WO2012178101A2 - Compositions and methods to remove genetic markers using counter-selection - Google Patents

Abstract

The present disclosure provides compositions and methods for genetic marker removal in cyanobacteria. Cyanobacteria are developed having a genetic marker capable of being selected against in the presence of a negatively selective condition, resulting in removal of a positively selective marker (e.g., an antibiotic resistance marker) from the host cell.

Description

COMPOSITIONS AND METHODS TO REMOVE GENETIC MARKERS USING

COUNTER-SELECTION

Inventors

Yu Xu

Brian D. Green

Cross-Reference to Related Applications

[0001] This application claims the benefit of U.S. Provisional patent Application No.

61/500,528, filed June 23, 2011, the disclosure of which is incorporated herein by reference.

Sequence Listing

[0002] The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on June 22, 2012, is named 21120PCT_CRF_sequencelisting.txt and is 52,831 bytes in size.

Field

[0003] The disclosure relates to counter-selection methods for removing genetic markers from photosynthetic microorganisms.

Background

[0004] To facilitate the development and utilization of genetically modified organisms (GMO), both selectable (in small scale) and screenable (in large scale) traits have been exploited. The most widely applied selectable traits are auxotrophies due to defects of metabolizing certain key nutrients, and resistance to antimicrobials, by expressing genes that confer certain antimicrobial resistance. An evolving GMO strain may carry several, if not dozens, of genetic modifications, and developers often encounter a situation where selectable traits for further strain development are limiting. In addition, when facing industrial scales, antimicrobial selection can be costly and environmentally troublesome. For these reasons and others, a site-specific marker removal process, i.e., a means of removing prior selected markers from organisms while keeping the desired genetic engineering, is desired, particularly in cyanobacteria. Summary

[0005] The present disclosure embodies a method for preparing a recombinant

cyanobacterium comprising: introducing a suicide plasmid into a host cyanobacterium, the suicide plasmid comprising a positively selective marker, a negatively selective marker, and a recombinant gene; selecting for primary recombinants incorporating the positively selective marker; from the primary recombinants, selecting for secondary recombinants that have lost the negatively selective marker and the positively selective marker; and isolating the secondary recombinants comprising the recombinant gene to obtain the recombinant cyanobacterium. The present disclosure also embodies a recombinant cyanobacterium prepared by an embodiment of the above method.

[0006] In another aspect, the disclosure embodies a method for preparing a recombinant cyanobacterium, comprising: introducing a suicide plasmid into a host cyanobacterium, the suicide plasmid comprising a positively selective marker, a negatively selective marker, and a recombinant gene, wherein the negatively selective marker confers host susceptibility to a selectable environmental condition; selecting for primary recombinants incorporating the positively selective marker; culturing the primary recombinants in the presence of the selectable environmental condition, thereby selecting for secondary recombinants that have lost the negatively selective marker and the positively selective marker; and isolating the secondary recombinants comprising the recombinant gene to obtain the recombinant cyanobacterium.

[0007] In one aspect, if the host cyanobacterium natively comprises a corresponding negatively selective marker, the method further comprises the step of removing or reducing expression of the corresponding negatively selective marker prior to introducing the suicide plasmid into the host cyanobacterium. In a further aspect, the step of removing or reducing expression of the corresponding negatively selective marker creates a host cyanobacterium having a null mutation, wherein the host cyanobacterium having the null mutation has a decreased sensitivity to a negatively selective condition as compared to a host

cyanobacterium without the null mutation. In another aspect, the negatively selective marker comprises a gene that has been knocked out in the recombinant cyanobacterium.

[0008] In one embodiment, the negatively selective marker comprises a gene expressing an enzyme capable of incorporating a cytotoxic compound. In a further embodiment, the cytotoxic compound is a nucleobase analog. In yet a further embodiment, the nucleobase anaog could be any one of a number of alogenic (i.e., F- / CI- / Br- / 1-) pyrimidines or purines or its precursor. In still a further embodiment, the base analog is 5-fluorouracil (5- FU), 5-fluoroorotic acid (5-FOA), 8-thioxanthine, 6-thioguanine, 8-aza-2,6-diaminopurine (8ADP), or 2-methylpurine (2MP). In another further embodiment, the gene is selected from pyrR, upp, pyrF/pyrE, pyrE, pyrF, gpt, glnQ, and pheSA294G. In yet another further embodiment, the gene is encoded by a polynucleotide comprising a sequence of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, or a polynucleotide sequence encoding SEQ ID NO: 16. In still another embodiment, the gene is encoded by a polynucleotide comprising a sequence at least 75%, at least 80%, at least 85%, at least 90%, or more preferably at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, or a polynucleotide sequence encoding SEQ ID NO: 16.

[0009] In one embodiment, a pyrR gene encodes an enzyme with an amino acid sequence of SEQ ID NO: 2 or a homo log thereof, wherein a homo log is a protein whose BLAST alignment (i) covers >90% length of SEQ ID NO: 2, (ii) covers >90% length of the matching protein, and (iii) has >50% identity with SEQ ID NO: 2 (when optimally aligned using parameters provided herein). In another embodiment, the pyrR gene is a homo log having substantial homology to SEQ ID NO: l, wherein a homo log is a gene whose BLAST alignment covers (i) covers >90% length of SEQ ID NO: 1, (ii) covers >90% length of the matching gene, and (iii) has >50% identity with SEQ ID NO: 1 (when optimally aligned using parameters provided herein).

[0010] In another embodiment, a upp gene encodes an enzyme with an amino acid sequence of SEQ ID NO: 5 or a homo log thereof, wherein a homo log is a protein whose BLAST alignment (i) covers >90% length of SEQ ID NO: 5, (ii) covers >90% length of the matching protein, and (iii) has >50% identity with SEQ ID NO: 5 (when optimally aligned using parameters provided herein). In another embodiment, the upp gene is a homo log having substantial homology to SEQ ID NO:4, wherein a homo log is a gene whose BLAST alignment covers (i) covers >90% length of SEQ ID NO: 4, (ii) covers >90% length of the matching gene, and (iii) has >50% identity with SEQ ID NO: 4 (when optimally aligned using parameters provided herein).

[0011] In one embodiment, a pyrF/pyrE gene encodes an enzyme with an amino acid sequence of SEQ ID NO: 11 or a homo log thereof, wherein a homo log is a protein whose BLAST alignment (i) covers >90% length of SEQ ID NO: 11, (ii) covers >90% length of the matching protein, and (iii) has >50% identity with SEQ ID NO: 11 (when optimally aligned using parameters provided herein). In another embodiment, the pyrF/pyrE gene is a homo log having substantial homology to SEQ ID NO: 10, wherein a homo log is a gene whose BLAST alignment covers (i) covers >90% length of SEQ ID NO: 10, (ii) covers >90% length of the matching gene, and (iii) has >50% identity with SEQ ID NO: 10 (when optimally aligned using parameters provided herein).

[0012] In one embodiment, a pyrE gene encodes an enzyme with an amino acid sequence of SEQ ID NO: 7 or a homo log thereof, wherein a homo log is a protein whose BLAST alignment (i) covers >90% length of SEQ ID NO: 7, (ii) covers >90% length of the matching protein, and (iii) has >50% identity with SEQ ID NO: 7 (when optimally aligned using parameters provided herein). In another embodiment, the pyrE gene is a homo log having substantial homology to SEQ ID NO:6, wherein a homolog is a gene whose BLAST alignment covers (i) covers >90% length of SEQ ID NO: 6, (ii) covers >90% length of the matching gene, and (iii) has >50% identity with SEQ ID NO: 6 (when optimally aligned using parameters provided herein).

[0013] In one embodiment, a pyrF gene encodes an enzyme with an amino acid sequence of SEQ ID NO: 9 or a homolog thereof, wherein a homolog is a protein whose BLAST alignment (i) covers >90% length of SEQ ID NO: 9, (ii) covers >90% length of the matching protein, and (iii) has >50% identity with SEQ ID NO: 9 (when optimally aligned using parameters provided herein). In another embodiment, the pyrF gene is a homolog having substantial homology to SEQ ID NO: 8, wherein a homolog is a gene whose BLAST alignment covers (i) covers >90% length of SEQ ID NO: 8, (ii) covers >90% length of the matching gene, and (iii) has >50% identity with SEQ ID NO: 8 (when optimally aligned using parameters provided herein).

[0014] In one embodiment, a gpt gene encodes an enzyme with an amino acid sequence of SEQ ID NO: 13 or a homolog thereof, wherein a homolog is a protein whose BLAST alignment (i) covers >90% length of SEQ ID NO: 13, (ii) covers >90% length of the matching protein, and (iii) has >50% identity with SEQ ID NO: 13 (when optimally aligned using parameters provided herein). In another embodiment, the gpt gene is a homolog having substantial homology to SEQ ID NO: 12, wherein a homolog is a gene whose BLAST alignment covers (i) covers >90% length of SEQ ID NO: 12, (ii) covers >90% length of the matching gene, and (iii) has >50% identity with SEQ ID NO: 12 (when optimally aligned using parameters provided herein).

[0015] In one embodiment, a pheSA294G gene encodes an enzyme with an amino acid sequence of SEQ ID NO: 16 or a homolog thereof, wherein a homolog is a protein whose BLAST alignment (i) covers >90% length of SEQ ID NO: 16, (ii) covers >90% length of the matching protein, and (iii) has >50% identity with SEQ ID NO: 16 (when optimally aligned using parameters provided herein).

[0016] In one aspect, the negatively selective marker is a recombinant gene encoding a uracil phosphoribosyltransferase. In a further aspect, the recombinant gene is pyrR. In still a further aspect, the recombinant pyrR gene is encoded by SEQ ID NO: 1. In another aspect, the recombinant pyrR gene is encoded by a sequence at least 75%, at least 80%, at least 85%, at least 90%, or more preferably at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1. In another aspect, the host cell is a pyrR-mA\ mutant. In still another aspect, the environmental condition is the presence of 5-fluorouracil or 5- fluoroorotic acid in a cell culture medium.

[0017] In one embodiment, the selectable environmental condition is the presence of a cytotoxic compound in a cell culture medium. In a further embodiment, the cytotoxic compound is a base analog. In still a further embodiment, the base analog is 5-fluorouracil (5-FU), 5-fluoroorotic acid, 8-thioxanthine, 6-thioguanine, 8-aza-2,6-diaminopurine (8ADP), or 2-methylpurine (2MP).

[0018] In one embodiment, the recombinant cyanobacterium is light dependent or fixes carbon. In another embodiment, the recombinant cyanobacterium further comprises a nucleic acid sequence encoding enzymatic pathways to synthesize a carbon-based product. In still another embodiment, the recombinant cyanobacterium releases, permeates, or exports the carbon-based product. In yet another embodiment, the carbon-based product is selected from alkanes, alkenes, aliphatic and aromatic alkane and alkene mixtures, alcohols, alkanals and alkenols, alkanoic and alkenoic acids, hydroxy alkanoic acids, keto acids, alkyl alkanoates, ethers, amino acids, lactams, organic polymers, isoprenoids and pharmaceuticals/multifunctional group molecules.

