Methods and compositions for producing hydrocarbons

0001The present invention relates to methods of producing an alkane or alkene from a genetically modified host cell.

BACKGROUND OF THE INVENTION

0002Petroleum is a limited, natural resource found in the Earth in liquid, gaseous, or solid forms. Petroleum is primarily composed of hydrocarbons, which are comprised mainly of carbon and hydrogen. It also contains significant amounts of other elements, such as, nitrogen, oxygen, or sulfur, in different forms.

0003Petroleum is a valuable resource, but petroleum products are developed at considerable costs, both financial and environmental. First, sources of petroleum must be discovered. Petroleum exploration is an expensive and risky venture. The cost of exploring deep water wells can exceed $100 million. Moreover, there is no guarantee that these wells will contain petroleum. It is estimated that only 40% of drilled wells lead to productive wells generating commercial hydrocarbons. In addition to the economic cost, petroleum exploration carries a high environmental cost. For example, offshore exploration disturbs the surrounding marine environments.

0004After a productive well is discovered, the petroleum must be extracted from the Earth at great expense. During primary recovery, the natural pressure underground is sufficient to extract about 20% of the petroleum in the well. As this natural pressure falls, secondary recovery methods are employed, if economical. Generally, secondary recovery involves increasing the well's pressure by, for example, water injection, natural gas injection, or gas lift. Using secondary recovery methods, an additional 5% to 15% of petroleum is recovered. Once secondary recovery methods are exhausted, tertiary recovery methods can be used, if economical. Tertiary methods involve reducing the viscosity of the petroleum to make it easier to extract. Using tertiary recovery methods, an additional 5% to 15% of petroleum is recovered. Hence, even under the best circumstances, only 50% of the petroleum in a well can be extracted. Petroleum extraction also carries an environmental cost. For example, petroleum extraction can result in large seepages of petroleum rising to the surface. Moreover, offshore drilling involves dredging the seabed which disrupts or destroys the surrounding marine environment.

0005Since petroleum deposits are not found uniformly throughout the Earth, petroleum must be transported over great distances from petroleum producing regions to petroleum consuming regions. In addition to the shipping costs, there is also the environmental risk of devastating oil spills.

0006In its natural form, crude petroleum extracted from the Earth has few commercial uses. It is a mixture of hydrocarbons (<i>e.g</i>., paraffins (or alkanes), olefins (or alkenes), alkynes, napthenes (or cylcoalkanes), aliphatic compounds, aromatic compounds, <i>etc</i>.) of varying length and complexity. In addition, crude petroleum contains other organic compounds (<i>e.g</i>., organic compounds containing nitrogen, oxygen, sulfur, <i>etc</i>.) and impurities (<i>e.g.,</i> sulfur, salt, acid, metals, <i>etc</i>.).

0007Hence, crude petroleum must be refined and purified before it can be used commercially. Due to its high energy density and its easy transportability, most petroleum is refined into fuels, such as transportation fuels (<i>e.g</i>., gasoline, diesel, aviation fuel, <i>etc</i>.), heating oil, liquefied petroleum gas, <i>etc.</i>

0008Crude petroleum is also a primary source of raw materials for producing petrochemicals. The two main classes of raw materials derived from petroleum are short chain olefins (<i>e.g</i>., ethylene and propylene) and aromatics (<i>e.g.</i>, benzene and xylene isomers). These raw materials are derived from longer chain hydrocarbons in crude petroleum by cracking it at considerable expense using a variety of methods, such as catalytic cracking, steam cracking, or catalytic reforming. These raw materials are used to make petrochemicals, which cannot be directly refined from crude petroleum, such as monomers, solvents, detergents, or adhesives.

0009One example of a raw material derived from crude petroleum is ethylene. Ethylene is used to produce petrochemicals such as, polyethylene, ethanol, ethylene oxide, ethylene glycol, polyester, glycol ether, ethoxylate, vinyl acetate, 1,2-dichloroethane, trichloroethylene, tetrachloroethylene, vinyl chloride, and polyvinyl chloride. An additional example of a raw material is propylene, which is used to produce isopropyl alcohol, acrylonitrile, polypropylene, propylene oxide, propylene glycol, glycol ethers, butylene, isobutylene, 1,3-butadiene, synthetic elastomers, polyolefins, alpha-olefins, fatty alcohols, acrylic acid, acrylic polymers, allyl chloride, epichlorohydrin, and epoxy resins.

0010These petrochemicals can then be used to make specialty chemicals, such as plastics, resins, fibers, elastomers, pharmaceuticals, lubricants, or gels. Particular specialty chemicals which can be produced from petrochemical raw materials are: fatty acids, hydrocarbons (<i>e.g</i>., long chain, branched chain, saturated, unsaturated, <i>etc</i>.), fatty alcohols, esters, fatty aldehydes, ketones, lubricants, <i>etc.</i>

0011Specialty chemicals have many commercial uses. Fatty acids are used commercially as surfactants, for example, in detergents and soaps. They can also be used as additives in fuels, lubricating oils, paints, lacquers, candles, salad oil, shortening, cosmetics, and emulsifiers. In addition, fatty acids are used as accelerator activators in rubber products. Fatty acids can also be used as a feedstock to produce methyl esters, amides, amines, acid chlorides, anhydrides, ketene dimers, and peroxy acids and esters.

0012Hydrocarbons have many commercial uses. For example, shorter chain alkanes are used as fuels. Methane and ethane are the main constituents of natural gas. Longer chain alkanes (<i>e.g</i>., from five to sixteen carbons) are used as transportation fuels (<i>e.g</i>., gasoline, diesel, or aviation fuel). Alkanes having more than sixteen carbon atoms are important components of fuel oils and lubricating oils. Even longer alkanes, which are solid at room temperature, can be used, for example, as a paraffin wax. Alkanes that contain approximately thirty-five carbons are found in bitumen, which is used for road surfacing. In addition, longer chain alkanes can be cracked to produce commercially useful shorter chain hydrocarbons.

