               
                   
               
               
                 
                   
                     GCGGCGCGGCTTAACTCAAGCGTTAGATGCACTAAGCACATAATTGCTCACAGCCAAACTATCAGG 
                   
                 
               
               
                   
               
               
                 
                   
                     TCAAGTCTGCTTTTATTATTTTTAAGCGTGCATAATAAGCCCTACACAAATTGGGAGATATATCATGA 
                   
                 
               
               
                   
               
               
                 GGCGCGCCacgagtgcggggaaatttcgggggcgatcgcccctatatcgcaaaaaggagttaccccatcagagctatagtcgagaagaaaacc 
               
               
                   
               
               
                 atcattcactcaacaaggctatgtcagaagagaaactagaccggatcgaagcagccctagagcaattggataaggatgtgcaaacgctccaaacag 
               
               
                   
               
               
                 agcttcagcaatcccaaaaatggcaggacaggacatgggatgttgtgaagtgggtaggcggaatctcagcgggcctagcggtgagcgcttccattg 
               
               
                   
               
               
                 ccctgttcgggttggtctttagattttctgtttccctgccataaaagcacattcttataagtcatacttgtttacatcaaggaacaaaaacggcat 
               
               
                   
               
               
                 tgtgccttgcaaggcacaatgtctttctcttatgcacagatggggactggaaaccacacgcacaattcccttaaaaagcaaccgcaaaaaataacc 
               
               
                   
               
               
                 atcaaaataaaactggacaaattctcatgtgGGCCGGCC 
               
               
                   
               
               
                 SEQ ID NO: 14 
                   
               
               
                 (UHR and DHR in lowercase; P aphII  underlined; tesA and fadD are in bold and underlined  
                   
               
               
                 and follow the promoter in order; aadA marker is italicized and underlined) 
               
               
                 CCTGCAGGGtcagcaagctctggaatttcccgattctctgatgggagatccaaaaattctcgcagtccctcaatcacgatatcggtcttggatcgc 
               
               
                   
               
               
                 cctgtagcttccgacaactgctcaattttttcgagcatctctaccgggcatcggaatgaaattaacggtgttttagccatgtgttatacagtgttt 
               
               
                   
               
               
                 acaacttgactaacaaatacctgctagtgtatacatattgtattgcaatgtatacgctattttcactgctgtctttaatggggattatcgcaagca 
               
               
                   
               
               
                 agtaaaaaagcctgaaaaccccaataggtaagggattccgagcttactcgataattatcacctttgagcgcccctaggaggaggcgaaaagctatg 
               
               
                   
               
               
                 tctgacaaggggtttgacccctgaagtcgttgcgcgagcattaaggtctgcggatagcccataacatacttttgttgaacttgtgcgcttttatca 
               
               
                   
               
               
                 accccttaagggcttgggagcgttttatGCGGCCGC GGGGGGGGGGGGGAAAGCCACGTTGTGTCTCAAAATCTCTGATGTTACATTGCACA   
               
               
                   
               
               
                 
                   AGATAAAAATATATCATCATGAACAATAAAACTGTCTGCTTACATAAACAGTAATACAAGG 
                 
               
               
                   
               
               
                   GGT CAT   ATGGCGGATACTCTGCTGATTCTGGGTGATTCTCTGTCTGCAGGCTACCGTAT     
               
               
                   
               
               
                 
                   
                     GTCCGCCTCCGCGGCCTGGCCAGCTCTGCTGAATGATAAGTGGCAGTCTAAGACGTCC 
                   
                 
               
               
                   
               
               
                 
                   
                     GTTGTGAACGCATCCATCTCTGGCGACACGAGCCAGCAGGGCCTGGCCCGTCTGCCTG 
                   
                 
               
               
                   
               
               
                 
                   
                     CACTGCTGAAACAGCACCAACCGCGCTGGGTCCTGGTGGAGCTGGGCGGTAACGACG 
                   
                 
               
               
                   
               
               
                 
                   
                     GTCTGCGCGGCTTCCAGCCGCAGCAGACCGAACAGACTCTGCGTCAGATTCTGCAGGA 
                   
                 
               
               
                   
               
               
                 
                   
                     CGTGAAAGCTGCTAACGCGGAACCGCTGCTGATGCAGATTCGTCTGCCAGCGAACTAT 
                   
                 
               
               
                   
               
               
                 
                   
                     GGCCGCCGTTACAACGAAGCGTTCTCTGCAATCTACCCAAAACTGGCGAAAGAGTTTG 
                   
                 
               
               
                   
               
               
                 
                   
                     ACGTCCCGCTGCTGCCGTTCTTCATGGAGGAAGTATACCTGAAACCGCAGTGGATGCA 
                   
                 
               
               
                   
               
               
                 
                   
                     AGATGACGGCATCCACCCGAACCGTGATGCGCAGCCGTTCATCGCTGACTGGATGGCG 
                   
                 
               
               
                   
               
               
                     AAGCAACTGCAGCCGCTGGTAAACCACGATTCCTAA   TTAAAGATCTGTAGTAGGATCCAT 
               
               
                   
               
               
                 GTAGGGTGAGGTTATAGCT   ATGAAGAAAGTTTGGCTGAACCGTTATCCGGCAGATGTAC     
               
               
                   
               
               
                 
                   
                     CGACTGAAATTAACCCAGATCGTTACCAGTCCCTGGTTGACATGTTCGAACAGTCCGTG 
                   
                 
               
               
                   
               
               
                 
                   
                     GCTCGCTACGCCGATCAGCCTGCTTTCGTCAACATGGGTGAGGTAATGACCTTTCGCA 
                   
                 
               
               
                   
               
               
                 
                   
                     AACTGGAGGAGCGTTCCCGTGCTTTCGCGGCATACCTGCAGCAGGGTCTGGGCCTGAA 
                   
                 
               
               
                   
               
               
                 
                   
                     GAAAGGCGACCGCGTGGCCCTGATGATGCCGAACCTGCTGCAATATCCTGTGGCGCTG 
                   
                 
               
               
                   
               
               
                 
                   
                     TTCGGTATCCTGCGTGCTGGTATGATCGTTGTCAATGTTAACCCTCTGTATACCCCTCG 
                   
                 
               
               
                   
               
               
                 
                   
                     TGAACTGGAGCACCAGCTGAATGACTCTGGTGCGTCTGCTATCGTTATCGTTTCCAATT 
                   
                 
               
               
                   
               
               
                 
                   
                     TCGCACATACGCTGGAGAAAGTGGTTGATAAAACCGCAGTGCAGCATGTCATTCTGAC 
                   
                 
               
               
                   
               
               
                 
                   
                     TCGCATGGGTGACCAGCTGTCCACCGCTAAAGGTACTGTAGTCAACTTCGTTGTGAAA 
                   
                 
               
               
                   
               
               
                 
                   
                     TACATTAAGCGCCTGGTTCCGAAATACCACCTGCCAGATGCAATTAGCTTTCGCTCTGC 
                   
                 
               
               
                   
               
               
                 
                   
                     ACTGCATAACGGTTACCGTATGCAGTACGTAAAACCAGAGCTGGTGCCGGAAGACCTG 
                   
                 
               
               
                   
               
               
                 
                   