[0019] In one aspect, the host cyanobacterium is selected from Chamaesiphon sp.,

Chroococcus sp., Cyanothece sp., Gloeothece sp., Gloeobacter sp., Microcystis sp.,

Prochlorococcus sp., Acaryochloris sp., Xenococcus sp., Dactylococcopsis sp., Prochloron sp., Chroogloeocystis sp., Coelosphaerium sp., Cyanodictyon sp., Geminocystis sp.,

Johannesbaptistia sp., Limnococcus sp., Radiocystis sp., Rhabdoderma sp., Rubidibacter sp., Snowella sp., Stanieria sp., Sphaerocavum sp., Synechococcus sp., Synechocystis spp., Cyanobacterium sp., Cyanobium sp., Gleocapsa sp., Thermosynechococcus sp.,

Dermocarpella sp., Chroococcidiopsis sp., Myxosarcina sp., Pleurocapsa sp., Borzia sp., Crinalium sp., Geitlerinemia sp., Limnothrix sp., Microcoleus sp., Pseudanabaena sp., Spirulina sp., Starria sp., Symploca sp., Trichodesmium sp., Tychonema sp., Anabaena sp., Anabaenopsis sp., Aphanizomenon sp., Cyanospira sp., Cylindrospermopsis sp., Cylindrospermum sp., Nodularia sp., Nostoc sp., Scytonema sp., Calothrix sp., Rivularia sp., Tolypothrix sp., Chlorogloeopsis sp., Fischer ella sp., Geitleria sp., Iyengariella sp.,

Nostochopsis sp., Stigonema sp., Arthrospira sp., Leptolyngbya sp., Lyngbya sp., Oscillatoria sp., Planktothrix sp., Prochlorothrix sp., and Microcoleus sp.

[0020] In one embodiment, the positively selective marker confers host resistance to a selectable environmental condition. In another embodiment, the positively selective marker is an antibiotic resistance gene. In a further embodiment, the antibiotic resistance gene is a kanamycin resistance gene or a gentamicin resistance gene. In still another embodiment, the positively selective marker is an auxotrophic selectable marker. In one aspect, the negatively selective marker confers host susceptibility to a selectable environmental condition.

[0021] In one aspect, the negatively selective marker is a recombinant gene encoding pyrE, pyrF or pyrF/pyrE. In another aspect, the pyrE gene comprises SEQ ID NO: 6. In still another aspect, the pyrE gene comprises a nucleotide sequence at least 75%, at least 80%, at least 85%o, at least 90%>, or more preferably at least 95%, at least 96%>, at least 97%, at least 98%), or at least 99% identical to SEQ ID NO: 6. In one aspect, the pyrF gene comprises SEQ ID NO: 8. In another aspect, the pyrF gene comprises a nucleotide sequence at least 75%, at least 80%, at least 85%, at least 90%, or more preferably at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 8. In one aspect, the pyrF /pyrE gene comprises SEQ ID NO: 10. In another aspect, the pyrF /pyrE gene comprises a nucleotide sequence at least 75%, at least 80%, at least 85%, at least 90%, or more preferably at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 10. In one embodiment, the host cell is a pyrE-null, pyrF-null or pyrF/pyrE- null mutant. In another embodiment, the selectable environmental condition is the presence of 5-fluoroorotic acid or 5-fluorouracil in the host cell medium.

[0022] In one embodiment, the negatively selective marker is a recombinant gene encoding upp. In one aspect, the upp gene comprises SEQ ID NO: 4. In another aspect, the upp gene comprises a nucleotide sequence at least 75%, at least 80%, at least 85%, at least 90%, or more preferably at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 4. In a further embodiment, the host cell is an upp-mA\ mutant. In a further aspect, the selectable environmental condition is the presence of 5-fluoroorotic acid in the host cell medium.

[0023] In one aspect, the negatively selective marker is a recombinant gene encoding gpt. In one aspect, the gpt gene comprises SEQ ID NO: 12. In another aspect, the gpt gene comprises a nucleotide sequence at least 75%, at least 80%, at least 85%, at least 90%, or more preferably at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 12. In a further aspect, the host cell is a gpt-mA\ mutant. In still another further embodiment, the selectable environmental condition is the presence of 8- thioxanthine in the host cell medium.

[0024] In another aspect, the negatively selective marker is a recombinant gene encoding glnQ. In a further aspect, the host cell is a glnQ- ull mutant. In one embodiment, the selectable environmental condition is the presence of gamma-glutamyl hydrazine in the host cell medium.

[0025] In one embodiment, the negatively selective marker is a recombinant gene encoding a pheS mutant. In another embodiment, the pheS mutant is pheSA294G. In one aspect, the pheS mutant gene comprises SEQ ID NO: 16. In another aspect, the pheS mutant gene comprises a nucleotide sequence at least 75%, at least 80%, at least 85%, at least 90%, or more preferably at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 16. In still another embodiment, the selectable environmental condition is the presence of /?-chloro-phenylalanine.

[0026] In one aspect, the positively selective marker is an antibiotic resistance marker or an auxotrophic marker. In another aspect, the presence of the recombinant gene in the secondary recombinants is identified by sequencing.

[0027] In one embodiment, also provided herein is a method for transforming a host cell, comprising obtaining a host cell whose genome has a null mutation for a gene encoding an enzyme capable of incorporating a toxic compound; introducing the gene into the host cell in combination with a positively selective marker; exposing the host cells to a condition that selects for primary recombinant host cells comprising the positively selective marker, and thus the gene; isolating the primary recombinants; exposing the primary recombinants to the toxic compound to select for secondary recombinants that have lost the gene; and isolating the secondary recombinants. In another embodiment, the positively selective marker is an antibiotic resistance marker. In yet another embodiment, the host cell is a cyanobacterium.

[0028] In one embodiment, a recombinant cyanobacterium is provided, prepared by a counter-selection method, wherein the counter-selection method comprises the steps of: introducing a suicide plasmid into a host cyanobacterium, the suicide plasmid comprising a positively selective marker, a negatively selective marker, and a recombinant gene; selecting for primary recombinants incorporating the positively selective marker; from the primary recombinants, selecting for secondary recombinants that have lost both the negatively selective marker and the positively selective marker; and isolating the secondary recombinants comprising the recombinant gene to obtain the recombinant cyanobacterium. In another embodiment, a recombinant cyanobacterium prepared by any of the methods discussed above is provided.

Brief Description of the Figures

[0029] FIG. 1 depicts two counter-selection strategies. A) Markers are integrated and excised via two single recombination events. B) Markers are integrated and excised via two double recombination events. WT, wild-type nucleotides. Recombinant, recombinant nucleotides to replace the wild-type nucleotides. PSM, positively selective marker. NSM, negatively selective marker. UHR, upstream homologous region. DHR, downstream homologous region. Repeat, two segments of nucleotides with identical sequences to facilitate double recombination. Recombination events and loci are represented by crosses.

[0030] FIG. 2 PheS protein sequence alignment from eight type cyanobacterial strains and E. coli PheS (SEQ ID NO: 24). Cce51142: Cyanothece sp. ATCC 51142 (SEQ ID NO: 17); Syn6803: Synechocystis sp. PCC 6803 (SEQ ID NO: 18); Syp7002: Synechococcus sp. PCC 7002 (SEQ ID NO: 15); Ana7120: Anabaena sp. PCC 7120 (SEQ ID NO: 19); AthNIES-39: Arthrospira platensis NIES-39 (SEQ ID NO: 20); Syp7942: Synechococcus sp. PCC 7942 (SEQ ID NO: 21); ThermoSypBP-1 : Thermosynechococcus elongatus BP-1 (SEQ ID NO: 22); ProchlMED4: Prochlorococcus marinus MED4 (SEQ ID NO: 23).

Detailed Description

Abbreviations and Terms

[0031] The following explanations of terms and methods are provided to better describe the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure. As used herein, "comprising" means "including" and the singular forms "a" or "an" or "the" include plural references unless the context clearly dictates otherwise. For example, reference to "comprising a cell" includes one or a plurality of such cells, and reference to "comprising the thioesterase" includes reference to one or more thioesterase peptides and equivalents thereof known to those of ordinary skill in the art, and so forth. The term "or" refers to a single element of stated alternative elements or a combination of two or more elements, unless the context clearly indicates otherwise. [0032] Unless explained otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Generally, nomenclatures used in connection with, and techniques of, biochemistry, enzymology, molecular and cellular biology, microbiology, genetics, metabolomics, genomics, metabolic engineering, protein engineering, protein and nucleic acid chemistry, computational biology and bioinformatics are those well known and commonly used in the art. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The methods and techniques of the present disclosure 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 specification unless otherwise indicated. See, for example, Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2^nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (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, NY (1990); and the like. The materials, methods, and examples are illustrative only and not intended to be limiting. Other features of the disclosure are apparent from the following detailed description and the claims.

[0033] Selective Marker: Refers to a gene whose presence or absence in the genome of a host cell can be determined by exposure of the host cell to a pre-defined condition. For example, for a selective marker encoding a resistance gene to an antibiotic, the presence of the marker in the genome of the host cell is indicated by survival of the host cell in the presence of the antibiotic. This is an example of a positively selective marker, or a marker whose presence in the genome of the host cell permits survival of the host cell in the presence of the correlated positively selective condition. For a negatively selective marker, its presence in the genome of the host cell will result in the death of the host cell in the presence of the correlated negatively selective condition, resulting in the selection for a host cell that does not have the negatively selective marker.

[0034] Accession Numbers: The accession numbers throughout this description are derived from the NCBI database (National Center for Biotechnology Information) maintained by the National Institute of Health, U.S.A. The accession numbers are as provided in the database on November 1^st, 2010. [0035] Amino acid: Triplets of nucleotides, referred to as codons, in DNA molecules which code for amino acid in a peptide. The term codon is also used for the corresponding (and complementary) sequences of three nucleotides in the mRNA into which the DNA sequence is transcribed. 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., 2nd 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-, a- disubstituted amino acids, N-alkyl amino acids, and other unconventional amino acids may also be suitable components for polypeptides of the present disclosure. Examples of unconventional amino acids include: 4-hydroxyproline, γ-carboxyglutamate, C- Ν,Ν,Ν-trimethyllysine, C -Nacetyllysine, O-phosphoserine, N-acetylserine, N- formylmethionine, 3-methylhistidine, 5 -hydroxy lysine, 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.

[0036] Antibody: 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, but not limited to, harvest from cell culture of native B lymphocytes, harvest from culture of hybridomas, recombinant expression systems and phage display.

[0037] Attenuate: The term 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 R A, ribozymes or antisense sequences that target the gene of interest, or through any other technique known in the art. Attenuation as applied to a nucleotide sequence encoding a gene or gene expression control sequence also refers to attenuation of the protein, and attenuation of a protein also refers to attenuation of the corresponding gene encoding the protein and/or the gene expression control sequence. 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.

[0038] Auxotroph: Auxotrophs (or auxotrophy or auxotrophic organisms) refers to organisms that do not have the ability to synthesize one or more particular compounds that are required for growth, and/or metabolic sustainability sufficient for the organism to maintain a living state or otherwise maintain viability, and is otherwise unable to synthesize or provide to itself intra-cellularly because of natural or genetic engineering means.

[0039] Biofuel: A biofuel refers to any fuel that is derived from a biological source.

Biofuel refers to one or more hydrocarbons, one or more alcohols, one or more fatty esters or a mixture thereof.

[0040] Carbon-based product of interest: A carbon-based product of interest (or carbon-based product) refers to, without limitation or implication that the scope of the claims are limited to the examples set forth herein, desirable end-products or metabolites produced by a biosynthetic pathway of an isolated host cell. The end products or metabolites include, but are not limited to, alkanes (propane, octane), alkenes (ethylene, 1,3-butadiene, propylene, olefins, alkenes, isoprene, lycopene, terpenes) aliphatic and aromatic alkane and alkene mixtures (diesel, jet propellant 8 (JP8)), alkanols and alkenols (ethanol, propanol,

isopropanol, butanol, fatty alcohols, 1,3 -propanediol, 1 ,4-butanediol, polyols, sorbitol, isopentenol), alkanoic and alkenoic acids (acrylate, acrylic acid, adipic acid, itaconic acid, itaconate, docosahexaenoic acid, (DHA), omega-3 DHA, malonic acid, succinate, omega fatty acids), hydroxy alkanoic acids (citrate, citric acid, malate, lactate, lactic acid, 3- hydroxypropionate, 3-hydroxypropionic acid (HP A), hydroxybutyrate), keto acid (levulinic acid, pyruvi acid), alkyl alkanoates (fatty acid esters, wax esters, ε-caprolactone, gamma butyrolactone, γ-valerolactone), ethers (THF), amino acids (glutamate, lysine, serine, aspartate, aspartic acid, glutamic acid, leucine, isoleucine, valine), lactams (pyrrolidones, caprolactam), organic polymers (terephthalate, polyhydroxyalkanoates (PHA), poly-beta- hydroxybutyrate (PHB), rubber), isoprenoids (lanosterol, isoprenoids, carotenoids, steroids), pharmaceuticals/multi-functional group molecules (ascorbate, ascorbic acid, paclitaxel, docetaxel, statins, erythromycin, polyketides, peptides, 7-aminodeacetoxycephalosporanic acid (7-ADCA)/cephalosporin) and metabolites (acetaldehyde).