0013Like short chain alkanes, short chain alkenes are used in transportation fuels. Longer chain alkenes are used in plastics, lubricants, and synthetic lubricants. In addition, alkenes are used as a feedstock to produce alcohols, esters, plasticizers, surfactants, tertiary amines, enhanced oil recovery agents, fatty acids, thiols, alkenylsuccinic anhydrides, epoxides, chlorinated alkanes, chlorinated alkenes, waxes, fuel additives, and drag flow reducers.

0014Fatty alcohols have many commercial uses. The shorter chain fatty alcohols are used in the cosmetic and food industries as emulsifiers, emollients, and thickeners. Due to their amphiphilic nature, fatty alcohols behave as nonionic surfactants, which are useful as detergents. In addition, fatty alcohols are used in waxes, gums, resins, pharmaceutical salves and lotions, lubricating oil additives, textile antistatic and finishing agents, plasticizers, cosmetics, industrial solvents, and solvents for fats.

0015Esters have many commercial uses. For example, biodiesel, an alternative fuel, is comprised of esters (<i>e.g</i>., fatty acid methyl ester, fatty acid ethyl esters, <i>etc</i>.). Some low molecular weight esters are volatile with a pleasant odor which makes them useful as fragrances or flavoring agents. In addition, esters are used as solvents for lacquers, paints, and varnishes. Furthermore, some naturally occurring substances, such as waxes, fats, and oils are comprised of esters. Esters are also used as softening agents in resins and plastics, plasticizers, flame retardants, and additives in gasoline and oil. In addition, esters can be used in the manufacture of polymers, films, textiles, dyes, and pharmaceuticals.

0016Aldehydes are used to produce many specialty chemicals. For example, aldehydes are used to produce polymers, resins (<i>e.g</i>., Bakelite), dyes, flavorings, plasticizers, perfumes, pharmaceuticals, and other chemicals. Some are used as solvents, preservatives, or disinfectants. Some natural and synthetic compounds, such as vitamins and hormones, are aldehydes. In addition, many sugars contain aldehyde groups.

0017Ketones are used commercially as solvents. For example, acetone is frequently used as a solvent, but it is also a raw material for making polymers. Ketones are also used in lacquers, paints, explosives, perfumes, and textile processing. In addition, ketones are used to produce alcohols, alkenes, alkanes, imines, and enamines.

0018In addition, crude petroleum is a source of lubricants. Lubricants derived petroleum are typically composed of olefins, particularly polyolefins and alpha-olefins. Lubricants can either be refined from crude petroleum or manufactured using raw materials refined from crude petroleum.

0019Obtaining these specialty chemicals from crude petroleum requires a significant financial investment as well as a great deal of energy. It is also an inefficient process because frequently the long chain hydrocarbons in crude petroleum are cracked to produce smaller monomers. These monomer are then used as the raw material to manufacture the more complex specialty chemicals.

0020In addition to the problems with exploring, extracting, transporting, and refining petroleum, petroleum is a limited and dwindling resource. One estimate of world petroleum consumption is 30 billion barrels per year. By some estimates, it is predicted that at current production levels, the world's petroleum reserves could be depleted before the year 2050.

0021Finally, the burning of petroleum based fuels releases greenhouse gases (<i>e.g.,</i> carbon dioxide) and other forms of air pollution (<i>e.g.</i>, carbon monoxide, sulfur dioxide, <i>etc.</i>). As the world's demand for fuel increases, the emission of greenhouse gases and other forms of air pollution also increases. The accumulation of greenhouse gases in the atmosphere leads to an increase global warming. Hence, in addition to damaging the environment locally (<i>e.g</i>., oil spills, dredging of marine environments, <i>etc.</i>), burning petroleum also damages the environment globally.

0022Due to the inherent challenges posed by petroleum, there is a need for a renewable petroleum source which does not need to be explored, extracted, transported over long distances, or substantially refined like petroleum. There is also a need for a renewable petroleum source that can be produced economically without creating the type of environmental damage produced by the petroleum industry and the burning of petroleum based fuels. For similar reasons, there is also a need for a renewable source of chemicals that are typically derived from petroleum. Database Entry UniProt Q54764 discloses a polypeptide sequence 100% identical to present SEQ ID NO: 2. <nplcit id="ncit0001" npl-type="s"><text>Fehler et al. (Biochemistry (1970), Vol. 9, No. 2, p. 418-22</text></nplcit>) describe the biosynthesis of hydrocarbons, in particular alkanes, in <i>Anabaena variabilis,</i> and mass spectrometry analysis thereof. <patcit id="pcit0001" dnum="WO2007136762A"><text>WO 2007/136762</text></patcit> relates to the production of fatty acid derivatives, including the production of alkanes and alkenes, in genetically modified organisms, including genetically modified <i>E. coli.</i><nplcit id="ncit0002" npl-type="s"><text>Phung et al. (Abstracts of the General Meeting of the American Society for Microbiology, 1 January 1995, p. 524, Abstract H-182, ISSN: 1060-2011</text></nplcit>) describe the identification and sequencing of four polypeptides involved in early steps of fatty acid biosynthesis in <i>Synechococcus sp.,</i> namely biotin carboxyl carrier protein (BCCP), a biotin carboxylase, the alpha subunit of carboxyltransferase, and a beta-ketoacyl transferase. <nplcit id="ncit0003" npl-type="s"><text>Ladygina et al. (Process Biochemistry (2006), Vol. 41, No. 5, pp. 1359-5113</text></nplcit>) are reviewing the microbial biosynthesis of hydrocarbons.

SUMMARY OF THE INVENTION

0023The invention is based, at least in part, on the identification of cyanobacterial genes that encode hydrocarbon biosynthetic polypeptides. The invention is defined by the claims. In particular, the invention provides a method of producing an alkane or alkene from a genetically modified host cell, the method comprising: (a) transforming a host cell to express by introducing (i) an aldehyde biosynthetic polynucleotide that encodes an aldehyde biosynthetic polypeptide, and (ii) an alkane or alkene biosynthetic polynucleotide that encodes an alkane or alkene biosynthetic polypeptide; and (b) culturing said genetically modified host cell in a culture medium containing a carbon source under conditions effective to produce an alkane or alkene in the culture medium, wherein said aldehyde biosynthetic polypeptide has acyl-ACP reductase activity and is from a cyanobacterium, and wherein said alkane or alkene biosynthetic polypeptide has decarbonylase activity and is from a cyanobacterium. In some embodiments of the disclosure, the method comprises producing in a host cell a polypeptide comprising the amino acid sequence of SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36, or a variant thereof, and isolating the hydrocarbon from the host cell.