                     GCCTTTCTGCAGTATACCGGCGGCACCACCGGCGTGGCAAAGGGCGCGATGCTGACCC 
                   
                 
               
               
                   
               
               
                 
                   
                     ATCGTAACATGCTGGCGAACCTGGAGCAGGTTAACGCAACGTACGGCCCGCTGCTGCA 
                   
                 
               
               
                   
               
               
                 
                   
                     CCCGGGTAAAGAACTGGTAGTTACGGCACTGCCTCTGTATCACATCTTTGCACTGACG 
                   
                 
               
               
                   
               
               
                 
                   
                     ATCAACTGTCTGCTGTTCATTGAACTGGGTGGTCAGAACCTGCTGATCACCAACCCGC 
                   
                 
               
               
                   
               
               
                 
                   
                     GTGACATTCCGGGCCTGGTAAAAGAGCTGGCTAAGTACCCGTTCACCGCCATTACTGG 
                   
                 
               
               
                   
               
               
                 
                   
                     CGTAAACACTCTGTTTAACGCGCTGCTGAACAACAAAGAGTTTCAGCAGCTGGACTTCT 
                   
                 
               
               
                   
               
               
                 
                   
                     CTAGCCTGCACCTGAGCGCTGGCGGTGGCATGCCGGTTCAGCAGGTTGTGGCAGAGC 
                   
                 
               
               
                   
               
               
                 
                   
                     GTTGGGTGAAACTGACCGGCCAGTATCTGCTGGAGGGTTATGGTCTGACCGAGTGTGC 
                   
                 
               
               
                   
               
               
                 
                   
                     ACCGCTGGTCAGCGTTAACCCGTATGATATTGATTACCACTCTGGTTCTATTGGTCTGC 
                   
                 
               
               
                   
               
               
                 
                   
                     CGGTTCCGTCCACGGAAGCCAAACTGGTGGACGATGACGACAACGAAGTACCTCCGGG 
                   
                 
               
               
                   
               
               
                 
                   
                     CCAGCCGGGTGAGCTGTGTGTCAAGGGTCCGCAGGTTATGCTGGGCTACTGGCAGCGC 
                   
                 
               
               
                   
               
               
                 
                   
                     CCGGACGCCACCGACGAAATCATTAAAAACGGTTGGCTGCATACCGGTGATATCGCTG 
                   
                 
               
               
                   
               
               
                 
                   
                     TAATGGACGAAGAAGGTTTCCTGCGTATCGTGGACCGTAAGAAAGATATGATTCTGGT 
                   
                 
               
               
                   
               
               
                 
                   
                     GAGCGGTTTCAACGTGTACCCGAACGAAATTGAGGACGTAGTTATGCAACACCCTGGC 
                   
                 
               
               
                   
               
               
                 
                   
                     GTGCAGGAGGTGGCAGCCGTGGGCGTGCCGTCCGGTTCTTCTGGTGAGGCTGTGAAA 
                   
                 
               
               
                   
               
               
                 
                   
                     ATCTTTGTCGTTAAAAAGGACCCGTCCCTGACCGAAGAATCTCTGGTGACGTTTTGCCG 
                   
                 
               
               
                   
               
               
                 
                   
                     CCGTCAACTGACTGGCTACAAAGTGCCGAAACTGGTCGAGTTCCGCGATGAGCTGCCA 
                   
                 
               
               
                   
               
               
                 
                   
                     AAATCTAACGTGGGTAAGATCCTGCGCCGCGAGCTGCGTGACGAGGCACGTGGCAAAG 
                   
                 
               
               
                   
               
               
                     TTGACAATAAAGCATAA   CAATTCGGTTTTCCGTCCTGTCTTGATTTTCAAGCAAACAATGCC 
               
               
                   
               
               
                 TCCGATTTCTAATCGGAGGCATTTGTTTTTGTTTATTGCAAAAACAAAAAATATTGTTACAAA 
               
               
                   
               
               
                 TTTTTACAGGCTATTAAGCCTACCGTCATAAATAATTTGCCATTTACTAGTTTTTAATTAA   CC     
               
               
                   
               
               
                 
                   
                     AGAACCTTGACCGAACGCAGCGGTGGTAACGGCGCAGTGGCGGTTTTCATGGCTTGTTATGACTG 
                   
                 
               
               
                   
               
               
                 
                   
                     TTTTTTTGGGGTACAGTCTATGCCTCGGGCATCCAAGCAGCAAGCGCGTTACGCCGTGGGTCGAT 
                   
                 
               
               
                   
               
               
                 
                   
                     GTTTGATGTTATGGAGCAGCAACGATGTTACGCAGCAGGGCAGTCGCCCTAAAACAAAGTTAAACA 
                   
                 
               
               
                   
               
               
                 
                   
                     TCATGAGGGAAGCGGTGATCGCCGAAGTATCGACTCAACTATCAGAGGTAGTTGGCGTCATCGAG 
                   
                 
               
               
                   
               
               
                 
                   
                     CGCCATCTCGAACCGACGTTGCTGGCCGTACATTTGTACGGCTCCGCAGTGGATGGCGGCCTGAA 
                   
                 
               
               
                   
               
               
                 
                   
                     GCCACACAGTGATATTGATTTGCTGGTTACGGTGACCGTAAGGCTTGATGAAACAACGCGGCGAGC 
                   
                 
               
               
                   
               
               
                 
                   
                     TTTGATCAACGACCTTTTGGAAACTTCGGCTTCCCCTGGAGAGAGCGAGATTCTCCGCGCTGTAGA 
                   
                 
               
               
                   
               
               
                 
                   
                     AGTCACCATTGTTGTGCACGACGACATCATTCCGTGGCGTTATCCAGCTAAGCGCGAACTGCAATT 
                   
                 
               
               
                   
               
               
                 
                   
                     TGGAGAATGGCAGCGCAATGACATTCTTGCAGGTATCTTCGAGCCAGCCACGATCGACATTGATCT 
                   
                 
               
               
                   
               
               
                 
                   
                     GGCTATCTTGCTGACAAAAGCAAGAGAACATAGCGTTGCCTTGGTAGGTCCAGCGGCGGAGGAAC 
                   
                 
               
               
                   
               
               
                 
                   
                     TCTTTGATCCGGTTCCTGAACAGGATCTATTTGAGGCGCTAAATGAAACCTTAACGCTATGGAACTC 
                   
                 
               
               
                   
               
               
                 
                   
                     GCCGCCCGACTGGGCTGGCGATGAGCGAAATGTAGTGCTTACGTTGTCCCGCATTTGGTACAGCG 
                   
                 
               
               
                   
               
               
                 
                   
                     CAGTAACCGGCAAAATCGCGCCGAAGGATGTCGCTGCCGACTGGGCAATGGAGCGCCTGCCGGC 
                   
                 
               
               
                   
               
               
                 
                   
                     CCAGTATCAGCCCGTCATACTTGAAGCTAGACAGGCTTATCTTGGACAAGAAGAAGATCGCTTGGC 
                   
                 
               
               
                   
               
               
                 
                   
                     CTCGCGCGCAGATCAGTTGGAAGAATTTGTCCACTACGTGAAAGGCGAGATCACCAAGGTAGTCG 
                   
                 
               
               
                   
               
               
                 
                   