[0041] Degenerate variant: A degenerate variant of a referenced nucleic acid sequence, as used herein, 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 nucleotide sequences that are not necessarily identical in sequence but that are homologous to one another within one or more particular segments.

[0042] Deletion: The removal of one or more nucleotides from a nucleic acid molecule or one or more amino acids from a protein, where 3 ' and 5 ' ends of the nucleotide sequence may be removed, or the carboxy (C) and amino (N) terminal ends of the protein sequence removed and the nucleotide ends and/or amino/carboxy ends are subsequently re- ligated. A deletion can also refer to the removal of an N- or C- terminal segment, or a 3 ' or 5 ' terminal end of a nucleotide sequence, wherein the translated or transcribed products are shorter in sequence length than the starting sequence.

[0043] Detectable: Capable of having an existence or presence ascertained using various analytical methods as described throughout the description or otherwise known to a person skilled in the art.

[0044] DNA: Deoxyribonucleic acid. DNA is a long chain polymer which includes the genetic material of most living organisms (some viruses have genes including ribonucleic acid, RNA). The repeating units in DNA polymers are four different nucleotides, each of which includes one of the four bases, adenine, guanine, cytosine and thymine bound to a deoxyribose sugar to which a phosphate group is attached.

[0045] Domain: 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, noncontiguous 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.

[0046] Down-regulation: Refers to when a gene is caused to be transcribed at a reduced rate compared to the endogenous gene transcription rate for that gene. In some examples, down-regulation additionally includes a reduced level of translation of the gene compared to the endogenous translation rate for that gene. Methods of testing for down- regulation are well known to those in the art. For example, the transcribed RNA levels can be assessed using RT-PCR, and protein levels can be assessed using SDS-PAGE analysis.

[0047] Downstream: Downstream, when describing the location of a nucleic acid sequence, refers to 1) the nucleic acid sequence 3 ' to a nucleic acid sequence described, and/or 2) the translation, transcription, regulation or other related activity performed on a second nucleic acid sequence occurring after the translation, transcription, regulation or other related activity performed on a first nucleic acid sequence.

[0048] Endogenous: As used herein with reference to a nucleic acid molecule and a particular cell or microorganism, refers to a nucleic acid sequence or peptide that is in the cell and was not introduced into the cell (or its progenitors) using recombinant engineering techniques. For example, a gene that was present in the cell when the cell was originally isolated from nature. A gene is still considered endogenous if the control sequences, such as a promoter or enhancer sequences that activate transcription or translation, have been altered through recombinant techniques.

[0049] Enzyme activity: As used herein, the term an "enzyme activity" refers to an indicated enzyme (e.g., an "alcohol dehydrogenase activity") having measurable attributes in terms of, e.g., substrate specific activity, pH and temperature optima, and other standard measures of enzyme activity as the activity encoded by a reference enzyme (e.g. , alcohol dehydrogenase). Furthermore, the enzyme is at least 60% identical at a nucleic or amino acid level to the sequence of the reference enzyme as measured by a BLAST search.

[0050] Enzyme Classification Numbers (EC): The EC numbers provided throughout this description are derived from the KEGG Ligand database, maintained by the Kyoto Encyclopedia of Genes and Genomics, sponsored in part by the University of Kyoto. The EC numbers are as provided in the database on February 1 , 2008.

[0051] Excise: As used herein, the term "excise" (or "excises" and "excision") with reference to a nucleic acid sequence, refers to the removal of a polynucleotide sequence from a host cell's plasmid or genome from an expressed recombinase protein. The excised polynucleotide sequence can be a complete promoter nucleotide sequence or a partial sequence thereof, a complete protein encoding nucleotide sequence or partial sequence thereof, or a combination of a complete promoter nucleotide sequence and a complete protein encoding nucleotide sequence or partial sequences thereof. Such excision results in the attenuation, disruption or complete absence of the trait conferred by the polynucleotide sequence (for example, as a nucleotide sequence recognized by a protein) or expression of the polynucleotide sequence.

[0052] Exogenous: As used herein, the term exogenous, when used with reference to a nucleic acid molecule and a particular cell or microorganism, refers to a nucleic acid sequence or peptide that was not present in the cell when the cell was originally isolated from nature. For example, a nucleic acid that originated in a different microorganism or synthesized de novo and was engineered into an alternate cell using recombinant DNA techniques or other methods for delivering said nucleic acid is exogenous. Exogenous with reference to a compound or organic compound refers to an extracellular compound or organic compound required for the growth, propagation, sustenance, viability or activity of any metabolic activity, without specific reference to any one metabolic activity. The exogenous compound or organic compound includes those that are subsequently converted by the microorganism to metabolites and/or intermediates necessary or useful for cellular function.

[0053] Expression: The process by which nucleic acid encoded information of a gene is converted into the structures and functions of a cell, such as a protein, transfer R A, or ribosomal RNA. Expressed genes include those that are transcribed into mRNA and then translated into protein and those that are transcribed into RNA but not translated into protein (for example, transfer and ribosomal RNAs).

[0054] Expression control sequence: An expression control sequence as used herein refers to nucleic acid 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.

[0055] Flanking: As used herein, the term "flanking nucleotide sequences" (or

"flanking" as used to describe an upstream and/or downstream nucleotide sequence) describes either the 5' or 3' targeted nucleotide sequences capable of being recognized by a recombinase protein, can be located upstream or downstream of a selectable marker gene, adjacent to a selectable marker gene, or embedded within and/or co-transcribed with the nucleotide sequences encoding the selectable marker gene.

[0056] Fusion Protein: 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 disclosure have particular utility. The heterologous polypeptide included within the fusion protein of the present disclosure 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.

[0057] Genetic element: A genetic element refers to any functional, regulatory or structural nucleic acid or nucleic acid sequence such as, without limitation, ribonucleic acid and deoxyribonucleic acid (RNA, DNA), whether originating from exogenous or endogenous sources, derived synthetically or originating from any organism or virus, including, without limitation, cDNA, genomic DNA, mRNA, RNAi, snRNA, siRNA, miRNA, ta-siRNA, tRNA, double stranded and/or single stranded, co-suppression molecules, ribozyme molecules or related nucleic acid constructs. [0058] Hydrocarbon: The term 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.

[0059] Isolated: An "isolated" nucleic acid or polynucleotide {e.g., R A, DNA or a mixed polymer) refers to 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. The term embraces a nucleic acid or polynucleotide 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 "isolated polynucleotide" 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 "isolated" or "substantially pure" also can be used in reference to recombinant or cloned DNA isolates, chemically synthesized polynucleotide analogs, or polynucleotide analogs that are biologically synthesized by heterologous systems. However, "isolated" does not necessarily require that the nucleic acid or polynucleotide so described has itself been physically removed from its native environment. For instance, an endogenous nucleic acid sequence in the genome of an organism is deemed "isolated" herein if a heterologous sequence (i.e., a sequence that is not naturally adjacent to this endogenous nucleic acid sequence) is placed adjacent to the endogenous nucleic acid sequence, such that the expression of this endogenous nucleic acid sequence is altered. By way of example, a non-native promoter sequence can be substituted {e.g. by homologous recombination) for the native promoter of a gene in the genome of a human cell, such that this gene has an altered expression pattern. This gene would now become "isolated" because it is separated from at least some of the sequences that naturally flank it. A nucleic acid is also considered "isolated" 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 "isolated" if it contains an insertion, deletion or a point mutation introduced artificially, e.g. by human intervention. An "isolated nucleic acid" also includes a nucleic acid integrated into a host cell chromosome at a heterologous site, as well as a nucleic acid construct present as an episome. Moreover, an "isolated nucleic acid" can be substantially free of other cellular material or substantially free of culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. The term also embraces nucleic acid molecules and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acid molecules and proteins.

[0060] Isolated protein or isolated polypeptide: 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.

[0061] Knock-out: Refers to a gene whose level of expression or activity has been reduced to zero. In some examples, a gene may be knocked-out with deletion of some or all of its coding sequence. In other examples, a gene may be knocked-out with an introduction of one or more nucleotides into its open-reading frame, which can result in translation of a non-sense or otherwise non-functional protein product. A type of mutation which results in a knock-out gene is a null mutation.

[0062] Null mutation: refers to a mutation in a gene that leads to its not being transcribed into RNA and/or translated into a functional protein product. For example, a null mutation in a gene that usually encodes a specific enzyme leads to the production of a nonfunctional enzyme or no enzyme at all. A null mutation can result in a a knock-out gene.

[0063] Modified derivative: 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

14 125 32 35 3

such as C, I, P, S, and 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).

[0064] Mutation or Mutated: 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 App lie. 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)).

[0065] Nucleic acid molecule: Nucleic acid molecule refers to both RNA and DNA molecules including, without limitation, cDNA, genomic DNA and mRNA, and also includes synthetic nucleic acid molecules, such as those that are chemically synthesized or

recombinantly produced. The nucleic acid molecule can be double-stranded or

single-stranded, circular or linear. If single-stranded, the nucleic acid molecule can be the sense strand or the antisense strand. 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: l" refers to a nucleic acid, at least a portion which has either (i) the sequence of SEQ. ID NO: l, or (ii) a sequence complimentary to SEQ. ID NO: l . The choice between the two is dictated by the context in which SEQ. ID NO: 1 is used. For instance, if the nucleic acid is used as a probe, the choice between the two is dicated by the requirement that the probe be complimentary to the desired target. Nucleic acid sequences of the present disclosure 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 naturally occurring nucleotides with an analog, inter-nucleotide modifications such as uncharged linkages (for example, methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged linkages (for example, phosphorothioates, phosphorodithioates, etc.), pendant moieties, (for example, polypeptides), intercalators (for example, acridine, psoralen, etc.), chelators, alkylators, and modified linkages (for example, 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 a molecule. Other modifications can include, for example, analogs in which the ribose ring contains a bridging moiety or other structure such as modifications found in "locked" nucleic acids.

[0066] Operably linked: A first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame. Configurations of separate genes that are transcribed in tandem as a single messenger RNA are denoted as operons. Thus placing genes in close proximity, for example in a plasmid vector, under the transcriptional regulation of a single promoter, constitutes a synthetic operon.

[0067] Overexpression: When a gene is caused to be transcribed at an elevated rate compared to the endogenous transcription rate for that gene. In some examples,

overexpression additionally includes an elevated rate of translation of the gene compared to the endogenous translation rate for that gene. Methods of testing for overexpression are well known in the art, for example transcribed RNA levels can be assessed using reverse transcriptase polymerase chain reaction (RT-PCR) and protein levels can be assessed using sodium dodecyl sulfate polyacrylamide gel elecrophoresis (SDS-PAGE) analysis.

Furthermore, a gene is considered to be overexpressed when it exhibits elevated activity compared to its endogenous activity, which may occur, for example, through reduction in concentration or activity of its inhibitor, or via expression of mutant version with elevated activity. In preferred embodiments, when the host cell encodes an endogenous gene with a desired biochemical activity, it is useful to over-express an exogenous gene, which allows for more explicit regulatory control during growth and a means to potentially mitigate the effects of indigenous regulation, which is focused around the native genes explicitly.

[0068] Peptide: 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.