0024In some embodiments of the disclosure, the polypeptide comprises an amino acid sequence having at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to SEQ ID NO:2, 4, 6, 8, 10, 12, 14,16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36.

0025In some embodiments of the disclosure, the polypeptide comprises the amino acid sequence of SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36 with one or more amino acid substitutions, additions, insertions, or deletions. The polypeptide has decarbonylase activity and is from a cyanobacterium. In yet other embodiments of the disclosure, the polypeptide comprises the amino acid sequence of SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36, with one or more conservative amino acid substitutions. For example, the polypeptide comprises one or more of the following conservative amino acid substitutions: replacement of an aliphatic amino acid, such as alanine, valine, leucine, and isoleucine, with another aliphatic amino acid; replacement of a serine with a threonine; replacement of a threonine with a serine; replacement of an acidic residue, such as aspartic acid and glutamic acid, with another acidic residue; replacement of a residue bearing an amide group, such as asparagine and glutamine, with another residue bearing an amide group; exchange of a basic residue, such as lysine and arginine, with another basic residue; and replacement of an aromatic residue, such as phenylalanine and tyrosine, with another aromatic residue. In some embodiments of the disclosure, the polypeptide has about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more amino acid substitutions, additions, insertions, or deletions. The polypeptide has decarbonylase activity and is from a cyanobacterium.

0026In other embodiments of the disclosure, the polypeptide comprises the amino acid sequence of: (i) SEQ ID NO:37 or SEQ ID NO:38 or SEQ ID NO:39; or (ii) SEQ ID NO:40 and any one of (a) SEQ ID NO:37, (b) SEQ ID NO:38, and (c) SEQ ID NO:39; or (iii) SEQ ID NO:41 or SEQ ID NO:42 or SEQ ID NO:43 or SEQ ID NO:44. The polypeptide has decarbonylase activity and is from a cyanobacterium.

0027In some embodiments of the disclosure, the method comprises expressing in a host cell a polynucleotide comprising a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, or 35. In some embodiments of the disclosure, the nucleotide sequence is SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, or 35. In some embodiments of the disclosure, the method further comprises isolating the hydrocarbon from the host cell.

0028In other embodiments of the disclosure, the nucleotide sequence hybridizes to a complement of SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, or 35, or to a fragment thereof, for example, under low stringency, medium stringency, high stringency, or very high stringency conditions.

0029In other embodiments of the disclosure, the nucleotide sequence encodes a polypeptide comprising: (i) the amino acid sequence of SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36; or (ii) the amino acid sequence of SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36 with one or more amino acid substitutions, additions, insertions, or deletions. In some embodiments of the disclosure, the polypeptide comprises the amino acid sequence of SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36 with one or more conservative amino acid substitutions. The polypeptide has decarbonylase activity and is from a cyanobacterium.

0030In other embodiments of the disclosure, the nucleotide sequence encodes a polypeptide having the same biological activity as a polypeptide comprising the amino acid sequence of SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36. In some embodiments, the nucleotide sequence is SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, or 35 or a fragment thereof. In other embodiments of the disclosure, the nucleotide sequence hybridizes to a complement of SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, or 35 or to a fragment thereof, for example, under low stringency, medium stringency, high stringency, or very high stringency conditions. The biological activity is decarbonylase activity and the encoded polypeptide is from a cyanobacterium.

0031In some embodiments of the disclosure, the method comprises transforming a host cell with a recombinant vector comprising a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, or 35. In some embodiments of the disclosure, the recombinant vector further comprises a promoter operably linked to the nucleotide sequence. In some embodiments of the disclosure, the promoter is a developmentally-regulated, an organelle-specific, a tissue-specific, an inducible, a constitutive, or a cell-specific promoter. In particular embodiments of the disclosure, the recombinant vector comprises at least one sequence selected from the group consisting of (a) a regulatory sequence operatively coupled to the nucleotide sequence; (b) a selection marker operatively coupled to the nucleotide sequence; (c) a marker sequence operatively coupled to the nucleotide sequence; (d) a purification moiety operatively coupled to the nucleotide sequence; (e) a secretion sequence operatively coupled to the nucleotide sequence; and (f) a targeting sequence operatively coupled to the nucleotide sequence. In certain embodiments of the disclosure, the nucleotide sequence is stably incorporated into the genomic DNA of the host cell, and the expression of the nucleotide sequence is under the control of a regulated promoter region.

0032In any of the aspects described herein, the host cell can be selected from the group consisting of a mammalian cell, plant cell, insect cell, yeast cell, fungus cell, filamentous fungi cell, and bacterial cell.

0033In some embodiments, the host cell is a Gram-positive bacterial cell. In other embodiments, the host cell is a Gram-negative bacterial cell.

0034In some embodiments, the host cell is selected from the genus <i>Escherichia, Bacillus, Lactobacillus, Rhodococcus, Pseudomonas, Aspergillus, Trichoderma, Neurospora, Fusarium, Humicola, Rhizomucor, Kluyveromyces, Pichia, Mucor, Myceliophtora, Penicillium, Phanerochaete, Pleurotus, Trametes, Chrysosporium, Saccharomyces, Stenotrophamonas, Schizosaccharomyces, Yarrowia,</i> or <i>Streptomyces.</i>

0035In particular embodiments, the host cell is a <i>Bacillus lentus</i> cell, a <i>Bacillus brevis</i> cell, a <i>Bacillus stearothermophilus</i> cell, a <i>Bacillus licheniformis</i> cell, a <i>Bacillus alkalophilus</i> cell, a <i>Bacillus coagulans</i> cell, a <i>Bacillus circulans</i> cell, a <i>Bacillus pumilis</i> cell, a <i>Bacillus thuringiensis</i> cell, a <i>Bacillus clausii</i> cell, a <i>Bacillus megaterium</i> cell, a <i>Bacillus subtilis</i> cell, or a <i>Bacillus amyloliquefaciens</i> cell.