                     GCAAATAATGTCTAACAATTCGTTCAAGCCGACGCCGCTTCGCGGCGCGGCTTAACTCAAGCGTTA 
                   
                 
               
               
                   
               
               
                 
                   
                     GATGCACTAAGCACATAATTGCTCACAGCCAAACTATCAGGTCAAGTCTGCTTTTATTATTTTTAAGC 
                   
                 
               
               
                   
               
               
                     GTGCATAATAAGCCCTACACAAATTGGGAGATATATCATGA   GGCGCGCCacgagtgcggggaaatttcgggggc 
               
               
                   
               
               
                 gatcgcccctatatcgcaaaaaggagttaccccatcagagctatagtcgagaagaaaaccatcattcactcaacaaggctatgtcagaagagaaac 
               
               
                   
               
               
                 tagaccggatcgaagcagccctagagcaattggataaggatgtgcaaacgctccaaacagagcttcagcaatcccaaaaatggcaggacaggacat 
               
               
                   
               
               
                 gggatgttgtgaagtgggtaggcggaatctcagcgggcctagcggtgagcgcttccattgccctgttcgggttggtctttagattttctgtttccc 
               
               
                   
               
               
                 tgccataaaagcacattcttataagtcatacttgtttacatcaaggaacaaaaacggcattgtgccttgcaaggcacaatgtctttctcttatgca 
               
               
                   
               
               
                 cagatggggactggaaaccacacgcacaattcccttaaaaagcaaccgcaaaaaataaccatcaaaataaaactggacaaattctcatgtgGGCCG 
               
               
                   
               
               
                 GCC 
               
               
                   
               
               
                 SEQ ID NO: 15 
                   
               
               
                 pJB161 
                   
               
               
                 (vector contains bla cassette, pUC ori and transcription terminators flanking the homology 
               
               
                 regions; UHR and DHR are lowercase; P aphII  promoter is underlined; adhII terminator is in 
               
               
                 bold; kan R  marker is italicized and underlined) 
               
               
                 ACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTG 
               
               
                   
               
               
                 CCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGCGCT 
               
               
                   
               
               
                 GCGATGATACCGCGAGAACCACGCTCACCGGCTCCGGATTTATCAGCAATAAACCAGCCAG 
               
               
                   
               
               
                 CCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAAT 
               
               
                   
               
               
                 TGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCAT 
               
               
                   
               
               
                 CGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCA 
               
               
                   
               
               
                 ACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTC 
               
               
                   
               
               
                 CTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTG 
               
               
                   
               
               
                 CATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACC 
               
               
                   
               
               
                 AAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGA 
               
               
                   
               
               
                 TAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGC 
               
               
                   
               
               
                 GAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCC 
               
               
                   
               
               
                 AACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCA 
               
               
                   
               
               
                 AAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATATTCTTCCTTT 
               
               
                   
               
               
                 TTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTA 
               
               
                   
               
               
                 TTTAGAAAAATAAACAAATAGGGGTCAGTGTTACAACCAATTAACCAATTCTGAACATTATC 
               
               
                   
               
               
                 GCGAGCCCATTTATACCTGAATATGGCTCATAACACCCCTTGTTTGCCTGGCGGCAGTAGCG 
               
               
                   
               
               
                 CGGTGGTCCCACCTGACCCCATGCCGAACTCAGAAGTGAAACGCCGTAGCGCCGATGGTAG 
               
               
                   
               
               
                 TGTGGGGACTCCCCATGCGAGAGTAGGGAACTGCCAGGCATCAAATAAAACGAAAGGCTCA 
               
               
                   
               
               
                 GTCGAAAGACTGGGCCTTTCGCCCGGGCTAATTATGGGGTGTCGCCCTTATTCGACTCTATA 
               
               
                   
               
               
                 GTGAAGTTCCTATTCTCTAGAAAGTATAGGAACTTCTGAAGTGGGGCCTGCAGGgccaccacagcc 
               
               
                   
               
               
                 aaattcatcgttaatgtggacttgccgacgcccccttttcgactaacaatcgcaatttttttcatagacatttcccacagaccacatcaaattaca 
               
               
                   
               
               
                 gcaattgatctagctgaaagtttaacccacttccccccagacccagaagaccagaggcgcttaagcttccccgaacaaactcaactgaccgagggg 
               
               
                   
               
               
                 gagggagccgtagcggcgttggtgttggcgtaaatgacaggccgagcaaagagcgatgagattttcccgacgattgtcttcggggatgtaattttt 
               
               
                   
               
               
                 taaaacagcccgcaggtgacgatcaatgcctttgaccttcacatccgacggaatacaaaccaagccacagagttcacagcgccagtctgcatcctctttta 
               
               
                   
               
               
                 gtggtggacgcttaaggtcttgtaaggcgatcgcctgccaatcatcagaatatcgagaagaatgtttcatctaaacctagcgccgcaagataatcctgaaa 
               
               
                   
               
               
                 tcgctacagtattaaaaaattctggccaacatcacagccaatactGCGGCCGC GGGGGGGGGGGGGAAAGCCACGTTGTGTCTCAAAATC   
               
               
                   
               
               
                 
                   TCTGATGTTACATTGCACAAGATAAAAATATATCATCATGAACAATAAAACTGTCTGCTTAC 
                 
               
               
                   
               
               
                   ATAAACAGTAATACAAGGGGTCAT ATGTAACAGGAATTC GGTTTTCCGTCCTGTCTTGATT   
               
               
                   
               
               
                 
                   TTCAAGCAAACAATGCCTCCGATTTCTAATCGGAGGCATTTGTTTTTGTTTATTGCAAA 
                 
               
               
                   
               
               
                 
                   AACAAAAAATATTGTTACAAATTTTTACAGGCTATTAAGCCTACCGTCATAAATAATTT 
                 
               
               
                   
               
               
                   GCCATTT ACTAGTTTTTAATTAAA   CCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCC     
               
               
                   
               
               
                 
                   
                     GCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGATTGAACAA 
                   
                 
               
               
                   
               
               
                 
                   
                     GATGGCCTGCATGCTGGTTCTCCGGCTGCTTGGGTGGAACGCCTGTTTGGTTACGACTGGGCTCA 
                   
                 
               
               
                   
               
               
                 
                   
                     GCTGACTATTGGCTGTAGCGATGCAGCGGTTTTCCGTCTGTCTGCACAGGGTCGTCCGGTTCTGTT 
                   
                 
               
               
                   
               
               
                 
                   
                     TGTGAAAACCGACCTGTCCGGCGCACTGAACGAACTGCAGGACGAAGCGGCCCGTCTGTCCTGG 
                   
                 
               
               
                   
               
               
                 
                   
                     CTCGCGACGACTGGTGTTCCGTGCGCGGCAGTTCTGGACGTAGTTACTGAAGCCGGTCGCGATTG 
                   
                 
               
               
                   
               
               
                 
                   
                     GCTGCTGCTGGGTGAAGTTCCGGGTCAGGATCTGCTGAGCAGCCACCTCGCTCCGGCAGAAAAAG 
                   
                 
               
               
                   
               
               
                 
                   
                     TTTCCATCATGGCGGACGCGATGCGCCGTCTGCACACCCTGGACCCGGCAACTTGCCCGTTTGAC 
                   
                 
               
               
                   
               
               
                 
                   