[0069] Percent Sequence Identity: As used herein, 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 nine nucleotides, usually about 20 nucleotides, more usually at least 24 nucleotides, typically at least about 28 nucleotides, more typically at least 32 nucleotides, and preferably at least about 36 or more nucleotides. There a re 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, WI. FASTA provides alignments and percent sequence identity of the regions of the best overlap between query and search sequences. Pearson, Methods. Enzymology. 183:63-98 (1990) (and 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 as provided in GCG Version 6.1 , herein incorporated by reference. Alternatively, sequences can be compared using the computer program Basic Local Alignment Search Tool ("BLAST"; Altschul, et al, J. Mol. Biol.

215:403-410 (1990); Gish and States, Nature Genetics 3:266-272 (1993); Madden, et al, Meth. Enzym. 266: 131-141 (1996); Altschul, et al, Nuc. Acids Res. 25:3389-3402 (1997); Zhang and Madden, Genome Res. 7:649-656 (1997), especially blastx, blastn, tblastx or tblastn (Altschul, et al, Nuc. Acids Res. 25:3389-3402 (1997)).

[0070] 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, blastx, tblastx 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.

[0071] 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).

[0072] 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.

[0073] 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.

[0074] In general, "stringent hybridization" is performed at about 25°C below the thermal melting point (Tm) for the specific DNA hybrid under a particular set of conditions.

"Stringent washing" is performed at temperatures about 5°C lower than the Tm for the specific DNA hybrid under a particular set of conditions. The Tm 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 6xSSC (where 20xSSC 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 0.2xSSC, 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.

[0075] The nucleic acids (also referred to as polynucleotides) of this present disclosure 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,

intemucleotide 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.

[0076] Polypeptide: 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.

[0077] Polypeptide fragment: 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.

[0078] Polypeptide mutant or mutein: 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 naturallyoccurring protein. 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. 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).

[0079] Protomer: As used herein, the term "protomer" refers to a polymeric form of amino acids forming a subunit of a larger oligomeric protein structure. Protomers of an oligomeric structure may be identical or non-identical. Protomers can combine to form an oligomeric subunit, which can combine further with other identical or non-identical protomers to form a larger oligomeric protein.

[0080] Purified: The term purified does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified product preparation, is one in which the product is more concentrated than the product is in its environment within a cell. For example, a purified wax is one that is substantially separated from cellular components (nucleic acids, lipids, carbohydrates, and other peptides) that can accompany it. In another example, a purified wax preparation is one in which the wax is substantially free from contaminants, such as those that might be present following fermentation.

[0081] Recombinant: A recombinant nucleic acid molecule or protein is one that has a sequence that is not naturally occurring, has a sequence that is made by an artificial combination of two otherwise separated segments of sequence, or both. This artificial combination can be achieved, for example, by chemical synthesis or by the artificial manipulation of isolated segments of nucleic acid molecules or proteins, such as genetic engineering techniques. Recombinant is also used to describe nucleic acid molecules that have been artificially manipulated, but contain the same regulatory sequences and coding regions that are found in the organism from which the nucleic acid was isolated.

[0082] The terms "recombinant host cell" ("expression host cell," "expression host system," "expression system," or simply "host cell" or "strain"), as used herein, refers to a cell into which a recombinant vector has been introduced, e.g., a vector comprising acyl-CoA synthase. 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.

[0083] Release: The movement of a compound from inside a cell (intracellular) to outside a cell (extracellular). The movement can be active or passive. When release is active it can be facilitated by one or more transporter peptides and in some examples it can consume energy. When release is passive, it can be through diffusion through the membrane facilitated or not by porters and can be facilitated by continually collecting the desired compound from the extracellular environment, thus promoting further diffusion. Release of a compound can also be accomplished by lysing a cell.

[0084] Segregation: As used herein, segregation refers to the process of enriching a certain allelic locus (e.g. a disrupted or wild-type gene, a modified or intact non-coding genomic region) on microbial genome (including both chromosome and indigenous plasmids) until the allelic locus completely replaces other alleles, by imposing selection pressures such as antibiotic resistance, auxotrophy, etc. Segregation process, as a necessity to stabilize intended genetic traits, is usually applied to microorganisms that have multiple copies of genomic DNA (polyploids). For instance, many cyanobacteria are polypoids.

[0085] Sequential plating: As used herein, sequential plating(s) or sequential host cell plating(s) refers to the process of culturing a host cell in or on one sterile medium, then selecting an isolated colony from the culture medium to culture on a next sterile medium. The culture medium can be, for example, an agar plate. The culture medium may or may not have an integral selective agent, such as an antibiotic, that allows only the culturing of

microorganisms that express an appropriate antibiotic resistance gene. The process can be continued indefinitely to sequentially culture an inoculum from a preceding inoculated growth culture to a next sterile growth culture.

[0086] Specific binding: "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).

[0087] Substantially pure: As used herein, a composition that is a "substantially pure" compound is substantially free of one or more other compounds, i.e., the composition contains greater than 80 vol.%, greater than 90 vol.%, greater than 95 vol.%, greater than 96 vol.%, greater than 97 vol.%, greater than 98 vol.%, greater than 99 vol.%, greater than 99.5 vol.%), greater than 99.6 vol.%>, greater than 99.7 vol.%>, greater than 99.8 vol.%>, or greater than 99.9 vol.%> of the compound; or less than 20 vol.%>, less than 10 vol.%>, less than 5 vol.%), less than 3 vol.%>, less than 1 vol.%>, less than 0.5 vol.%>, less than 0.1 vol.%>, or less than 0.01 vol.% of the one or more other compounds, based on the total volume of the composition. [0088] Suitable fermentation conditions. The term generally refers to fermentation media and conditions adjustable with, H, temperature, levels of aeration, etc., preferably optimum conditions that allow microorganisms to produce carbon-based products of interest. To determine if culture conditions permit product production, the microorganism can be cultured for about 24 hours to one week after inoculation and a sample can be obtained and analyzed. The cells in the sample or the medium in which the cells are grown are tested for the presence of the desired product.

[0089] Up-regulation: Refers to when a gene is caused to be transcribed at an increased rate compared to the endogenous gene transcription rate for that gene. In some examples, up- regulation additionally includes an increased level of translation of the gene compared to the endogenous translation rate for that gene. Methods of testing for up-regulation are well known to those in the art. For example, the transcribed RNA levels can be assessed using RT-PCR, and protein levels can be assessed using SDS-PAGE analysis.

[0090] Vector: The term "vector" as used herein refers 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 refers to a circular double-stranded DNA loop into which additional DNA segments may be ligated. Other vectors include cosmids, bacterial artificial chromosomes (BACs) and yeast artificial chromosomes (YACs). 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 ("integrative vectors") can be integrated wholly or partially into the genome of a host cell upon introduction into the host cell via intended recombination, 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"). A vector can also include one or more selectable marker genes and other genetic elements known in the art. Additionally, a vector can be all of integrative, recombinant and expression vectors. One type of vector is a plasmid suicide vector (or "suicide plasmid") which refers to a plasmid that cannot replicate in a particular host.

[0091] "Wash out": The term wash, wash out or washing out, as used herein in the context of cell cultures and cell lines, refers to the process of segregational loss of plasmids in host cells through the propagation of the host cells over successive generations. The process may require removing selection agent from the cell culture for which resulting selective pressure allows maintenance of the plasmid in the host cells. For example, in the absence of such a selective agent, a wild-type host cell plasmid may preferentially propagate through cell generations over a similar plasmid differing only in having an exogenous engineered gene. Thus, the engineered plasmid will be "washed out" of the cell line and be replaced by the wild type plasmid.

Antibiotic Selectable Genetic Markers

[0092] Expression systems and vectors encoding a target peptide transformed typically include a selection marker. Often, the selection marker is a gene whose product is required for survival during growth cycle of the host cell under selective pressure. Host cells lacking the selection marker, such as cells that have reverted back to the non-transformed or wild type state, are unable to survive. The use of selection markers is intended to ensure that only bacteria containing the expression systems and vectors survive, eliminating competition between the revertants and transformants. The most commonly used selection markers are antibiotic resistance genes. Host cells are grown in a medium supplemented with an antibiotic capable of being degraded by the selected antibiotic resistance gene product. Cells that do not contain the expression vector with the antibiotic resistance gene are killed by the antibiotic. Therefore, in an embodiment of the present disclosure, a selectable genetic marker is an antibiotic resistance gene.

[0093] It is contemplated that selectable antibiotic markers for which resistance genes may be used in the host cells of the present disclosure include amikacin, gentamicin, kanamycin, neomycin, netilmicin, tobramycin, paromomycin, geldanamycin, herbimycin, loracarbef, ertapenem, doripenim, imipenem/cilastatin, meropenem, cefadroxil, cefazolin, cefalotin, cefalexin, cefaclor, cefamandole, cefoxitin, cefprozil, cefuroxime, cefixime, cefdinir, cefditoren, cefoperazone, cefotaxime, cefpodoxime, ceftazidime, ceftibuten, ceftizoxime, ceftriaxone, cefepime, ceftobiprole, teicoplanin, vancomycin, telavancin, clindamycin, lincomycin, daptomycin, azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, troleandomycin, telithromycin, spectinomycin, aztreonam, furazolidone, nitrofurantoin, amoxicillin, ampicillin, azlocillin, carbenicillin, cloxacillin, dicloxacillin, flucloxacillin, mezlocillin, methicillin, nafcillin, oxacillin, penicillin, piperacillin, temocillin, ticarcillin, ciprofloxacin, enoxacin, gatifloxacin, levofloxacin, lomefloxacin, moxifloxacin, nalidixic acid, norfloxacin, ofloxacin, trovafloxacin, grepafloxacin, sparfloxacin,

temafloxacin, mafenide, sulfonamidochrysoiodine, sulfacetamide, sulfadiazine, silver sulfadiazine, sulfamethizole, sulfamethoxazole, sulfanilamide, sulfasalazine, sulfisoxazole, trimethoprim, trimethoprim-sulfamethoxazole, demeclocycline, doxycycline, minocycline, oxytetracycline, tetracycline, clofazimine, dapsone, capreomycin, cycloserine, ethambutol, ethionamide, isoniazid, pyrazinamide, rifampicin, rifabutin, rifapentine, streptomycin, arsphenamine, chloramphenicol, fosfomycin, fusidic acid, linezolid, metronidazole, mupriocin, platensimycin, quinupristin/dalfopristin, rifaximin, thiamphenicol, tigecycline and imidazole.

Auxotrophic Selectable Genetic Markers

[0094] Selection markers may include those that express a metabolite for which the host cell is auxotrophic. Auxotrophy may be engineered into the cell, for example by knock out or attenuation of essential genes, or the wild-type host cell may be auxotrophic. One or more than one metabolic activity may be selected for knock-out or replacement. In the case of native auxotrophy(ies), additional metabolic knockouts or replacements can be provided. Where multiple activities are selected, the auxotrophy-restoring selection markers can be of a biosynthetic-type (anabolic), of a utilization-type (catabolic), or may be chosen from both types. For example, one or more than one activity in a given biosynthetic pathway for the selected compound may be knocked-out; or more than one activity, each from different biosynthetic pathways, may be knocked-out. The corresponding activity or activities are then provided by at least one recombinant vector which, upon transformation into the cell, restores prototrophy to the cell. Thus, in another embodiment of the present disclosure, the selectable genetic marker is an auxotrophic selectable marker.

[0095] It is contemplated for the present disclosure that compounds and molecules whose biosynthesis or utilization can be targeted to produce auxotrophic host cells include: lipids, including, for example, fatty acids; mono- and disaccharides and substituted derivatives thereof, including, for example, glucose, fructose, sucrose, glucose-6-phosphate, and glucuronic acid, as well as Entner-Doudoroff, Pentose Phosphate, Calvin cycle and Kreb's cycle pathway intermediates and products; nucleosides, nucleotides, dinucleotides, including, for example, ATP, dNTP, FMN, FAD, NAD, NADP, nitrogenous bases, including, for example, pyridines, purines, pyrimidines, pterins, and hydro-, dehydro-, and/or substituted nitrogenous base derivatives, such as cofactors, for example, biotin, cobamamide, riboflavine, thiamine; organic acids, glycolysis, Kreb's cycle and amino acid biosynthesis intermediates and products, including, for example, hydroxyacids and amino acids; storage carbohydrates and storage poly(hydroxyalkanoate) polymers, including, for example, cellulose, starch, amylose, amylopectin, glycogen, poly-hydroxybutyrate, and polylactate. In one example, cyanobacteria auxotrophic for vitamin !½ may use a selection marker comprise gene(s) necessary for the production of vitamin B12.