0036In other embodiments, the host cell is a <i>Trichoderma koningii</i> cell, a <i>Trichoderma viride</i> cell, a <i>Trichoderma reesei</i> cell, a <i>Trichoderma longibrachiatum</i> cell, an <i>Aspergillus awamori</i> cell, an <i>Aspergillus fumigates</i> cell, an <i>Aspergillus foetidus</i> cell, an <i>Aspergillus nidulans</i> cell, an <i>Aspergillus niger</i> cell, an <i>Aspergillus oryzae</i> cell, a <i>Humicola insolens</i> cell, a <i>Humicola lanuginose</i> cell, a <i>Rhodococcus opacus</i> cell, a <i>Rhizomucor miehei</i> cell, or a <i>Mucor michei</i> cell.

0037In yet other embodiments, the host cell is a <i>Streptomyces lividans</i> cell or a <i>Streptomyces murinus</i> cell. In other embodiments, the host cell is an <i>Actinomycetes</i> cell.

0038In some embodiments of the disclosure, the host cell is a CHO cell, a COS cell, a VERO cell, a BHK cell, a HeLa cell, a Cv1 cell, an MDCK cell, a 293 cell, a 3T3 cell, or a PC12 cell.

0039In particular embodiments, the host cell is an <i>E. coli</i> cell, such as a strain B, a strain C, a strain K, or a strain W <i>E. coli</i> cell.

0040In other embodiments, the host cell is a cyanobacterial host cell. In particular embodiments, the cyanobacterial host cell is a cell listed in Table 1.

0041In some embodiments, the alkanes and alkenes are secreted from by the host cell.

0042In certain embodiments of the disclosure, the host cell overexpresses a substrate described herein. In some embodiments of the disclosure, the method further includes transforming the host cell with a nucleic acid that encodes an enzyme described herein, and the host cell overexpresses a substrate described herein. In other embodiments of the disclosure, the method further includes culturing the host cell in the presence of at least one substrate described herein. In some embodiments of the disclosure, the substrate is a fatty acid derivative, an acyl-ACP, a fatty acid, an acyl-CoA, a fatty aldehyde, a fatty alcohol, or a fatty ester.

0043In some embodiments of the disclosure, the fatty acid derivative substrate is an unsaturated fatty acid derivative substrate, a monounsaturated fatty acid derivative substrate, or a saturated fatty acid derivative substrate. In other embodiments, the fatty acid derivative substrate is a straight chain fatty acid derivative substrate, a branched chain fatty acid derivative substrate, or a fatty acid derivative substrate that includes a cyclic moiety.

0044In certain embodiments of the aspects described herein, the hydrocarbon produced is an alkane. In some embodiments, the alkane is a C₃-C₂₅ alkane. For example, the alkane is a C₃, C₄, C₅, C₆, C₇, C₈, C₉, C₁₀, C₁₁, C₁₂, C₁₃, C₁₄, C₁₅, C₁₆, C₁₇, C₁₈, C₁₉, C₂₀, C_21, C₂₂, C₂₃, C₂₄, or C₂₅ alkane. In some embodiments, the alkane is tridecane, methyltridecane, nonadecane, methylnonadecane, heptadecane, methylheptadecane, pentadecane, or methylpentadecane.

0045In some embodiments, the alkane is a straight chain alkane, a branched chain alkane, or a cyclic alkane.

0046In certain embodiments, the method further comprises culturing the host cell in the presence of a saturated fatty acid derivative, and the hydrocarbon produced is an alkane. In certain embodiments, the saturated fatty acid derivative is a C₆-C₂₆ fatty acid derivative substrate. For example, the fatty acid derivative substrate is a C₆, C₇, C₈, C₉, C₁₀, C₁₁, C₁₂, C₁₃, C₁₄, C₁₅, C₁₆, C₁₇, C₁₈, C₁₉, C₂₀, C_21, C₂₂, C₂₃, C₂₄, C₂₅, or a C₂₆ fatty acid derivative substrate. In particular embodiments, the fatty acid derivative substrate is 2-methylicosanal, icosanal, octadecanal, tetradecanal, 2-methyloctadecanal, stearaldehyde, or palmitaldehyde.

0047In some embodiments, the method further includes isolating the alkane from the host cell or from the culture medium. In other embodiments of the disclosure, the method further includes cracking or refining the alkane.

0048In certain embodiments of the aspects described herein, the hydrocarbon produced is an alkene. In some embodiments, the alkene is a C₃-C₂₅ alkene. For example, the alkene is a C₃, C₄, C₅, C₆, C₇, C₈, C₉, C₁₀, C₁₁, C₁₂, C₁₃, C₁₄, C₁₅, C₁₆, C₁₇, C₁₈, C₁₉, C₂₀, C_21, C₂₂, C₂₃, C₂₄, or C₂₅ alkene. In some embodiments, the alkene is pentadecene, heptadecene, methylpentadecene, or methylheptadecene.

0049In some embodiments, the alkene is a straight chain alkene, a branched chain alkene, or a cyclic alkene.

0050In certain embodiments, the method further comprises culturing the host cell in the presence of an unsaturated fatty acid derivative, and the hydrocarbon produced is an alkene. In certain embodiments, the unsaturated fatty acid derivative is a C₆-C₂₆ fatty acid derivative substrate. For example, the fatty acid derivative substrate is a C₆, C₇, C₈, C₉, C₁₀, C₁₁, C₁₂, C₁₃, C₁₄, C₁₅, C₁₆, C₁₇, C₁₈, C₁₉, C₂₀, C_21, C₂₂, C₂₃, C₂₄, C₂₅, or a C₂₆ unsaturated fatty acid derivative substrate. In particular embodiments, the fatty acid derivative substrate is octadecenal, hexadecenal, methylhexadecenal, or methyloctadecenal.

0051In another aspect, described herein is a genetically engineered microorganism comprising an exogenous control sequence stably incorporated into the genomic DNA of the microorganism. In one embodiment, the control sequence is integrated upstream of a polynucleotide comprising a nucleotide sequence having at least about 70% sequence identity to SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, or 35. In some aspects, the nucleotide sequence has at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, or 35. In some aspects, the nucleotide sequence is SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, or 35.