                     CATCAGGCTAAACACCGTATTGAACGTGCACGCACTCGTATGGAAGCGGGTCTGGTTGATCAGGA 
                   
                 
               
               
                   
               
               
                 
                   
                     CGACCTGGATGAAGAGCACCAGGGCCTCGCACCGGCGGAACTGTTTGCACGTCTGAAAGCCCGC 
                   
                 
               
               
                   
               
               
                 
                   
                     ATGCCGGACGGCGAAGACCTGGTGGTAACGCATGGCGACGCTTGTCTGCCAAACATTATGGTGGA 
                   
                 
               
               
                   
               
               
                 
                   
                     AAACGGCCGCTTCTCTGGTTTTATTGACTGTGGCCGTCTGGGTGTAGCTGATCGCTATCAGGATAT 
                   
                 
               
               
                   
               
               
                 
                   
                     CGCCCTCGCTACCCGCGATATTGCAGAAGAACTGGGTGGTGAATGGGCTGACCGTTTCCTGGTGC 
                   
                 
               
               
                   
               
               
                 
                   
                     TGTACGGTATCGCAGCGCCGGATTCTCAGCGCATTGCCTTCTACCGTCTGCTGGATGAGTTCTTCT 
                   
                 
               
               
                   
               
               
                     AA   GGCGCGCCgaaactgcgccaagaatagctcacttcaaatcagtcacggttttgtttagggcttgtctggcgattttggtgacatagacagtcaca 
               
               
                   
               
               
                 gcaacagtagccacaaaaccaagaatccggatcgaccactgggcaatggggttggcgctggtgctttctgtgccgagggtcgcaagatttccggccag 
               
               
                   
               
               
                 ggagccaatgtagacatacatgatggtgccagggatcatccccacagagccgaggacatagtcttttagggaaacgcccgtgaccccataggcatagtt 
               
               
                   
               
               
                 aagcagattaaagggaaatacaggtgagagacgcgtcaggagaacaatcttcaggccttccttgcccacagcttcgtcgatggcgcgaaatttcgggttg 
               
               
                   
               
               
                 tcggcgattttttggctcacccattggcgggccagataacgacccactaggaaagcagcgatcgctcctagggttgcgccaacaaagacgtaaattgatc 
               
               
                   
               
               
                 ctaaagcgacaccaaaaacaaccccggctcccaaggtcagaatcgaccccggtagaaaagccaccgtcgccaccacataaagcaccataaaggcga 
               
               
                   
               
               
                 tGGCCGGCCAAAATGAAGTGAAGTTCCTATACTTTCTAGAGAATAGGAACTTCTATAGTGAG 
               
               
                   
               
               
                 TCGAATAAGGGCGACACAAAATTTATTCTAAATGCATAATAAATACTGATAACATCTTATAG 
               
               
                   
               
               
                 TTTGTATTATATTTTGTATTATCGTTGACATGTATAATTTTGATATCAAAAACTGATTTTCCCT 
               
               
                   
               
               
                 TTATTATTTTCGAGATTTATTTTCTTAATTCTCTTTAACAAACTAGAAATATTGTATATACAAA 
               
               
                   
               
               
                 AAATCATAAATAATAGATGAATAGTTTAATTATAGGTGTTCATCAATCGAAAAAGCAACGTA 
               
               
                   
               
               
                 TCTTATTTAAAGTGCGTTGCTTTTTTCTCATTTATAAGGTTAAATAATTCTCATATATCAAGCA 
               
               
                   
               
               
                 AAGTGACAGGCGCCCTTAAATATTCTGACAAATGCTCTTTCCCTAAACTCCCCCCATAAAAA 
               
               
                   
               
               
                 AACCCGCCGAAGCGGGTTTTTACGTTATTTGCGGATTAACGATTACTCGTTATCAGAACCGC 
               
               
                   
               
               
                 CCAGGGGGCCCGAGCTTAAGACTGGCCGTCGTTTTACAACACAGAAAGAGTTTGTAGAAAC 
               
               
                   
               
               
                 GCAAAAAGGCCATCCGTCAGGGGCCTTCTGCTTAGTTTGATGCCTGGCAGTTCCCTACTCTC 
               
               
                   
               
               
                 GCCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATC 
               
               
                   
               
               
                 AGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAAC 
               
               
                   
               
               
                 ATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTT 
               
               
                   
               
               
                 TCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCG 
               
               
                   
               
               
                 AAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTC 
               
               
                   
               
               
                 CTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGC 
               
               
                   
               
               
                 TTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCT 
               
               
                   
               
               
                 GTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAG 
               
               
                   
               
               
                 TCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCA 
               
               
                   
               
               
                 GAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGGCTAACTACGGCTACACT 
               
               
                   
               
               
                 AGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGG 
               
               
                   
               
               
                 TAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGC 
               
               
                   
               
               
                 AGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGAC 
               
               
                   
               
               
                 GCTCAGTGGAACGACGCGCGCGTAACTCACGTTAAGGGATTTTGGTCATGAGCTTGCGCCGT 
               
               
                   
               
               
                 CCCGTCAAGTCAGCGTAATGCTCTGCTTTT 
               
               
                   
               
               
                 SEQ ID NO: 16 
                   
               
               
                 P psaA -tolC-P tsr2142 -acrAB insert with flanking homology regions 
                   
               
               
                 This sequence inserted into pJB161 to create PJB1074 
               
               
                 (UHR and DHR in lowercase and not underlined; P psaA  and P tsr2142  are underlined and capitalized; 
               
               
                 tolC, acrA and acrB are in bold, lowercase, and underlined and follow the promoter in order; 
               
               
                 kan R  marker is italicized and underlined) 
               
               
                 CCTGCAGGgccaccacagccaaattcatcgttaatgtggacttgccgacgcccccttttcgactaacaatcgcaatttttttcatagacatttcccaca 
               
               
                   
               
               
                 gaccacatcaaattacagcaattgatctagctgaaagtttaacccacttccccccagacccagaagaccagaggcgcttaagcttccccgaacaaactca 
               
               
                   
               
               
                 actgaccgagggggagggagccgtagcggcgttggtgttggcgtaaatgacaggccgagcaaagagcgatgagattttcccgacgattgtcttcgggg 
               
               
                   
               
               
                 atgtaatttttgtggtggacgcttaaggttaaaacagcccgcaggtgacgatcaatgcctttgaccttcacatccgacggaatacaaaccaagccacagag 
               
               
                   
               
               
                 ttcacagcgccagtctgcatcctcttttacttgtaaggcgatcgcctgccaatcatcagaatatcgagaagaatgtttcatctaaacctagcgccgcaaga 
               
               
                   
               
               
                 taatcctgaaatcgctacagtattaaaaaattctggccaacatcacagccaatactGCGGCCGC GCCCCTATATTATGCATTTATA   
               
               
                   
               
               
                 
                   CCCCCACAATCATGTCAAGAATTCAAGCATCTTAAATAATGTTAATTATCGGCAAAGTCTGT 
                 
               
               
                   
               
               
                 
                   GCTCCCCTTCTATAATGCTGAATTGAGCATTCGCCTCCTGAACGGTCTTTATTCTTCCATTGT 
                 
               
               
                   
               