Microbial Host Cells

[0096] Microorganisms include 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.

[0097] A variety of host organisms can be transformed to produce a product of interest. The engineered cell provided by the present disclosure may be derived from eukaryotic plants, industrially important organisms including, but not limited to, Xanthomonas spp.,

Escherichia coli, Corynebacterium spp., Lactobacillus spp., Aspergillus spp., Streptomyces spp., Acetobacter spp., Penicillin spp., Bacillus spp., Pseudomonad spp., Clostridium spp., Zymomonas spp., Salmonella spp., Serratia spp., Erwinia spp., Klebsiella spp., Shigella spp., Enteroccoccus spp., Alcaligenes spp., Paenibacillus spp., Arthrobacter spp., Brevibacterium spp., algae, cyanobacteria, green-sulfur bacteria, green non-sulfur bacteria, purple sulfur bacteria, purple non-sulfur bacteria, extremophiles, yeast, fungi, engineered organisms thereof, and synthetic organisms. In certain related embodiments, the cell is light dependent or fixes carbon. In other related embodiments, the cell has autotrophic activity or photoautotrophic activity. In other embodiments, the cell is photoautotrophic in the presence of light and heterotrophic or mixotrophic in the absence of light. In other related

embodiments, the engineered cell is a plant cell selected from the group consisting of Arabidopsis, Beta, Glycine, Jatropha, Miscanthus, Panicum, Phalaris, Populus, Saccharum, Salix, Simmondsia and Zea. In still other related embodiments, the engineered cell of the present disclosure is an algae and/or cyanobacterial organism selected from the group consisting of 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, Campy lodiscus, Capsosiphon, Carteria, Catena, Cavinula, Centritr actus,

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, Hemitonia, 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, Poly goniochloris, 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, Thermosynechococcus,

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.

[0098] In yet other related embodiments, the engineered cell provided by the present disclosure is derived from a Chloroflexus, Chloronema, Oscillochloris, Heliothrix,

Herpetosiphon, Roseiflexus, and Thermomicrobium cell; a green sulfur bacteria selected from: Chlorobium, Clathrochloris, and Prosthecochloris; a purple sulfur bacteria is selected from: Allochromatium, Chromatium, Halochromatium, Isochromatium, Marichromatium, Rhodovulum, Thermochromatium, Thiocapsa, Thiorhodococcus, and Thiocystis; a purple non-sulfur bacteria is selected from: Phaeospirillum, Rhodobaca, Rhodobacter,

Rhodomicrobium, Rhodopila, Rhodopseudomonas, Rhodothalassium, Rhodospirillum, Rodovibrio, and Roseospira; an aerobic chemolithotrophic bacteria selected from: nitrifying bacteria. 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,

Hydrogenobacter sp., iron and manganese-oxidizing and/or depositing bacteria, Siderococcus sp., and magnetotactic bacteria, Aquaspirillum sp; an archaeobacteria selected from:

methanogenic archaeobacteria, 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 sulfur-Metabolizers such as Thermoproteus sp., Pyrodictium sp., Sulfolobus sp., Acidianus sp., Bacillus subtilis, Saccharomyces cerevisiae, Streptomyces sp., Ralstonia sp., Rhodococcus sp., Cory neb acteria sp., Brevibacteria sp., Mycobacteria sp., and oleaginous yeast.

[0099] In other related embodiments, the engineered cell provided by the present disclosure is derived from an extremophile that can withstand various environmental parameters such as temperature, radiation, pressure, gravity, vacuum, desiccation, salinity, pH, oxygen tension, and chemicals. These include hyperthermophiles, which grow at or above 80°C such as Pyrolobus fumarii; thermophiles, which grow between 60-80°C such as Synechococcus lividis. As used herein, the term thermophilic, thermophile, hyperthermophile or

hyperthermophilic generally refers to any microorganism adapted to have the ability to survive in environments of elevated or extreme temperatures.

[00100] Additionally, there are 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 or barophiles which tolerate pressure of 130 MPa. Hypergravity (e.g., >lg) hypogravity (e.g., <lg) 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.

[00101] Salt tolerant organisms include halophiles (e.g., 2-5 M NaCl) Halobacteriacea and Dunaliella salina. As used and described herein, halophiles or halophilic generally refers to any microorganism adapted to have the ability to survive in environments of elevated or extreme salinity. [00102] H tolerant organisms include alkaliphiles such as Natronobacterium, Bacillus firmus OF4, Spirulina spp. (e.g., pH > 9) and acidophiles such as Cyanidium caldarium, Ferroplasma sp. (e.g., low pH).

[00103] Anaerobes, which cannot tolerate 0₂ such as Methanococcus jannaschii;

microaerophils, which tolerate some 0₂ such as Clostridium and aerobes, which require 0₂ are also contemplated. Gas tolerant organisms, which tolerate pure C0₂, 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) are also contemplated.

[00104] In yet other embodiments, the host cell provided by the present disclosure is derived from 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, and 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).

[00105] In still other embodiments, the engineered cell provided by the present disclosure is a Clostridium ljungdahlii, Clostridium thermocellum, Penicillium chrysogenum, Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pseudomonas fluorescens, or Zymomonas mobilis cell.

[00106] In a more preferred embodiment of the present disclosure, the host cell is selected from Cyanobium sp., Dunaliella sp., Chlamydomonas sp., Spirulina sp., Cyanocethe sp., Chlorella sp., Botryococcus sp., Hamatococcus sp., Chamaesiphon sp., Chlorococcus sp., Gloeothece sp., Gloeobacter sp., Prochlorococcus sp., Acaryochloris sp., Xenococcus sp., Dactylococcopsis sp., Prochloron sp., Chroogloeocystis sp., Coelosphaerium sp.,

Cyanodictyon sp., Geminocystis sp., Johannesbaptistia sp., Limnococcus sp., Radiocystis sp., Rhabdoderma sp., Rubidibacter sp., Snowella sp., Sphaerocavum sp., Synechococcus sp., Synechocystis spp., Cyanobacterium sp., Cyanobium sp., Gleocapsa sp. and

Thermosynechococcus sp.

[00107] In certain embodiments, the host cell provided by the present disclosure is capable of conducting or regulating at least one metabolic pathway selected from the group consisting of photosynthesis, sulfate reduction, methanogenesis, acetogenesis, reductive TCA cycle, Calvin cycle, 3-HPA cycle and 3HP/4HB cycle. [00108] A common theme in selecting or engineering a suitable organism is autotrophic fixation of carbon, such as C0₂ to products. This would cover photosynthesis and methanogenesis. Acetogenesis, encompassing the three types of C0₂ fixation; Calvin cycle, acetyl CoA pathway and reductive TCA pathway is also covered. The capability to use carbon dioxide as the sole source of cell carbon (autotrophy) is found in almost all major groups ofprokaryotes. The C0₂ 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 CO₂ 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 C0₂ fixation pathway in almost all aerobic autotrophic bacteria, for example, the cyanobacteria.

Production of Carbon-Based Products

[00109] In various embodiments of the present disclosure, desired hydrocarbons and/or alcohols of certain chain length or a mixture thereof can be produced . In certain aspects, the host cell produces at least one of the following carbon-based products of interest: I~ dodecanol, 1- letradecanol, 1 -pentadecanol, n~tridecane, n-tetradecane, 15:1 n~pentadecane, n-pentadecane, 16: 1 /7-hexadecene, n-hexadecane, 17: 1 n-heptadeeene, n-heptadecane, 16:1 n-hexadecen-ol, /7-hexadecan-l-ol and n-octadecen-l-ol, as show in the Examples herein. In other aspects, the carbon chain length ranges from C10 to C¾>. Accordingly, the present disclosure provides production of various chain lengths of alkanes, alkenes and alkanols suitable for use as fuels & chemicals,

[00110] In other embodiments of the present disclosure, a carbon-based product of interest (or carbon-based product) is produced by a biosynthetic pathway of an isolated host cell. The end products or metabolites include, but are not limited to, alkanes (propane, octane), alkenes (ethylene, 1,3-butadiene, propylene, olefins, alkenes, isoprene, lycopene, terpenes) aliphatic and aromatic alkane and alkene mixtures (diesel, jet propellant 8 (JP8)), alkanols and alkenols (ethanol, propanol, isopropanol, butanol, fatty alcohols,

1,3-propanediol, 1 ,4-butanediol, polyols, sorbitol, isopentenol), alkanoic and alkenoic acids (acrylate, acrylic acid, adipic acid, itaconic acid, itaconate, docosahexaenoic acid, (DHA), omega-3 DHA, malonic acid, succinate, omega fatty acids), hydroxy alkanoic acids (citrate, citric acid, malate, lactate, lactic acid, 3-hydroxypropionate, 3-hydroxypropionic acid (HP A), hydroxybutyrate), keto acid (levulinic acid, pyruvi acid), alkyl alkanoates (fatty acid esters, wax esters, ε-caprolactone, gamma butyrolactone, γ-valerolactone), ethers (THF), amino acids (glutamate, lysine, serine, aspartate, aspartic acid, glutamic acid, leucine, isoleucine, valine), lactams (pyrrolidones, caprolactam), organic polymers (terephthalate,

polyhydroxyalkanoates (PHA), poly-beta-hydroxybutyrate (PHB), rubber), isoprenoids (lanosterol, isoprenoids, carotenoids, steroids), pharmaceuticals/multi-functional group molecules (ascorbate, ascorbic acid, paclitaxel, docetaxel, statins, erythromycin, polyketides, peptides, 7-aminodeacetoxycephalosporanic acid (7-ADCA)/cephalosporin) and metabolites (acetaldehyde).

[00111] In preferred aspects, the methods provide culturmg 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 present disclosure enables production of various defined chain length of hydrocarbons and alcohols, the secreted products are easily recovered or separated. The products of the present disclosure, therefore, can be used directly or used with minimal processing.

[00112] In various embodiments, compositions produced by the methods of the disclosure are used as fuels. Such fuels comply with ASTM standards, for instance, standard specifications for diesel fuel oils D 975-0%. and Jet A, Jet A-I 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 present disclosure produce an array of hydrocarbons, such as Co-Cr; or C10-C15 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.

Propagation of Selected Microoganisms

[00113] Methods for cultivation of photosynthetic organisms in liquid media and on agarose-containing plates are well known to those skilled in the art (see, e.g. , US 7,785,861 , and various websites associated with ATCC and with the Institute Pasteur). For example, Synechococcus sp. PCC 7002 cells (available from the Pasteur Culture Collection of

Cyanobacteria) are cultured in BG-1 1 medium (17.65 mM NaN0₃, 0.18 mM K₂HP04, 0.3 mM MgS0₄, 0.25 mM CaCl₂, 0.03 mM citric acid, 0.03 mM ferric ammonium citrate, 0.003 mM EDTA, 0.19 mM Na₂C0₃, 2.86 mg/L H₃B0₃, 1.81 mg/L MnCl₂, 0.222 mg/L ZnS0₄, 0.390 mg/L Na₂Mo0₄, 0.079 mg/L CuS0₄, and 0.049 mg/L Co(N0₃)2, pH 7.4)

supplemented with 16 μg/L biotin, 20 mM MgS0₄, 8 mM KCl, and 300 mM NaCl (see, e.g., website associated with the Institute Pasteur, and Price GD, Woodger FJ, Badger MR, Howitt SM, Tucker L. "Identification of a SulP-type bicarbonate transporter in marine

cyanobacteria. Proc Natl. Acad. Sci. USA (2004) 101(52): 18228-33). Typically, cultures are maintained at 28°C and bubbled continuously with 5% C0₂ under a light intensity of 120 μιηοΐ photons/m²/s. Alternatively, Synechococcus sp. PCC 7002 cells are cultured in A⁺ medium as previously described [Frigaard NU et al. (2004) "Gene inactivation in the cyanobacterium Synechococcus sp. PCC 7002 and the green sulfur bacterium Chlorobium tepidum using in vitro-made DNA constructs and natural transformation," Methods Mol. Biol, 274:325-340].