0052In some embodiments of the disclosure, the microorganism expresses an increased level of a hydrocarbon relative to a wild-type cyanobacterium.

0053In another aspect, described herein is a method of making a hydrocarbon, the method comprising culturing a genetically engineered microorganism described herein under conditions suitable for gene expression, and isolating the hydrocarbon.

0054In some embodiments of the disclosure, the polypeptide (i) has at least 70% identity to the amino acid sequence of SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36, or a variant thereof; (ii) is encoded by a nucleotide sequence having at least 70% identity to SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, or 35, or a variant thereof; (iii) comprises the amino acid sequence of SEQ ID NO:37, 38, or 39; (iv) comprises the amino acid sequence of SEQ ID NO:40 and any one of (a) SEQ ID NO:37, (b) SEQ ID NO:38, and (c) SEQ ID NO:39; or (v) is SEQ ID NO:41, 42, 43, or 44. The polypeptide has decarbonylase activity and is from a cyanobacterium.

0055In some embodiments of the disclosure, the polypeptide has at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36. In some embodiments, the polypeptide has the amino acid sequence of SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36.

0056In some embodiments of the disclosure, the polypeptide is encoded by a nucleotide sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, or 35. In some embodiments of the disclosure, the polypeptide is encoded by a nucleotide sequence having SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, or 35.

0057In some embodiments, the biological substrate is a fatty acid derivative, an acyl-ACP, a fatty acid, an acyl-CoA, a fatty aldehyde, a fatty alcohol, or a fatty ester.

0058In some embodiments, the substrate is a saturated fatty acid derivative, and the hydrocarbon is an alkane, for example, a C₃-C₂₅ alkane. For example, the alkane is a C₃, C₄, C₅, C₆, C₇, C₈, C₉, C₁₀, C₁₁, C₁₂, C₁₃, C₁₄, C₁₅, C₁₆, C₁₇, C₁₈, C₁₉, C₂₀, C_21, C₂₂, C₂₃, C₂₄, or C₂₅ alkane. In some embodiments, the alkane is tridecane, methyltridecane, nonadecane, methylnonadecane, heptadecane, methylheptadecane, pentadecane, or methylpentadecane.

0059In some embodiments, the alkane is a straight chain alkane, a branched chain alkane, or a cyclic alkane.

0060In some embodiments, the saturated fatty acid derivative is 2-methylicosanal, icosanal, octadecanal, tetradecanal, 2-methyloctadecanal, stearaldehyde, or palmitaldehyde.

0061In other embodiments, the biological substrate is an unsaturated fatty acid derivative, and the hydrocarbon is an alkene, for example, a C₃-C₂₅ alkene. For example, the alkene is a C₃, C₄, C₅, C₆, C₇, C₈, C₉, C₁₀, C₁₁, C₁₂, C₁₃, C₁₄, C₁₅, C₁₆, C₁₇, C₁₈, C₁₉, C₂₀, C_21, C₂₂, C₂₃, C₂₄, or C₂₅ alkene. In some embodiments, the alkene is pentadecene, heptadecene, methylpentadecene, or methylheptadecene.

0062In some embodiments, the alkene is a straight chain alkene, a branched chain alkene, or a cyclic alkene.

0063In some embodiments, the unsaturated fatty acid derivative is octadecenal, hexadecenal, methylhexadecenal, or methyloctadecenal.

0064In another aspect, described herein is a hydrocarbon produced by any of the methods or microorganisms described herein. The hydrocarbon may be an alkane or an alkene having a δ¹³C of about -15.4 or greater. For example, the alkane or alkene has a δ¹³C of about -15.4 to about -10.9, for example, about -13.92 to about -13.84. The alkane or alkene may have an f_M¹⁴C of at least about 1.003. For example, the alkane or alkene has an f_M¹⁴C of at least about 1.01 or at least about 1.5. The alkane or alkene may have an f_M¹⁴C of about 1.111 to about 1.124.

0065In another aspect, described herein is a biofuel that includes a hydrocarbon produced by any of the methods or microorganisms described herein. The hydrocarbon may be an alkane or alkene having a δ¹³C of about -15.4 or greater. For example, the alkane or alkene has a δ¹³C of about -15.4 to about -10.9, for example, about -13.92 to about -13.84. The alkane or alkene may have an f_M¹⁴C of at least about 1.003. For example, the alkane or alkene has an f_M¹⁴C of at least about 1.01 or at least about 1.5. The alkane or alkene may have an f_M¹⁴C of about 1.111 to about 1.124. The biofuel may be diesel, gasoline, or jet fuel.

0066In another aspect, described herein is an isolated nucleic acid consisting of no more than about 500 nucleotides of SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, or 35. The nucleic acid may consist of no more than about 300 nucleotides, no more than about 350 nucleotides, no more than about 400 nucleotides, no more than about 450 nucleotides, no more than about 550 nucleotides, no more than about 600 nucleotides, or no more than about 650 nucleotides, of SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, or 35. The nucleic acid encodes a polypeptide having decarbonylase activity and being from a cyanobacterium.

0067In another aspect, described herein is an isolated nucleic acid consisting of no more than about 99%, no more than about 98%, no more than about 97%, no more than about 96%, no more than about 95%, no more than about 94%, no more than about 93%, no more than about 92%, no more than about 91%, no more than about 90%, no more than about 85%, or no more than about 80% of the nucleotides of SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, or 35. The nucleic acid encodes a polypeptide having decarbonylase activity and being from a cyanobacterium.

0068In another aspect, described herein is an isolated polypeptide consisting of no more than about 200, no more than about 175, no more than about 150, or no more than about 100 of the amino acids of SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36. The polypeptide has decarbonylase activity and is from a cyanobacterium.

0069In another aspect, described herein is an isolated polypeptide consisting of no more than about 99%, no more than about 98%, no more than about 97%, no more than about 96%, no more than about 95%, no more than about 94%, no more than about 93%, no more than about 92%, no more than about 91%, no more than about 90%, no more than about 85%, or no more than about 80% of the amino acids of SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36. The polypeptide has decarbonylase activity and is from a cyanobacterium.