               
                   GGGTCTTTAGATTCACGATTCTTCACAATCATTGATCTAAAGATCTTTCTAGATTCTCGAGG C 
               
               
                   
               
               
                 AT   atgaagaaattgctccccattcttatcggcctgagcctttctgggttcagttcgttgagccaggccgagaacctgatgcaagtttatcagcaa     
               
               
                   
               
               
                 
                   
                     gcacgccttagtaacccggaattgcgtaagtctgccgccgatcgtgatgctgcctttgaaaaaattaatgaagcgcgcagtccattactgccaca 
                   
                 
               
               
                   
               
               
                 
                   
                     gctaggtttaggtgcagattacacctatagcaacggctaccgcgacgcgaacggcatcaactctaacgcgaccagtgcgtccttgcagttaact 
                   
                 
               
               
                   
               
               
                 
                   
                     caatccatttttgatatgtcgaaatggcgtgcgttaacgctgcaggaaaaagcagcagggattcaggacgtcacgtatcagaccgatcagcaaa 
                   
                 
               
               
                   
               
               
                 
                   
                     ccttgatcctcaacaccgcgaccgcttatttcaacgtgttgaatgctattgacgttctttcctatacacaggcacaaaaagaagcgatctaccgtc 
                   
                 
               
               
                   
               
               
                 
                   
                     aattagatcaaaccacccaacgttttaacgtgggcctggtagcgatcaccgacgtgcagaacgcccgcgcacagtacgataccgtgctggcga 
                   
                 
               
               
                   
               
               
                 
                   
                     acgaagtgaccgcacgtaataaccttgataacgcggtagagcagctgcgccagatcaccggtaactactatccggaactggctgcgctgaatg 
                   
                 
               
               
                   
               
               
                 
                   
                     tcgaaaactttaaaaccgacaaaccacagccggttaacgcgctgctgaaagaagccgaaaaacgcaacctgtcgctgttacaggcacgcttga 
                   
                 
               
               
                   
               
               
                 
                   
                     gccaggacctggcgcgcgagcaaattcgccaggcgcaggatggtcacttaccgactctggatttaacggcttctaccgggatttctgacacctct 
                   
                 
               
               
                   
               
               
                 
                   
                     tatagcggttcgaaaacccgtggtgccgctggtacccagtatgacgatagcaatatgggccagaacaaagttggcctgagcttctcgctgccga 
                   
                 
               
               
                   
               
               
                 
                   
                     tttatcagggcggaatggttaactcgcaggtgaaacaggcacagtacaactttgtcggtgccagcgagcaactggaaagtgcccatcgtagcgt 
                   
                 
               
               
                   
               
               
                 
                   
                     cgtgcagaccgtgcgttcctccttcaacaacattaatgcatctatcagtagcattaacgcctacaaacaagccgtagtttccgctcaaagctcatt 
                   
                 
               
               
                   
               
               
                 
                   
                     agacgcgatggaagcgggctactcggtcggtacgcgtaccattgttgatgtgttggatgcgaccaccacgttgtacaacgccaagcaagagctg 
                   
                 
               
               
                   
               
               
                 
                   
                     gcgaatgcgcgttataactacctgattaatcagctgaatattaagtcagctctgggtacgttgaacgagcaggatctgctggcactgaacaatgc 
                   
                 
               
               
                   
               
               
                 
                   
                     gctgagcaaaccggtttccactaatccggaaaacgttgcaccgcaaacgccggaacagaatgctattgctgatggttatgcgcctgatagcccg 
                   
                 
               
               
                   
               
               
                     gcaccagtcgttcagcaaacatccgcacgcactaccaccagtaacggtcataaccctttccgtaactga   GGATCC AAGGTGGCTA   
               
               
                   
               
               
                 
                   CTTCAACGATAGCTTAAACTTCGCTGCTCCAGCGAGGGGATTTCACTGGTTTGAATGCTTCA 
                 
               
               
                   
               
               
                 
                   ATGCTTGCCAAAAGAGTGCTACTGGAACTTACAAGAGTGACCCTGCGTCAGGGGAGCTAGC 
                 
               
               
                   
               
               
                 
                   ACTCAAAAAAGACTCCTCCAATTCCGTCC atgaacaaaaacagagggtttacgcctctggcggtcgttctgatgctctca   
                 
               
               
                   
               
               
                 
                   
                     ggcagcttagccctaacaggatgtgacgacaaacaggcccaacaaggtggccagcagatgcccgccgttggcgtagtaacagtcaaaactga 
                   
                 
               
               
                   
               
               
                 
                   
                     acctctgcagatcacaaccgagcttccgggtcgcaccagtgcctaccggatcgcagaagttcgtcctcaagttagcgggattatcctgaagcgta 
                   
                 
               
               
                   
               
               
                 
                   
                     atttcaaagaaggtagcgacatcgaagcaggtgtctctctctatcagattgatcctgcgacctatcaggcgacatacgacagtgcgaaaggtga 
                   
                 
               
               
                   
               
               
                 
                   
                     tctggcgaaagcccaggctgcagccaatatcgcgcaattgacggtgaatcgttatcagaaactgctcggtactcagtacatcagtaagcaagag 
                   
                 
               
               
                   
               
               
                 
                   
                     tacgatcaggctctggctgatgcgcaacaggcgaatgctgcggtaactgcggcgaaagctgccgttgaaactgcgcggatcaatctggcttaca 
                   
                 
               
               
                   
               
               
                 
                   
                     ccaaagtcacctctccgattagcggtcgcattggtaagtcgaacgtgacggaaggcgcattggtacagaacggtcaggcgactgcgctggcaa 
                   
                 
               
               
                   
               
               
                 
                   
                     ccgtgcagcaacttgatccgatctacgttgatgtgacccagtccagcaacgacttcctgcgcctgaaacaggaactggcgaatggcacgctgaa 
                   
                 
               
               
                   
               
               
                 
                   
                     acaagagaacggcaaagccaaagtgtcactgatcaccagtgacggcattaagttcccgcaggacggtacgctggaattctctgacgttaccgtt 
                   
                 
               
               
                   
               
               
                 
                   
                     gatcagaccactgggtctatcaccctacgcgctatcttcccgaacccggatcacactctgctgccgggtatgttcgtgcgcgcacgtctggaaga 
                   
                 
               
               
                   
               
               
                 
                   
                     agggcttaatccaaacgctattttagtcccgcaacagggcgtaacccgtacgccgcgtggcgatgccaccgtactggtagttggcgcggatgac 
                   
                 
               
               
                   
               
               
                 
                   
                     aaagtggaaacccgtccgatcgttgcaagccaggctattggcgataagtggctggtgacagaaggtctgaaagcaggcgatcgcgtagtaata 
                   
                 
               
               
                   
               
               
                 
                   
                     agtgggctgcagaaagtgcgtcctggtgtccaggtaaaagcacaagaagttaccgctgataataaccagcaagccgcaagcggtgctcagcct 
                   
                 
               
               
                   
               
               
                     gaacagtccaagtcttaa   cttaaacaggagccgttaagac   atgcctaatttctttatcgatcgcccgatttttgcgtgggtgatcgccattatcatcat     
               
               
                   
               
               
                 
                   
                     gttggcaggggggctggcgatcctcaaactgccggtggcgcaatatcctacgattgcaccgccggcagtaacgatctccgcctcctaccccggc 
                   