[00114] Thermosynechococcus elongatus BP-1 (available from the Kazusa

DNAResearch Institute, Japan) is propagated in BG11 medium supplemented with 20 mM TES-KOH (pH 8.2) as previously described [Iwai M, Katoh H, Katayama M, Ikeuchi M. "Improved genetic transformation of the thermophilic cyanobacterium,

Thermosynechococcus elongatus BP-1." Plant Cell Physiol (2004). 45(2): 171-175)].

Typically, cultures are maintained at 50°C and bubbled continuously with 5% C0₂ under a light intensity of 38 μιηοΐ photons m^~2 s^"1. T. elongatus BP-1 can be grown in A⁺ medium also.

[00115] Chlamydomonas reinhardtii (available from the Chlamydomonas Center culture collection maintained by Duke University, Durham, North Carolina,) are grown in minimal salt medium consisting of 143 mg/L K₂HP0₄, 73 mg/L KH₂P0₄, 400 mg/L

NH₄N0₃, 100 mg/L MgS0₄-7H₂0, 50 mg/L CaCl₂-2 H₂0, 1 mL/L trace elements stock, and 10 mL/L 2.0 M MOPS titrated with Tris base to pH 7.6 as described (Geraghty AM,

Anderson JC, Spalding MH. "A 36 kilodalton limiting-C0₂ induced polypeptide of

Chlamydomonas is distinct from the 37 kilodalton periplasmic anhydrase." Plant Physiol (1990). 93: 116-121). Typically, cultures are maintained at 24°C and bubbled with 5% C0₂ in air, under a light intensity of 60 μιηοΐ photons m^~2 s^"1.

[00116] The above define typical propagation conditions. As appropriate, incubations are performed using alternate media or gas compositions, alternate temperatures (5 - 75°C), and/or light fluxes (0-5500 μιηοΐ photons m^"2 s^"1). [00117] Light is delivered through a variety of mechanisms, including natural illumination (sunlight), standard incandescent, fluorescent, or halogen bulbs, or via propagation in specially-designed illuminated growth chambers (for example Model LI 15 Illuminated Growth Chamber (Sheldon Manufacturing, Inc. Cornelius, OR). For experiments requiring specific wavelengths and/or intensities, light is distributed via light emitting diodes (LEDs), in which wavelength spectra and intensity can be carefully controlled (Philips).

[00118] Carbon dioxide is supplied via inclusion of solid media supplements (i.e., sodium bicarbonate) or as a gas via its distribution into the growth incubator or media. Most experiments are performed using concentrated carbon dioxide gas, at concentrations between 1 and 30%, which is directly bubbled into the growth media at velocities sufficient to provide mixing for the organisms. When concentrated carbon dioxide gas is utilized, the gas originates in pure form from commercially-available cylinders, or preferentially from concentrated sources including off-gas or flue gas from coal plants, refineries, cement production facilities, natural gas facilities, breweries, and the like.

Transformation of Selected Microorganisms

[00119] Preferably, Synechococcus sp. PCC 7002 cells are transformed according to the optimized protocol previously described [Essich ES, Stevens Jr., E, Porter RD

"Chromosomal Transformation in the Cyanobacterium Agmenellum quadruplicatum". J Bacteriol (1990). 172(4): 1916-1922]. Cells are grown in Medium A (18 g/L NaCl, 5 g/L MgS0₄. 7 H₂0, 30 mg/L Na₂EDTA, 600 mg/L KC1, 370 mg/L CaCl₂. 2 H₂0, 1 g/L NaN0₃, 50 mg/L KH₂P0₄, 1 g/L Trizma base pH 8.2, 4 μg/L Vitamin B_i2, 3.89 mg/L FeCl₃. 6 H₂0, 34.3 mg/L H₃B0₃, 4.3 mg/L MnCl₂. 4 H20, 315 μg/L ZnCl₂, 30 μg/L Mo0₃, 3 μg/L CuS0₄. 5 H₂0, 12.2 μg/L CoCl₂. 6 H₂0) [Stevens SE, Patterson COP, and Myers J. "The production of hydrogen peroxide by green algae: a survey." J. Phycology (1973). 9:427-430] plus 5g/L of NaN0₃ to approximately 108 cells/ mL. Nine volumes of cells are mixed with 1 volume of 1-10 μg/mL DNA in 0.15 M NaCl/0.015 M Na₃citrate and incubated at 27-30 °C for 3 hours before addition of 1 volume of DNasel to a final concentration of 10 μg/mL. The cells are plated in 2.5mL of 0.6% medium A overlay agar that was tempered at 45°C and incubated. Cells are challenged with antibiotic by under-laying 2.0 mL of 0.6% medium A agar containing appropriate concentration of antibiotic with a sterile Pasteur pipette.

Transformants are picked 3-4 days later. Selections are typically performed using 200 μg/ml kanamycin, 8 μg/ml chloramphenicol, 10 μg/ml spectinomycin on solid media, whereas 150 μg/ml kanamycin, 7 μg/ml chloramphenicol, and 5 μg/ml spectinomycin are employed in liquid media. [00120] More preferably, T. elongatus BP-1 cells are transformed according to the optimized protocol previously described {vide supra).

[00121] E. coli are transformed using standard techniques known to those skilled in the art, including heat shock of chemically competent cells and electroporation (Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology volume 152 Academic Press, Inc., San Diego, Calif; Sambrook et al. (1989) Molecular Cloning—A Laboratory Manual (2nd ed.) Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, N.Y.; and Current Protocols in Molecular Biology, F. M. Ausubel et al, eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (through and including the 1997 Supplement)).

[00122] The biosynthetic pathways as described herein are first tested and optimized using episomal plasmids described above. Non-limiting optimizations include promoter swapping and tuning, ribosome binding site manipulation, alteration of gene order {e.g., gene ABC versus BAC, CBA, CAB, BCA), co-expression of molecular chaperones, random or targeted mutagenesis of gene sequences to increase or decrease activity, folding, or allosteric regulation, expression of gene sequences from alternate species, codon manipulation, addition or removal of intracellular targeting sequences such as signal sequences, and the like.

[00123] Each gene is optimized individually, or alternately, in parallel. Functional promoter and gene sequences are subsequently integrated into the E. coli chromosome to enable stable propagation in the absence of selective pressure (i.e., inclusion of antibiotics) using standard techniques known to those skilled in the art.

[00124] The examples below are provided herein for illustrative purposes and are not intended to be restrictive.

Examples

Example 1 : pyrR/5-FU counter-selection system:

[00125] A unicellular coastal/marine cyanobacterium Synechococcus sp. PCC 7002

(JCC138) is used in this example for demonstration purposes. The example could also be extended to the entire cyanobacteria phylum, including fresh- water, marine, unicellular, filamentous, heterocystous, and non-heterocystous cyanobacteria.

[00126] The pyrR gene {i.e., A1692) (SEQ ID NO: 1) from Synechococcus sp. strain

PCC 7002, encoding a putative uracil phosphoribosyltransferase (SEQ ID NO: 2), was selected as a potential counterselective marker. Phosphoribosyltransferases incorporate free uracil, cytosine, thymine, guanine and adenine bases, as well as cytotoxic base analogs, such as 5-fluorouracil (5-FU), 8-thioxanthine, 6-thioguanine, 8-aza-2,6-diaminopurine (8ADP), 2- methylpurine (2MP), etc. Due to the essential function of these enzymes in

pyrimidine/purine salvage pathways, they are well conserved in all cyanobacteria (Table 2).

[00127] The pyrRI5-¥ system was developed using gene mutants defective in uracil base salvage through pyrR. Gene mutations in these pathways confer resistance to cytotoxic uracil base analogs in the null mutants. The AA1692 Synechococcus sp. strain PCC 7002 mutant with deletional inactivation of A 1692 was constructed according to standard recombinant techniques and fully segregated. Wild-type (i.e. , WT) and ΔΑ1692 mutant strains of Synechococcus sp. PCC 7002 were probed for their sensitivities to the toxic uracil analog, 5-fluorouracil (i.e., 5-FU). The WT strain was determined to be sensitive to 5-FU with a minimum inhibitory concentration (i.e., MIC) at 1 μg/ml. In contrast, the AA1692 mutant strain was determined to be relatively insensitive to 5-FU with a MIC greater than 50 μg/ml (Table 1). The result indicates that A 1692 functions as a genuine uracil

phosphoribosyltransferase to incorporate toxic 5-FU. Since the MIC window of 5-FU between the WT strain and the AA1692 mutant strain is large, a modular Al 692 expression cassette with promoter driven A 1692 expression was tested for its capability to serve as a strong counterselective marker when 5-FU is present at a concentration above ^g/ml (i.e. , the MIC of the WT strain) as described below.

Table 1

[00128] In the next step, A1692 was expressed by a strong constitutive promoter

(P_CPCB) at the Idh locus in the AA1692 mutant background to create the strain

AA1692::kan _Aldh::[P_cpcB-A1692]genf. The strain was fully segregated. A plate-based 5- FU sensitivity test was conducted to investigate whether expression of A 1692 in the AA1692 mutant would make the strain regain 5-FU sensitivity. 10⁶, 10⁵ and 10⁴ fresh cells of WT, AA1692 mutant and three isolates (#1 , #2, #6) of the strain AA1692: :kan _Aldh: : [P_CPCB- A1692]genf wQXQ placed on plates of A⁺, A⁺ with 20 μg/ml 5-FU and A⁺ with 40 μg/ml 5- FU. All strains grew on the non-selective A⁺ plate while only the AA1692 mutant grew on 5- FU containing A⁺ plates. The WT and three isolates of strain AA1692::kan _Aldh::[P_CpcB- A1692]genf did not grow on 5-FU containing A⁺ plates. The result shows that an A 1692 expression cassette functions as an effective counterselective marker in the presence of 5-FU.

[00129] Integrative or non-integrative suicide vectors such as those in Figure 1 will be constructed to contain homologous regions for targeted recombination, a positively selective marker, and a cassette for ectopic expression of a negatively selective marker A 1692. The vectors will be introduced into AA1692 mutants. Targeted recombination occurs via either a single crossover or a double crossover event, depending on vector designs, to create mutants with the gene of interest disrupted. Mutants having undergone recombination with the suicide vector are selected using the resistance (e.g. , antibiotic resistance) conferred by the positively selective marker. Segregated mutants are confirmed by PCR and DNA

sequencing.

[00130] Complementation of the inactivated genes by ectopic expression of corresponding functional copies re-sensitizes cells to the pyrimidine/purine base analogs, creating a cell sensitive to a negatively selective condition. A cytotoxic base analog, 5-FU, is added to the culture medium as a counterselective condition to select against cyanobacterial expressing an A 1692 selective marker. This will simultaneously lead to removal of the positively selective marker (e.g., the antibiotic marker), due to recombination occurred at either a single homologous site or two flanking homologous sites. Segregated markerless mutants will be confirmed by PCR and DNA sequencing.

[00131] In summary, an ectopically expressed A1692 gene will serve as a

counterselectable marker being forced out in a AA1692 background under 5-FU selection. Inactivation of the A 1692 gene in a background of the A 1692 -null mutant will increase cell resistance to 5-FU, and will facilitate the counter-selective marker removal process.

Example 2: upp/5-FXJ counter-selection system

[00132] Other putative phosphoribosyltransferases , such as the upp gene (SEQ ID

NO: 4) from Synechococcus sp. PCC 7002 may also be knocked out and inserted

recombinantly as a counterselectable marker.