Definitions

0070Throughout the specification, a reference may be made using an abbreviated gene name or polypeptide name, but it is understood that such an abbreviated gene or polypeptide name represents the genus of genes or polypeptides. Such gene names include all genes encoding the same polypeptide and homologous polypeptides having the same physiological function. Polypeptide names include all polypeptides that have the same activity (e.g., that catalyze the same fundamental chemical reaction).

0071The accession numbers referenced herein are derived from the NCBI database (National Center for Biotechnology Information) maintained by the National Institute of Health, U.S.A. Unless otherwise indicated, the accession numbers are as provided in the database as of April 2009.

0072EC numbers are established by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB) (available at http://www.chem.qmul.ac.uk/iubmb/enzyme/). The EC numbers referenced herein are derived from the KEGG Ligand database, maintained by the Kyoto Encyclopedia of Genes and Genomics, sponsored in part by the University of Tokyo. Unless otherwise indicated, the EC numbers are as provided in the database as of March 2008.

0073The articles "a" and "an" are used herein to refer to one or to more than one (<i>i.e</i>., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

0074The term "about" is used herein to mean a value ± 20% of a given numerical value. Thus, "about 60%" means a value of between 60 ± (20% of 60) (<i>i.e</i>., between 48 and 70).

0075As used herein, the term "aldehyde" means a hydrocarbon having the formula RCHO characterized by an unsaturated carbonyl group (C=O). In a preferred embodiment, the aldehyde is any aldehyde made from a fatty acid or fatty acid derivative. In one embodiment, the R group is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbons in length.

0076As used herein, an "aldehyde biosynthetic gene" or an "aldehyde biosynthetic polynucleotide" is a nucleic acid that encodes an aldehyde biosynthetic polypeptide.

0077As used herein, an "aldehyde biosynthetic polypeptide" is a polypeptide that is a part of the biosynthetic pathway of an aldehyde. Such polypeptides can act on a biological substrate to yield an aldehyde. The aldehyde biosynthetic polypeptide has reductase activity and is from a cyanobacterium.

0078As used herein, the term "alkane" means a hydrocarbon containing only single carbon-carbon bonds.

0079As used herein, an "alkane biosynthetic gene" or an "alkane biosynthetic polynucleotide" is a nucleic acid that encodes an alkane biosynthetic polypeptide.

0080As used herein, an "alkane biosynthetic polypeptide" is a polypeptide that is a part of the biosynthetic pathway of an alkane. Such polypeptides can act on a biological substrate to yield an alkane. The alkane biosynthetic polypeptide has decarbonylase activity and is from a cyanobacterium.

0081As used herein, an "alkene biosynthetic gene" or an "alkene biosynthetic polynucleotide" is a nucleic acid that encodes an alkene biosynthetic polypeptide.

0082As used herein, an "alkene biosynthetic polypeptide" is a polypeptide that is a part of the biosynthetic pathway of an alkene. Such polypeptides can act on a biological substrate to yield an alkene. The alkene biosynthetic polypeptide has decarbonylase activity and is from a cyanobacterium.

0083As used herein, the term "attenuate" means to weaken, reduce or diminish. For example, a polypeptide can be attenuated by modifying the polypeptide to reduce its activity (<i>e.g</i>., by modifying a nucleotide sequence that encodes the polypeptide).

0084As used herein, the term "biodiesel" means a biofuel that can be a substitute of diesel, which is derived from petroleum. Biodiesel can be used in internal combustion diesel engines in either a pure form, which is referred to as "neat" biodiesel, or as a mixture in any concentration with petroleum-based diesel. Biodiesel can include esters or hydrocarbons, such as aldehydes and alkanes.

0085As used therein, the term "biofuel" refers to any fuel derived from biomass. Biofuels can be substituted for petroleum based fuels. For example, biofuels are inclusive of transportation fuels (<i>e.g.,</i> gasoline, diesel, jet fuel, <i>etc</i>.), heating fuels, and electricity-generating fuels. Biofuels are a renewable energy source.

0086As used herein, the term "biomass" refers to a carbon source derived from biological material. Biomass can be converted into a biofuel. One exemplary source of biomass is plant matter. For example, corn, sugar cane, or switchgrass can be used as biomass. Another non-limiting example of biomass is animal matter, for example cow manure. Biomass also includes waste products from industry, agriculture, forestry, and households. Examples of such waste products that can be used as biomass are fermentation waste, straw, lumber, sewage, garbage, and food leftovers. Biomass also includes sources of carbon, such as carbohydrates (<i>e.g</i>., monosaccharides, disaccharides, or polysaccharides).

0087As used herein, the phrase "carbon source" refers to a substrate or compound suitable to be used as a source of carbon for prokaryotic or simple eukaryotic cell growth. Carbon sources can be in various forms, including, but not limited to polymers, carbohydrates, acids, alcohols, aldehydes, ketones, amino acids, peptides, and gases (<i>e.g.</i>, CO and CO₂). These include, for example, various monosaccharides, such as glucose, fructose, mannose, and galactose; oligosaccharides, such as fructo-oligosaccharide and galacto-oligosaccharide; polysaccharides such as xylose and arabinose; disaccharides, such as sucrose, maltose, and turanose; cellulosic material, such as methyl cellulose and sodium carboxymethyl cellulose; saturated or unsaturated fatty acid esters, such as succinate, lactate, and acetate; alcohols, such as ethanol or mixtures thereof. The carbon source can also be a product of photosynthesis, including, but not limited to, glucose. A preferred carbon source is biomass. Another preferred carbon source is glucose.

0088As used herein, a "cloud point lowering additive" is an additive added to a composition to decrease or lower the cloud point of a solution.

0089As used herein, the phrase "cloud point of a fluid" means the temperature at which dissolved solids are no longer completely soluble. Below this temperature, solids begin precipitating as a second phase giving the fluid a cloudy appearance. In the petroleum industry, cloud point refers to the temperature below which a solidified material or other heavy hydrocarbon crystallizes in a crude oil, refined oil, or fuel to form a cloudy appearance. The presence of solidified materials influences the flowing behavior of the fluid, the tendency of the fluid to clog fuel filters, injectors, <i>etc.,</i> the accumulation of solidified materials on cold surfaces (<i>e.g.,</i> a pipeline or heat exchanger fouling), and the emulsion characteristics of the fluid with water.