                 
               
               
                   
               
               
                 
                   
                     gctgatgcgaaaacagtgcaggacacggtgacacaggttatcgaacagaatatgaacggtatcgataacctgatgtacatgtcctctaacagt 
                   
                 
               
               
                   
               
               
                 
                   
                     gactccacgggtaccgtgcagatcaccctgacctttgagtctggtactgatgcggatatcgcgcaggttcaggtgcagaacaaactgcagctgg 
                   
                 
               
               
                   
               
               
                 
                   
                     cgatgccgttgctgccgcaagaagttcagcagcaaggggtgagcgttgagaaatcatccagcagcttcctgatggttgtcggcgttatcaacac 
                   
                 
               
               
                   
               
               
                 
                   
                     cgatggcaccatgacgcaggaggatatctccgactacgtggcggcgaatatgaaagatgccatcagccgtacgtcgggcgtgggtgatgttca 
                   
                 
               
               
                   
               
               
                 
                   
                     gttgttcggttcacagtacgcgatgcgtatctggatgaacccgaatgagctgaacaaattccagctaacgccggttgatgtcattaccgccatca 
                   
                 
               
               
                   
               
               
                 
                   
                     aagcgcagaacgcccaggttgcggcgggtcagctcggtggtacgccgccggtgaaaggccaacagcttaacgcctctattattgctcagacgc 
                   
                 
               
               
                   
               
               
                 
                   
                     gtctgacctctactgaagagttcggcaaaatcctgctgaaagtgaatcaggatggttcccgcgtgctgctgcgtgacgtcgcgaagattgagctg 
                   
                 
               
               
                   
               
               
                 
                   
                     ggtggtgagaactacgacatcatcgcagagtttaacggccaaccggcttccggtctggggatcaagctggcgaccggtgcaaacgcgctggat 
                   
                 
               
               
                   
               
               
                 
                   
                     accgctgcggcaatccgtgctgaactggcgaagatggaaccgttcttcccgtcgggtctgaaaattgtttacccatacgacaccacgccgttcgt 
                   
                 
               
               
                   
               
               
                 
                   
                     gaaaatctctattcacgaagtggttaaaacgctggtcgaagcgatcatcctcgtgttcctggttatgtatctgttcctgcagaacttccgcgcgacg 
                   
                 
               
               
                   
               
               
                 
                   
                     ttgattccgaccattgccgtaccggtggtattgctcgggacctttgccgtccttgccgcctttggcttctcgataaacacgctaacaatgttcgggat 
                   
                 
               
               
                   
               
               
                 
                   
                     ggtgctcgccatcggcctgttggtggatgacgccatcgttgtggtagaaaacgttgagcgtgttatggcggaagaaggtttgccgccaaaagaa 
                   
                 
               
               
                   
               
               
                 
                   
                     gctacccgtaagtcgatggggcagattcagggcgctctggtcggtatcgcgatggtactgtcggcggtattcgtaccgatggccttctttggcggt 
                   
                 
               
               
                   
               
               
                 
                   
                     tctactggtgctatctatcgtcagttctctattaccattgtttcagcaatggcgctgtcggtactggtggcgttgatcctgactccagctctttgtgcca 
                   
                 
               
               
                   
               
               
                 
                   
                     ccatgctgaaaccgattgccaaaggcgatcacggggaaggtaaaaaaggcttcttcggctggtttaaccgcatgttcgagaagagcacgcacc 
                   
                 
               
               
                   
               
               
                 
                   
                     actacaccgacagcgtaggcggtattctgcgcagtacggggcgttacctggtgctgtatctgatcatcgtggtcggcatggcctatctgttcgtgc 
                   
                 
               
               
                   
               
               
                 
                   
                     gtctgccaagctccttcttgccagatgaggaccagggcgtgtttatgaccatggttcagctgccagcaggtgcaacgcaggaacgtacacagaa 
                   
                 
               
               
                   
               
               
                 
                   
                     agtgctcaatgaggtaacgcattactatctgaccaaagaaaagaacaacgttgagtcggtgttcgccgttaacggcttcggctttgcgggacgtg 
                   
                 
               
               
                   
               
               
                 
                   
                     gtcagaataccggtattgcgttcgtttccttgaaggactgggccgatcgtccgggcgaagaaaacaaagttgaagcgattaccatgcgtgcaac 
                   
                 
               
               
                   
               
               
                 
                   
                     acgcgctttctcgcaaatcaaagatgcgatggttttcgcctttaacctgcccgcaatcgtggaactgggtactgcaaccggctttgactttgagctg 
                   
                 
               
               
                   
               
               
                 
                   
                     attgaccaggctggccttggtcacgaaaaactgactcaggcgcgtaaccagttgcttgcagaagcagcgaagcaccctgatatgttgaccagc 
                   
                 
               
               
                   
               
               
                 
                   
                     gtacgtccaaacggtctggaagataccccgcagtttaagattgatatcgaccaggaaaaagcgcaggcgctgggtgtttctatcaacgacatta 
                   
                 
               
               
                   
               
               
                 
                   
                     acaccactctgggcgctgcatggggcggcagctatgtgaacgactttatcgaccgcggtcgtgtgaagaaagtttatgtcatgtcagaagcgaa 
                   
                 
               
               
                   
               
               
                 
                   
                     ataccgtatgctgccggatgatatcggcgactggtatgttcgtgctgctgatggtcagatggtgccattctcggcgttctcctcttctcgttgggagt 
                   
                 
               
               
                   
               
               
                 
                   
                     acggttcgccgcgtctggaacgttacaacggcctgccatccatggaaatcttaggccaggcggcaccgggtaaaagtaccggtgaagcaatgg 
                   
                 
               
               
                   
               
               
                 
                   
                     agctgatggaacaactggcgagcaaactgcctaccggtgttggctatgactggacggggatgtcctatcaggaacgtctctccggcaaccagg 
                   
                 
               
               
                   
               
               
                 
                   
                     caccttcactgtacgcgatttcgttgattgtcgtgttcctgtgtctggcggcgctgtacgagagctggtcgattccgttctccgttatgctggtcgttc 
                   
                 
               
               
                   
               
               
                 
                   
                     cgctgggggttatcggtgcgttgctggctgccaccttccgtggcctgaccaatgacgtttacttccaggtaggcctgctcacaaccattgggttgtc 
                   
                 
               
               
                   
               
               
                 
                   
                     ggcgaagaacgcgatccttatcgtcgaattcgccaaagacttgatggataaagaaggtaaaggtctgattgaagcgacgcttgatgcggtgcg 
                   
                 
               
               
                   
               
               
                 
                   
                     gatgcgtttacgtccgatcctgatgacctcgctggcgtttatcctcggcgttatgccgctggttatcagtactggtgctggttccggcgcgcagaac 
                   
                 
               
               
                   
               
               
                 
                   
                     gcagtaggtaccggtgtaatgggcgggatggtgaccgcaacggtactggcaatcttcttcgttccggtattctttgtggtggttcgccgccgcttta 
                   
                 
               
               
                   
               
               
                     gccgcaagaatgaagatatcgagcacagccatactgtcgatcatcattga   GAGCTCttGAATTCGGTTTTCCGTCCTGTCT 
               
               
                   
               
               