[00133] Null mutants of upp^~ in cyanobacteria are constructed. Gene mutations in these pathways confer resistance to cytotoxic pyrimidine/purine base analogs in the null mutants. Sensitivity of wild-type cells and insensitivity of mutant cells to cytotoxic base analogs will be confirmed. Integrative or non-integrative suicide vectors such as those in Figure 1 will be constructed to contain homologous regions for targeted recombination, a positively selective marker, and a cassette for ectopic expression of a negatively selective marker upp⁺ . The vectors will be introduced into the corresponding null mutants. Targeted recombination occurs via either a single crossover or a double crossover event, depending on vector designs, to create mutants with the gene of interest disrupted. Mutants having undergone recombination with the suicide vector are selected using the resistance {e.g. , antibiotic resistance) conferred by the positively selective marker. Segregated mutants are confirmed by PCR and DNA sequencing.

[00134] Complementation of the inactivated genes by ectopic expression of corresponding functional copies re-sensitizes cells to the pyrimidine/purine base analogs, creating a cell sensitive to a negatively selective condition. A cytotoxic base analog, 5-FU, is added to the culture medium as a counterselective condition to select against cyanobacterial expressing a upp⁺ selective marker. This will simultaneously lead to removal of the positively selective marker {e.g., the antibiotic marker), due to recombination occurred at either a single homologous site or two flanking homologous sites. Segregated markerless mutants will be confirmed by PCR and DNA sequencing.

[00135] In summary, an ectopically expressed upp⁺gQUQ will serve as counterselectable markers being forced out in a upp^~ background under 5-FU selection. Inactivation of the upp in background of the upp- ull mutant will increase cell resistance to 5-FU, and will facilitate the counter-selective marker removal process. It is noticed that, besides upp genes, cyanobacteria possess additional putative uracil phosphoribosyltransferase genes that may be used in the system above.

Example 3: pyrEF/5-FOA counter-selection system

[00136] A unicellular coastal/marine cyanobacterium Synechococcus sp. PCC 7002

(JCC138) could be used in the example for demonstration purposes. The example could also be extended to the entire cyanobacteria phylum, including fresh-water, marine, unicellular, filamentous, heterocystous, and non-heterocystous cyanobacteria.

[00137] The pyrEF/5-FOA system is developed using gene mutants defective in pyrimidine/purine base salvage pathways. The pyrF gene (e.g., SEQ ID NO: 8) {URA3 in yeast) encodes an orotidine 5 '-phosphate decarboxylase (e.g., SEQ ID NO: 9), which catalyzes decarboxylation of orotidine monophosphate (OMP) to uridine monophosphate (UMP). The pyrE gene (e.g., SEQ ID NO: 6) {URA5 in yeast) encodes orotate

phosphoribosyltransferase (OPT) (e.g., SEQ ID NO: 7), which salvages orotic acid to form OMP. The orotic acid analog 5-fluoroorotic acid (5-FOA) is a toxic substrate for PyrF/PyrE (e.g., SEQ ID NO: 11), thus cells having the wild-type pyrF/pyrE (e.g., SEQ ID NO: 10) is sensitive to growth inhibition rendered by medium containing 5-FOA, whereas pyrE or pyrF mutant becomes incapable of uptaking exogenously uracil derivatives and thus become resistant to 5-FOA. Due to essential functions of these genes involved in pyrimidine/purine salvage pathways, they are quite conserved in all cyanobacteria (Table 1).

[00138] Null mutants of pyrE ^', pyrF, or pyrF/pyrE in cyanobacteria are constructed.

Gene mutations in these pathways confer resistance to cytotoxic pyrimidine/purine base analogs in the null mutants. Sensitivity of wild-type cells and insensitivity of mutant cells to cytotoxic base analogs will be confirmed. Integrative or non-integrative suicide vectors such as those in Figure 1 will be constructed to contain homologous regions for targeted recombination, a positively selective marker, and a cassette for ectopic expression of a negatively selective marker pyrE⁺, pyrF⁺ , or pyrF/pyrE⁺ . The vectors will be introduced into the corresponding null mutants. Targeted recombination occurs via either a single crossover or a double crossover event, depending on vector designs, to create mutants with the gene of interest disrupted. Mutants having undergone recombination with the suicide vector are selected using the resistance {e.g., antibiotic resistance) conferred by the positively selective marker. Segregated mutants are confirmed by PCR and DNA sequencing.

[00139] Complementation of the inactivated genes by ectopic expression of corresponding functional copies re-sensitizes cells to the pyrimidine/purine base analogs, creating a cell sensitive to a negatively selective condition. A cytotoxic base analog, 5-FOA, is added to the culture medium as a counterselective condition to select against

cyanobacterial expressing a pyrE⁺, pyrF⁺ , or pyrF/pyrE⁺ selective marker. This will simultaneously lead to removal of the positively selective marker {e.g., the antibiotic marker), due to recombination occurred at either a single homologous site or two flanking homologous sites. Segregated markerless mutants will be confirmed by PCR and DNA sequencing.

[00140] In summary, ectopically expressed pyrE⁺ or pyrF⁺ gene will serve as counterselectable markers being forced out in a pyrE^' or pyrF background under 5-FOA selection. Inactivation of the pyrF/pyrE in background of the pyrF or pyrE-mA\ mutant will increase cell resistance to 5-FOA, and will facilitate the counter-selective marker removal process.

Example 4: gj?t/8-thioxanthine counter-selection system

[00141] A unicellular coastal/marine cyanobacterium Synechococcus sp. PCC 7002

(JCC138) could be used in the example for demonstration purposes. The example could also be extended to the entire cyanobacteria phylum, including fresh-water, marine, unicellular, filamentous, heterocystous, and non-heterocystous cyanobacteria.

[00142] The g/?t/8-thioxanthine system is developed using gene mutants defective in pyrimidine/purine base salvage pathways. The gpt gene (e.g., SEQ ID NO: 12) encodes purine phosphoribosyltransferase (e.g., SEQ ID NO: 13). This phosphoribosyltransferase incorporates free uracil, guanine and adenine bases, as well as their cytotoxic base analogs, such as 5-fluorouracil (5-FU), 8-thioxanthine, 6-thioguanine, 8-aza-2,6-diaminopurine (8ADP), 2-methylpurine (2MP), etc. As a result, the gpf mutants become insensitive to the corresponding cytotoxic base analogs. Due to essential functions of these genes involved in pyrimidine/purine salvage pathways, they are well conserved in all cyanobacteria (Table 1).

[00143] Null mutants of gpf in cyanobacteria are constructed. Gene mutations in these pathways confer resistance to cytotoxic pyrimidine/purine base analogs in the null mutants. Sensitivity of wild-type cells and insensitivity of mutant cells to cytotoxic base analogs will be confirmed. Integrative or non-integrative suicide vectors such as those in Figure 1 will be constructed to contain homologous regions for targeted recombination, a positively selective marker, and a cassette for ectopic expression of a negatively selective marker gpt⁺ . The vectors will be introduced into the corresponding null mutants. Targeted recombination occurs via either a single crossover or a double crossover event, depending on vector designs, to create mutants with the gene of interest disrupted. Mutants having undergone

recombination with the suicide vector are selected using the resistance {e.g. , antibiotic resistance) conferred by the positively selective marker. Segregated mutants are confirmed by PCR and DNA sequencing.

[00144] Complementation of the inactivated genes by ectopic expression of corresponding functional copies re-sensitizes cells to the pyrimidine/purine base analogs, creating a cell sensitive to a negatively selective condition. A cytotoxic base analog, 8- thioxanthine, is added to the culture medium as a counterselective condition to select against cyanobacterial expressing a gpt⁺ selective marker. This will simultaneously lead to removal of the positively selective marker {e.g., the antibiotic marker), due to recombination occurred at either a single homologous site or two flanking homologous sites. Segregated markerless mutants will be confirmed by PCR and DNA sequencing.

[00145] In summary, ectopically expressed gpt⁺ gene will serve as a counterselectable marker being forced out in a gpf background under 8-thioxanthine selection. Inactivation of the gpt in background of the gpt-mA\ mutant will increase cell resistance to 8-thioxanthine, and will facilitate the counter-selective marker removal process. Example 5: glnQ/GGH counter-selection system

[00146] A unicellular coastal/marine cyanobacterium Synechococcus sp. PCC 7002

(JCC138) could be used in the example for demonstration purposes. The example could also be extended to the entire cyanobacteria phylum, including fresh-water, marine, unicellular, filamentous, heterocystous, and non-heterocystous cyanobacteria.

[00147] The endogenous glnQ gene in Streptococci spp. encodes a glutamine transporter that has relaxed specificity to uptake certain glutamine analogs, such as gamma- glutamyl hydrazide (GGH), and is required for GGH-induced growth inhibition. Some cyanobacteria possess glnQ genes that share significant homology with Streptococci glnQ in amino acid sequences (Table 1). A glnQ/GGH counter-selection system will be developed in cyanobacterium.

[00148] Null mutants of glnQ^' in cyanobacteria are constructed. Gene mutations in these pathways confer resistance to cytotoxic glutamine analogs in the null mutants.

Sensitivity of wild-type cells and insensitivity of mutant cells to cytotoxic glutamine analogs will be confirmed. Integrative or non-integrative suicide vectors such as those in Figure 1 will be constructed to contain homologous regions for targeted recombination, a positively selective marker, and a cassette for ectopic expression of a negatively selective marker glnQ⁺ . The vectors will be introduced into the corresponding null mutants. Targeted recombination occurs via either a single crossover or a double crossover event, depending on vector designs, to create mutants with the gene of interest disrupted. Mutants having undergone recombination with the suicide vector are selected using the resistance {e.g. , antibiotic resistance) conferred by the positively selective marker. Segregated mutants are confirmed by PCR and DNA sequencing.

[00149] Complementation of the inactivated genes by ectopic expression of corresponding functional copies re-sensitizes cells to the glutamine analogs, creating a cell sensitive to a negatively selective condition. A cytotoxic glutamine analog, gamma-glutamyl hydrazide (GGH), is added to the culture medium as a counterselective condition to select against cyanobacterial expressing a glnQ⁺ selective marker. This will simultaneously lead to removal of the positively selective marker {e.g., the antibiotic marker), due to recombination occurred at either a single homologous site or two flanking homologous sites. Segregated markerless mutants will be confirmed by PCR and DNA sequencing.

[00150] In summary, ectopically expressed glnQ⁺ gene will serve as a

counterselectable marker being forced out in a glnQ^' background under GGH selection. Inactivation of the glnQ in background of the glnQ-mA\ mutant will increase cell resistance to GGH, and will facilitate the counter-selective marker removal process.

Example 6: pheS*/p-Cl-PhQ counter-selection system

[00151] A unicellular coastal/marine cyanobacterium Synechococcus sp. PCC 7002

(JCC 138) could be used in the example for demonstration purposes. The example could also be extended to the entire cyanobacteria phylum, including fresh-water, marine, unicellular, filamentous, heterocystous, and non-heterocystous cyanobacteria.

[00152] PheS protein sequence alignment from eight type cyanobacterial strains and E. coli PheS revealed that the critical Ala²⁹⁴ residues are conserved in cyanobacterial PheS (Figure 2, shown in a rectangle). A missense mutation introduced into the phenylalanyl- tR A synthetases at Ala²⁹⁴ (pheSA294G) will relax the enzyme to recognize and incorporate halogenized phenylalanine derivatives, such as /?-chloro-phenylalanine (p-Cl-Phe).

[00153] Integrative or non-integrative suicide vectors such as those in Figure 1 will be constructed to contain homologous regions for targeted recombination, a positively selective marker, and a cassette for ectopic expression of a negatively selective marker pheSA294G. The vectors will be introduced into the cyanobacteria. Targeted recombination occurs via either a single crossover or a double crossover event, depending on vector designs, to create mutants with the gene of interest disrupted. Mutants having undergone recombination with the suicide vector are selected using the resistance (e.g., antibiotic resistance) conferred by the positively selective marker. Segregated mutants are confirmed by PCR and DNA sequencing.