0090A nucleotide sequence is "complementary" to another nucleotide sequence if each of the bases of the two sequences matches (<i>i.e.,</i> is capable of forming Watson Crick base pairs). The term "complementary strand" is used herein interchangeably with the term "complement". The complement of a nucleic acid strand can be the complement of a coding strand or the complement of a non-coding strand.

0091As used herein, the term "conditions sufficient to allow expression" means any conditions that allow a host cell to produce a desired product, such as a polypeptide, aldehyde, or alkane described herein. Suitable conditions include, for example, fermentation conditions. Fermentation conditions can comprise many parameters, such as temperature ranges, levels of aeration, and media composition. Each of these conditions, individually and in combination, allows the host cell to grow. Exemplary culture media include broths or gels. Generally, the medium includes a carbon source, such as glucose, fructose, cellulose, or the like, that can be metabolized by a host cell directly. In addition, enzymes can be used in the medium to facilitate the mobilization (e.g., the depolymerization of starch or cellulose to fermentable sugars) and subsequent metabolism of the carbon source.

0092To determine if conditions are sufficient to allow expression, a host cell can be cultured, for example, for about 4, 8, 12, 24, 36, or 48 hours. During and/or after culturing, samples can be obtained and analyzed to determine if the conditions allow expression. For example, the host cells in the sample or the medium in which the host cells were grown can be tested for the presence of a desired product. When testing for the presence of a product, assays, such as, but not limited to, TLC, HPLC, GC/FID, GC/MS, LC/MS, MS, can be used.

0093It is understood that the polypeptides described herein may have additional conservative or non-essential amino acid substitutions, which do not have a substantial effect on the polypeptide functions. Whether or not a particular substitution will be tolerated (<i>i.e.,</i> will not adversely affect desired biological properties, such as decarboxylase activity) can be determined as described in <nplcit id="ncit0004" npl-type="s"><text>Bowie et al., Science (1990) 247:1306 1310</text></nplcit>. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (<i>e.g.</i>, lysine, arginine, histidine), acidic side chains (<i>e.g.,</i> aspartic acid, glutamic acid), uncharged polar side chains (<i>e.g</i>., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (<i>e.g</i>., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (<i>e.g</i>., threonine, valine, isoleucine), and aromatic side chains (<i>e.g</i>., tyrosine, phenylalanine, tryptophan, histidine).

0094As used herein, "control element" means a transcriptional control element. Control elements include promoters and enhancers. The term "promoter element," "promoter," or "promoter sequence" refers to a DNA sequence that functions as a switch that activates the expression of a gene. If the gene is activated, it is said to be transcribed or participating in transcription. Transcription involves the synthesis of mRNA from the gene. A promoter, therefore, serves as a transcriptional regulatory element and also provides a site for initiation of transcription of the gene into mRNA. Control elements interact specifically with cellular proteins involved in transcription (<nplcit id="ncit0005" npl-type="s"><text>Maniatis et al., Science 236:1237, 1987</text></nplcit>).

0095As used herein, the term "ester synthase" means a peptide capable of producing fatty esters. More specifically, an ester synthase is a peptide which converts a thioester to a fatty ester. In a preferred embodiment, the ester synthase converts a thioester (e.g., acyl-CoA) to a fatty ester.

0096In an alternate embodiment, an ester synthase uses a thioester and an alcohol as substrates to produce a fatty ester. Ester synthases are capable of using short and long chain thioesters as substrates. In addition, ester synthases are capable of using short and long chain alcohols as substrates.

0097Non-limiting examples of ester synthases are wax synthases, wax-ester synthases, acyl CoA:alcohol transacylases, acyltransferases, and fatty acyl-coenzyme A:fatty alcohol acyltransferases. Exemplary ester synthases are classified in enzyme classification number EC 2.3.1.75. Exemplary GenBank Accession Numbers are provided in <figref idref="f0037 f0038 f0039 f0040 f0041 f0042 f0043 f0044 f0045 f0046 f0047 f0048 f0049 f0050 f0051 f0052 f0053 f0054 f0055 f0056 f0057 f0058 f0059">Figure 40</figref>.

0098As used herein, the term "fatty acid" means a carboxylic acid having the formula RCOOH. R represents an aliphatic group, preferably an alkyl group. R can comprise between about 4 and about 22 carbon atoms. Fatty acids can be saturated, monounsaturated, or polyunsaturated. In a preferred embodiment, the fatty acid is made from a fatty acid biosynthetic pathway.

0099As used herein, the term "fatty acid biosynthetic pathway" means a biosynthetic pathway that produces fatty acids. The fatty acid biosynthetic pathway includes fatty acid enzymes that can be engineered, as described herein, to produce fatty acids, and in some embodiments can be expressed with additional enzymes to produce fatty acids having desired carbon chain characteristics.

0100As used herein, the term "fatty acid derivative" means products made in part from the fatty acid biosynthetic pathway of the production host organism. "Fatty acid derivative" also includes products made in part from acyl-ACP or acyl-ACP derivatives. The fatty acid biosynthetic pathway includes fatty acid synthase enzymes which can be engineered as described herein to produce fatty acid derivatives, and in some examples can be expressed with additional enzymes to produce fatty acid derivatives having desired carbon chain characteristics. Exemplary fatty acid derivatives include for example, fatty acids, acyl-CoA, fatty aldehyde, short and long chain alcohols, hydrocarbons, fatty alcohols, and esters (<i>e.g</i>., waxes, fatty acid esters, or fatty esters).

0101As used herein, the term "fatty acid derivative enzymes" means all enzymes that may be expressed or overexpressed in the production of fatty acid derivatives. These enzymes are collectively referred to herein as fatty acid derivative enzymes. These enzymes may be part of the fatty acid biosynthetic pathway. Non-limiting examples of fatty acid derivative enzymes include fatty acid synthases, thioesterases, acyl-CoA synthases, acyl-CoA reductases, alcohol dehydrogenases, alcohol acyltransferases, fatty alcohol-forming acyl-CoA reductase, ester synthases, aldehyde biosynthetic polypeptides, and alkane biosynthetic polypeptides. Fatty acid derivative enzymes convert a substrate into a fatty acid derivative. In some examples, the substrate may be a fatty acid derivative which the fatty acid derivative enzyme converts into a different fatty acid derivative.