                 TGATTTTCAAGCAAACAATGCCTCCGATTTCTAATCGGAGGCATTTGTTTTTGTTTATTGCAA 
               
               
                   
               
               
                 AAACAAAAAATATTGTTACAAATTTTTACAGGCTATTAAGCCTACCGTCATAAATAATTTGC 
               
               
                   
               
               
                 CATTTACTAGTTTTTAATTAAA   CCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCT     
               
               
                   
               
               
                 
                   
                     CATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGATTGAACAAGAT 
                   
                 
               
               
                   
               
               
                 
                   
                     GGCCTGCATGCTGGTTCTCCGGCTGCTTGGGTGGAACGCCTGTTTGGTTACGACTGGGCTCAGCT 
                   
                 
               
               
                   
               
               
                 
                   
                     GACTATTGGCTGTAGCGATGCAGCGGTTTTCCGTCTGTCTGCACAGGGTCGTCCGGTTCTGTTTGT 
                   
                 
               
               
                   
               
               
                 
                   
                     GAAAACCGACCTGTCCGGCGCACTGAACGAACTGCAGGACGAAGCGGCCCGTCTGTCCTGGCTC 
                   
                 
               
               
                   
               
               
                 
                   
                     GCGACGACTGGTGTTCCGTGCGCGGCAGTTCTGGACGTAGTTACTGAAGCCGGTCGCGATTGGCT 
                   
                 
               
               
                   
               
               
                 
                   
                     GCTGCTGGGTGAAGTTCCGGGTCAGGATCTGCTGAGCAGCCACCTCGCTCCGGCAGAAAAAGTTT 
                   
                 
               
               
                   
               
               
                 
                   
                     CCATCATGGCGGACGCGATGCGCCGTCTGCACACCCTGGACCCGGCAACTTGCCCGTTTGACCAT 
                   
                 
               
               
                   
               
               
                 
                   
                     CAGGCTAAACACCGTATTGAACGTGCACGCACTCGTATGGAAGCGGGTCTGGTTGATCAGGACGA 
                   
                 
               
               
                   
               
               
                 
                   
                     CCTGGATGAAGAGCACCAGGGCCTCGCACCGGCGGAACTGTTTGCACGTCTGAAAGCCCGCATG 
                   
                 
               
               
                   
               
               
                 
                   
                     CCGGACGGCGAAGACCTGGTGGTAACGCATGGCGACGCTTGTCTGCCAAACATTATGGTGGAAAA 
                   
                 
               
               
                   
               
               
                 
                   
                     CGGCCGCTTCTCTGGTTTTATTGACTGTGGCCGTCTGGGTGTAGCTGATCGCTATCAGGATATCGC 
                   
                 
               
               
                   
               
               
                 
                   
                     CCTCGCTACCCGCGATATTGCAGAAGAACTGGGTGGTGAATGGGCTGACCGTTTCCTGGTGCTGT 
                   
                 
               
               
                   
               
               
                     ACGGTATCGCAGCGCCGGATTCTCAGCGCATTGCCTTCTACCGTCTGCTGGATGAGTTCTTCTAA   G 
               
               
                   
               
               
                 GCGCGCCgaaactgcgccaagaatagctcacttcaaatcagtcacggttttgtttagggcttgtctggcgattttggtgacatagacagtcacagcaa 
               
               
                   
               
               
                 cagtagccacaaaaccaagaatccggatcgaccactgggcaatggggttggcgctggtgctttctgtgccgagggtcgcaagatttccggccagggag 
               
               
                   
               
               
                 ccaatgtagacatacatgatggtgccagggatcatccccacagagccgaggacatagtcttttagggaaacgcccgtgaccccataggcatagttaagc 
               
               
                   
               
               
                 agattaaagggaaatacaggtgagagacgcgtcaggagaacaatcttcaggccttccttgcccacagcttcgtcgatggcgcgaaatttcgggttgtcgg 
               
               
                   
               
               
                 cgattttttggctcacccattggcgggccagataacgacccactaggaaagcagcgatcgctcctagggttgcgccaacaaagacgtaaattgatcctaa 
               
               
                   
               
               
                 agcgacaccaaaaacaaccccggctcccaaggtcagaatcgaccccggtagaaaagccaccgtcgccaccacataaagcaccataaaggcgatGG 
               
               
                   
               
               
                 CCGGCC 
               
               
                   
               
               
                 SEQ ID NO: 17 
                   
               
               
                 P(nirA):  S .  elongatus  PCC 7942 
                   
               
               
                 TCCCTCTCAGCTCAAAAAGTATCAATGATTACTTAATGTTTGTTCTGCGCAAACTTCT 
               
               
                   
               
               
                 TGCAGAACATGCATGATTTACAAAAAGTTGTAGTTTCTGTTACCAATTGCGAATCGA 
               
               
                   
               
               
                 GAACTGCCTAATCTGCCGAGTATGCAAGCTGCTTTGTAGGCAGATGAATCCCAT 
               
               
                   
               
               
                 SEQ ID NO: 18 
                   
               
               
                 P(nir07):  S .  elongatus  PCC 7942 +  Synechococcus  sp. PCC 7002 rbcL altered ribosome binding 
                   
               
               
                 site (RBS) 
               
               
                 GCTTGTAGCAATTGCTACTAAAAACTGCGATCGCTGCTGAAATGAGCTGGAATTTTG 
               
               
                   
               
               
                 TCCCTCTCAGCTCAAAAAGTATCAATGATTACTTAATGTTTGTTCTGCGCAAACTTCT 
               
               
                   
               
               
                 TGCAGAACATGCATGATTTACAAAAAGTTGTAGTTTCTGTTACCAATTGCGAATCGA 
               
               
                   
               
               
                 GAACTGCCTAATCTGCCGAGTATGCGATCCTTTAGCAGGAGGAAAACCAT 
               
               
                   
               
               
                 SEQ ID NO: 19 
                   
               
               
                 P(nir09):  Anabaena  sp. PCC 7120 +  Synechococcus  sp. PCC 7002 rbcL RBS 
                   
               
               
                 GCTACTCATTAGTTAAGTGTAATGCAGAAAACGCATATTCTCTATTAAACTTACGCA 
               
               
                   
               
               
                 TTAATACGAGAATTTTGTAGCTACTTATACTATTTTACCTGAGATCCCGACATAACCT 
               
               
                   
               
               
                 TAGAAGTATCGAAATCGTTACATAAACATTCACACAAACCACTTGACAAATTTAGCC 
               
               
                   
               
               
                 AATGTAAAAGACTACAGTTTCTCCCCGGTTTAGTTCTAGAGTTACCTTCAGTGAAAC 
               
               
                   
               
               
                 ATCGGCGGCGTGTCAGTCATTGAAGTAGCATAAATCAATTCAAAATACCCTGCGGG 
               
               
                   
               
               
                 AAGGCTGCGCCAACAAAATTAAATATTTGGTTTTTCACTATTAGAGCATCGATTCAT 
               
               
                   
               
               
                 TAATCAAAAACCTTACCCCCCAGCCCCCTTCCCTTGTAGGGAAGTGGGAGCCAAACT 
               
               
                   
               
               
                 CCCCTCTCCGCGTCGGAGCGAAAAGTCTGAGCGGAGGTTTCCTCCGAACAGAACTTT 
               
               
                   