[00154] Ectopic expression of the pheSA294G copies sensitizes cells to the

halogenized phenylalanine derivatives, creating a cell sensitive to a negatively selective condition. Ectopic expression of pheSA294G in a cyanobacterial strain on medium containing /?-Cl-Phe will lead to growth inhibition, likely due to accumulative expression of non-functional proteins with the Phe residues massively replaced by /?-Cl-Phe. /?-Cl-Phe is added to the culture medium as a counterselective condition to select against cyanobacterial expressing a pheSA294G selective marker. This will simultaneously lead to removal of the positively selective marker (e.g., the antibiotic marker), due to recombination occurred at either a single homologous site or two flanking homologous sites. Segregated markerless mutants will be confirmed by PCR and DNA sequencing. [00155] In summary, the cyanobacterial strains having the pheSA294G analogs will then be used as a host-genotype-independent counterselective marker using /?-Cl-Phe as the counter-selection agent.

Table 2

Claims

What is claimed is:

1. A method for preparing a recombinant cyanobacterium, comprising:

a. introducing a suicide plasmid into a host cyanobacterium, said suicide plasmid comprising a positively selective marker, a negatively selective marker, and a recombinant gene, wherein said negatively selective marker confers host susceptibility to a selectable environmental condition;

b. selecting for primary recombinants incorporating the positively selective marker; c. culturing said primary recombinants in the presence of said selectable

environmental condition, thereby selecting for secondary recombinants that have lost the negatively selective marker and the positively selective marker; and d. isolating the secondary recombinants comprising the recombinant gene to obtain said recombinant cyanobacterium.

2. The method of claim 1, wherein if said host cyanobacterium natively comprises a corresponding negatively selective marker, the method further comprises the step of removing or reducing expression of said corresponding negatively selective marker prior to introducing said suicide plasmid into said host cyanobacterium.

3. The method of claim 2, wherein said step of removing or reducing expression of said corresponding negatively selective marker creates a modified cyanobacterium having a null mutation, wherein said host cyanobacterium having the null mutation has a decreased sensitivity to a negatively selective condition as compared to a host cyanobacterium without said null mutation.

4. The method of claim 1, wherein said negatively selective marker comprises a gene that has been knocked out in the recombinant cyanobacterium.

5. The method of any of claims 1-4, wherein said negatively selective marker comprises a gene expressing an enzyme capable of incorporating a cytotoxic compound.

6. The method of claim 5, wherein said cytotoxic compound is a nucleobase analog.

7. The method of claim 6, wherein said nucleobase analog is a halogenic pyrimidine or purine, or a precursor thereof.

8. The method of claim 6, wherein said nucleobase analog is selected from: 5- fluorouracil (5-FU), 5-fluoroorotic acid (5-FOA), 8-thioxanthine, 6-thioguanine, 8- aza-2,6-diaminopurine (8ADP), 2-methylpurine (2MP).

9. The method of claim 5, wherein said gene expresses an enzyme from the enzyme category EC 2.4.2.x, EC 3.6.3.x, or EC 6.1.1.20.

55

10. The method of claim 5, wherein said gene is selected from: pyrR, upp, pyrF/pyrE, pyrE, pyrF, gpt, glnQ, and pheSA294G.

11. The method of claim 5, wherein said gene is encoded by a polynucleotide comprising a sequence selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, and a polynucleotide sequence encoding SEQ ID NO: 16.

12. The method of claim 5, wherein said gene expresses a homo log of a polypeptide sequence selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, and SEQ ID NO: 16.

13. The method of claim 5, wherein said negatively selective marker is a recombinant gene encoding a uracil phosphoribosyltransferase.

14. The method of claim 13, wherein said recombinant gene is pyrR.

15. The method of claim 14, wherein said recombinant gene is encoded by SEQ ID NO:

1.

16. The method of claim 5, wherein said rebombinant gene is encoded by a homo log of SEQ ID NO: 1.

17. The method of claim 14, wherein said host cell is a pyrR-mA\ mutant.

18. The method of claim 13, wherein said selectable environmental condition is the

presence of 5-fluorouracil or 5-fluoroorotic acid in a cell culture medium

19. The method of any of claims 1-4, wherein said selectable environmental condition is the presence of a cytotoxic compound in a cell culture medium.

20. The method of claim 19, wherein said cytotoxic compound is a base analog.

21. The method of claim 20, wherein said base analog is selected from the group

consisting of: 5-fluorouracil (5-FU), 5-fluoroorotic acid, 8-thioxanthine, 6- thioguanine, 8-aza-2,6-diaminopurine (8ADP), and 2-methylpurine (2MP).

22. The method of claim 1, wherein said recombinant cyanobacterium is light dependent or fixes carbon.

23. The method of claim 1, wherein said recombinant cyanobacterium further comprises a nucleic acid sequence encoding enzymatic pathways to synthesize a carbon-based product.

24. The method of claim 23, wherein said recombinant cyanobacterium releases,

permeates, or exports said carbon-based product.

25. The method of claim 23, wherein said carbon-based product is selected from alkanes, alkenes, aliphatic and aromatic alkane and alkene mixtures, alcohols, alkanals and

56 alkenols, alkanoic and alkenoic acids, hydroxy alkanoic acids, keto acids, alkyl alkanoates, ethers, amino acids, lactams, organic polymers, isoprenoids and pharmaceuticals/multi-functional group molecules.

26. The method of claim 1, wherein said host cyanobacterium is selected from

Chamaesiphon sp., Chroococcus sp., Cyanothece sp., Gloeothece sp., Gloeobacter sp., Microcystis sp., Prochlorococcus sp., Acaryochloris sp., Xenococcus sp., Dactylococcopsis sp., Prochloron sp., Chroogloeocystis sp., Coelosphaerium sp., Cyanodictyon sp., Geminocystis sp., Johannesbaptistia sp., Limnococcus sp., Radiocystis sp., Rhabdoderma sp., Rubidibacter sp., Snowella sp., Stanieria sp., Sphaerocavum sp., Synechococcus sp., Synechocystis spp., Cyanobacterium sp., Cyanobium sp., Gleocapsa sp., Thermosynechococcus sp., Dermocarpella sp., Chroococcidiopsis sp., Myxosarcina sp., Pleurocapsa sp., Borzia sp., Crinalium sp., Geitlerinemia sp., Limnothrix sp., Microcoleus sp., Pseudanabaena sp., Spirulina sp., Starria sp., Symploca sp., Trichodesmium sp., Tychonema sp., Anabaena sp., Anabaenopsis sp., Aphanizomenon sp., Cyanospira sp., Cylindrospermopsis sp., Cylindrospermum sp., Nodularia sp., Nostoc sp., Scytonema sp., Calothrix sp., Rivularia sp., Tolypothrix sp., Chlorogloeopsis sp., Fischer ella sp., Geitleria sp., lyengariella sp., Nostochopsis sp., Stigonema sp., Arthrospira sp., Leptolyngbya sp., Lyngbya sp., Oscillatoria sp., Planktothrix sp., Prochlorothrix sp., and Microcoleus sp.

27. The method of claim 1, wherein said positively selective marker confers host

resistance to a selectable environmental condition.

28. The method of claim 1, wherein said positively selective marker is an antibiotic

resistance gene.

29. The method of claim 28, wherein said antibiotic resistance gene is selected from

genes conferring resistance to at least one of the group consisting of: kanamycin, gentamicin, spectinomycin, streptomycin, erythromycin, chloramphenicol, zeocin, ampicillin, and carbinicillin.

30. The method of claim 1, wherein said positively selective marker is an auxotrophic selectable marker.

31. The method of claim 1 , wherein said negatively selective marker is a recombinant gene encoding /r rE, /r r or pyrF/pyrE.

32. The method of claim 31, wherein said /r rE gene comprises SEQ ID NO: 6.

57

33. The method of claim 31, wherein said pyrE gene comprises a homolog of SEQ ID NO: 6.

34. The method of claim 31, wherein said pyrF gene comprises SEQ ID NO: 8.

35. The method of claim 31, wherein said pyrF gene comprises a homolog of SEQ ID NO: 8.

36. The method of claim 31, wherein said pyrF/pyrE gene comprises SEQ ID NO: 10.

37. The method of claim 31 , wherein said pyrF/pyrE gene comprises a homolog of SEQ ID NO: 10

38. The method of claim 31, wherein said host cell is pyrE-mA\, pyrF-mA\, or pyrF/pyrE- null mutant.

39. The method of claim 31, wherein said selectable environmental condition is the

presence of 5-fluoroorotic acid or 5-fluorouracil in a cell culture medium.

40. The method of claim 1, wherein said negatively selective marker is a recombinant gene encoding upp.

41. The method of claim 40, wherein said recombinant gene comprises SEQ ID NO: 4.

42. The method of claim 40, wherein said recombinant gene comprises a homolog of SEQ ID NO: 4.

43. The method of claim 40, wherein said host cell is a upp-mA\ mutant.

44. The method of claim 40, wherein said selectable environmental condition is the

presence of 5-fluoroorotic acid or 5-fluorouracil in a cell culture medium.

45. The method of claim 1, wherein said negatively selective marker is a recombinant gene encoding gpt.

46. The method of claim 45, wherein said recombinant gene comprises SEQ ID NO: 12.

47. The method of claim 45, wherein said recombinant gene comprises a homolog of SEQ ID NO: 12.

48. The method of claim 45, wherein said host cell is gpt-mA\ mutant.

49. The method of claim 45, wherein said selectable environmental condition is the

presence of 8-thioxanthine in a cell culture medium.

50. The method of claim 1, wherein said negatively selective marker is a recombinant gene encoding glnQ.

51. The method of claim 50, wherein said host cell is glnQ-mA\ mutant.

52. The method of claim 50, wherein said selectable environmental condition is the

presence of gamma-glutamyl hydrazine in a cell culture medium.

58

53. The method of claim 1, wherein said negatively selective marker is a recombinant gene encoding a pheS mutant.

54. The method of claim 53, wherein said pheS mutant is pheSA294G.

55. The method of claim 54, wherein said pheSA294G encodes an enzyme comprising SEQ ID NO: 16.

56. The method of claim 54, wherein said pheSA294G encodes an enzyme comprising a homolog of SEQ ID NO: 16.

57. The method of claim 53, wherein said selectable environmental condition is the

presence of /?-chloro-phenylalanine.

58. The method of claim 1, wherein said positively selective marker is an antibiotic

resistance marker or an auxotrophic marker.

59. The method of claim 1, wherein the presence of said recombinant gene in said

secondary recombinants is identified by sequencing.

60. A method for transforming a host cell, comprising:

a. obtaining a host cell whose genome has a null mutation for a gene encoding an enzyme capable of incorporating a toxic compound;

b. introducing said gene into said host cell in combination with a positively

selective marker;

c. exposing said host cells to a condition that selects for primary recombinant host cells comprising said positively selective marker, and thus said gene; d. isolating said primary recombinants;

e. exposing said primary recombinants to said toxic compound to select for secondary recombinants that have lost said gene; and

f. isolating said secondary recombinants.

61. The method of claim 60, wherein said positively selective marker is an antibiotic resistance marker.

62. The method of claim 60, wherein said host cell is a cyanobacterium.

63. A recombinant cyanobacterium prepared by a counter-selection method, said counter- selection method comprising the following steps:

a. introducing a suicide plasmid into a host cyanobacterium, said suicide plasmid comprising a positively selective marker, a negatively selective marker, and a recombinant gene;

b. selecting for primary recombinants incorporating the positively selective marker;

59 c. from said primary recombinants, selecting for secondary recombinants that have lost the negatively selective marker and the positively selective marker; and d. isolating the secondary recombinants comprising the recombinant gene to obtain said recombinant cyanobacterium.

64. A recombinant cyanobacterium prepared by any of the methods recited in claims 1- 62.

60