               
               
                 TAAAGAGAGAGGGGTTGGGGGAGAGGTTCTTTCAAGATTACTAAATTGCTATCACT 
               
               
                   
               
               
                 AGACCTCGTAGAACTAGCAAAGACTACGGGTGGATTGATCTTGAGCAAAAAAACTT 
               
               
                   
               
               
                 TATGAGAACTTTAGCAGGAGGAAAACCAT 
               
               
                   
               
               
                 SEQ ID NO: 20 
                   
               
               
                 nrsS-nrsR - P(nrsB):  Synechocystis  sp. PCC 6803 s110798-s110797 Pslr0793 + 
                   
               
               
                   Synechococcus  sp. PCC 7002 rbcL RBS 
               
               
                 GATTACCCTATATCGGGCTTTTCTCAATAAAATCTTTATTTTTTGAGGTGCTTTTTAG 
               
               
                   
               
               
                 CCATAAATAATCACTTTAGTATAAAATTTTGACGGCGTAAAGTTGATAAAATAGAAT 
               
               
                   
               
               
                 TAAGAATGGACTATCGGTACAGAAAAAATGGGTAACTGGATGGTGAATAAACTTCC 
               
               
                   
               
               
                 CTTACCCAATGCACTCTCCACCGTTAAAGACCCCCTATGCTTAACGGTGATCACCTG 
               
               
                   
               
               
                 GGCAATGGCGAGTCCCAACCCTGTCCCCCCCGTTTTGCGCGAACGATCTCGATTAAC 
               
               
                   
               
               
                 TCGGTAAAAACGCTCAAAAATGTGTTCCTGTTGGTCGGGGGCAATGCCGATGCCGGT 
               
               
                   
               
               
                 ATCTTGCACGGTGATGATAGCCATCTGTTCATGGGATGTCAGGGTAATATCAACACG 
               
               
                   
               
               
                 TCCCCCAGCAGTTGTGTATTGAATGGCGTTGGCAATTAGGTTTGAGACCAGTCGATA 
               
               
                   
               
               
                 GAGTTGGGATTCATTACCCCAGGCGTAAACTTCCCCTGAACTCAGATCACTGCTGAG 
               
               
                   
               
               
                 ATCAATGTGGGCGGCGATCGCTAATTCTAAAAACTCTTCGGTGAGGTCACTGACTAA 
               
               
                   
               
               
                 ATCATTTAAACAACAAAGCCGCCAATCTTCGGCGGTGGTTTCCTGCTCTAAGCGACT 
               
               
                   
               
               
                 TAGTAGCAATAAATCCGTAATCAATTGGCTTAATCGCCTTCCCTGTCGTTCAACGGT 
               
               
                   
               
               
                 ATGTAGCATGGTGTTAATTTCTGGGGAATGGCTTGAGTCGATGCGTAATACCGCTTC 
               
               
                   
               
               
                 CACCGTGGCCAACAGACTAGCCAATGGCGATCGTAATTCATGGGCTGCATTCGCGGT 
               
               
                   
               
               
                 GAATTGTTGTTGTTGTTGGTAGGACTGGTAAATGGGACGCATGGCTAACCCCGCTAA 
               
               
                   
               
               
                 GCCCCAACTGGAGAAGGCGACCAAACCCAGGGCAATGGGAAAACTAAGCCCTAAA 
               
               
                   
               
               
                 ATCCAAAGAATACGTTTATTTTCGGCATCAAAGGCTGCCAGGCTCCGGCCAATTTGT 
               
               
                   
               
               
                 AGATAGCCCCAGGAAGATTTGTCTGTATTACCGGCGCTATGCAAAATGGTGGTGAAT 
               
               
                   
               
               
                 TGTCGATACCGATCGCCGGTTGGGGGGTGAATAGTCTGCCAAGTTTCCTGGTTAAAA 
               
               
                   
               
               
                 ATGGAGGATAGGGAAGCCGGTTGATTAGGCGAAAAAGCCAGCAGGTTGCCTTGATA 
               
               
                   
               
               
                 ATCAAATAAACGAATGTAATATAAACTGCGATCACTAATGCCCAACGTGTGACGTTC 
               
               
                   
               
               
                 AATCAGGGTGGGGTTGACCTGGCAGGGTTGGTTGACCAAACACAGATCGGGCAACA 
               
               
                   
               
               
                 TTTTTTGTAATACTCCGGTGGGACTAGCATTACTCGGCAACATCGGCTCTAAACTGTC 
               
               
                   
               
               
                 ATGCAACGTCCCGGCGATCGACTCCACTTCTCGCTCCAACGCCATCCAGTTGGCCTG 
               
               
                   
               
               
                 CACAATGGCACGATAAACCCCCAACCCCAACAGGGTAAGAATTCCCCCCATTACTA 
               
               
                   
               
               
                 GGGCATACCAGAAAGCCAATTGCAGACGACTACGGGCAAAGAGGCGACGGGTATTC 
               
               
                   
               
               
                 ATGGCGATAGGGTGAACCGATAGCCTTGACCGGGAACTGTTTTAATTGGGCAAGGA 
               
               
                   
               
               
                 CAATTTTGTTGAGCTAGCTTGCGTCGTATCAAACGCATTTGGGCCGCCACCACATTA 
               
               
                   
               
               
                 CTCATGGGCTCCTCATCAAGATCCCACAGTTGTTGCCGGATCTTGCTACCGGAAATG 
               
               
                   
               
               
                 ATCCGCTCTGGGTTTTGCATCAGATATTGAAAAATTTGAAATTCTCTTACGGTTAAA 
               
               
                   
               
               
                 GCAATTTCCTGTCTTTCTAGGTTTAGTGGCTCCGAGATAGTTACCGATAACAGATTAT 
               
               
                   
               
               
                 TACTGGGATCAAGGCTGAAGTTGCCCAAAGTTAAAATTTGCGGTTGGAATTGTGGCG 
               
               
                   
               
               
                 ATCGCCGTTGTAGTGCCCGCAGTCTTGCTAATAGCTCTGCCATCACAAACGGTTTTGT 
               
               
                   
               
               
                 TAGATAGTCATCTGCCCCGGCATCTAGTCCTTCGACACGGTTTTCCGGTTCTCCTAAC 
               
               
                   
               
               
                 GCTGTTAACATCAACACCGGCAAGGAATTACCCTGGGTTCTCAGTTTTTGACAGAGT 
               
               
                   
               
               
                 TCCAAACCCGATAATCCCGGCAGTAACCAATCCACAATGGCAAGGGTGTATTCCGTC 
               
               
                   
               
               
                 CATTGATTTTCCAAATAATCCCAAGCTTGGGAGCCATCCGTCACCCAATCCACCACA 
               
               
                   
               
               
                 TACTTTTCACTAACTAGCACTTTCTTAATAGCCATTCCCAAATCCGTCTCATCTTCCA 
               
               
                   
               
               
                 CCAGCAAAATTCGCATCGCCTCTGCCTTTTTTATAACGGTCTGATCTTAGCGGGGGA 
               
               
                   
               
               
                 AGGAGATTTTCACCTGAATTTCATACCCCCTTTGGCAGACTGGGAAAATCTTGGACA 
               
               
                   
               
               
                 AATTAGGAGGAAAACCAT