0102As used herein, the term "fatty alcohol forming peptides" means a peptide capable of catalyzing the conversion of acyl-CoA to fatty alcohol, including fatty alcohol forming acyl-CoA reductase (FAR, EC 1.1.1.*), acyl-CoA reductase (EC 1.2.1.50), or alcohol dehydrogenase (EC 1.1.1.1). Additionally, one of ordinary skill in the art will appreciate that some fatty alcohol forming peptides will catalyze other reactions as well. For example, some acyl-CoA reductase peptides will accept other substrates in addition to fatty acids. Such non-specific peptides are, therefore, also included. Nucleic acid sequences encoding fatty alcohol forming peptides are known in the art, and such peptides are publicly available. Exemplary GenBank Accession Numbers are provided in <figref idref="f0037 f0038 f0039 f0040 f0041 f0042 f0043 f0044 f0045 f0046 f0047 f0048 f0049 f0050 f0051 f0052 f0053 f0054 f0055 f0056 f0057 f0058 f0059">Figure 40</figref>.

0103As used herein, "fatty acid enzyme" means any enzyme involved in fatty acid biosynthesis. Fatty acid enzymes can be expressed or overexpressed in host cells to produce fatty acids. Non-limiting examples of fatty acid enzymes include fatty acid synthases and thioesterases.

0104As used herein, the term "fatty ester" means an ester. In a preferred embodiment, a fatty ester is any ester made from a fatty acid, for example a fatty acid ester. In one embodiment, a fatty ester contains an A side (<i>i.e</i>., the carbon chain attached to the carboxylate oxygen) and a B side (<i>i.e</i>., the carbon chain comprising the parent carboxylate). In a preferred embodiment, when the fatty ester is derived from the fatty acid biosynthetic pathway, the A side is contributed by an alcohol, and the B side is contributed by a fatty acid. Any alcohol can be used to form the A side of the fatty esters. For example, the alcohol can be derived from the fatty acid biosynthetic pathway. Alternatively, the alcohol can be produced through non-fatty acid biosynthetic pathways. Moreover, the alcohol can be provided exogenously. For example, the alcohol can be supplied in the fermentation broth in instances where the fatty ester is produced by an organism. Alternatively, a carboxylic acid, such as a fatty acid or acetic acid, can be supplied exogenously in instances where the fatty ester is produced by an organism that can also produce alcohol.

0105The carbon chains comprising the A side or B side can be of any length. In one embodiment, the A side of the ester is at least about 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 14, 16, or 18 carbons in length. The B side of the ester is at least about 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, or 26 carbons in length. The A side and/or the B side can be straight or branched chain. The branched chains may have one or more points of branching. In addition, the branched chains may include cyclic branches. Furthermore, the A side and/or B side can be saturated or unsaturated. If unsaturated, the A side and/or B side can have one or more points of unsaturation.

0106In one embodiment, the fatty ester is produced biosynthetically. In this embodiment, first the fatty acid is "activated." Non-limiting examples of "activated" fatty acids are acyl-CoA, acyl-ACP, and acyl phosphate. Acyl-CoA can be a direct product of fatty acid biosynthesis or degradation. In addition, acyl-CoA can be synthesized from a free fatty acid, a CoA, or an adenosine nucleotide triphosphate (ATP). An example of an enzyme which produces acyl-CoA is acyl-CoA synthase

0107After the fatty acid is activated, it can be readily transferred to a recipient nucleophile. Exemplary nucleophiles are alcohols, thiols, or phosphates.

0108In one embodiment, the fatty ester is a wax. The wax can be derived from a long chain alcohol and a long chain fatty acid. In another embodiment, the fatty ester can be derived from a fatty acyl-thioester and an alcohol. In another embodiment, the fatty ester is a fatty acid thioester, for example fatty acyl Coenzyme A (CoA). In other embodiments, the fatty ester is a fatty acyl panthothenate, an acyl carrier protein (ACP), or a fatty phosphate ester. Fatty esters have many uses. For example, fatty esters can be used as a biofuel.

0109As used herein, "fraction of modern carbon" or "f_M" has the same meaning as defined by National Institute of Standards and Technology (NIST) Standard Reference Materials (SRMs) 4990B and 4990C, known as oxalic acids standards HOxI and HOxII, respectively. The fundamental definition relates to 0.95 times the ¹⁴C/¹²C isotope ratio HOxI (referenced to AD 1950). This is roughly equivalent to decay-corrected pre-Industrial Revolution wood. For the current living biosphere (plant material), f_M is approximately 1.1.

0110Calculations of "homology" between two sequences can be performed as follows. The sequences are aligned for optimal comparison purposes (<i>e.g</i>., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In a preferred embodiment, the length of a reference sequence that is aligned for comparison purposes is at least about 30%, preferably at least about 40%, more preferably at least about 50%, even more preferably at least about 60%, and even more preferably at least about 70%, at least about 80%, at least about 90%, or about 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein, amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology"). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

0111The comparison of sequences and determination of percent homology between two sequences can be accomplished using a mathematical algorithm. In a preferred embodiment, the percent homology between two amino acid sequences is determined using the <nplcit id="ncit0006" npl-type="s"><text>Needleman and Wunsch (1970), J. Mol. Biol. 48:444 453</text></nplcit>, algorithm that has been incorporated into the GAP program in the GCG software package, using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percent homology between two nucleotide sequences is determined using the GAP program in the GCG software package, using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. A particularly preferred set of parameters (and the one that should be used if the practitioner is uncertain about which parameters should be applied to determine if a molecule is within a homology limitation of the claims) are a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

0112As used herein, a "host cell" is a cell used to produce a product described herein (<i>e.g</i>., an aldehyde or alkane described herein). A host cell can be modified to express or overexpress selected genes or to have attenuated expression of selected genes. Non-limiting examples of host cells include plant, animal, human, bacteria, yeast, or filamentous fungi cells.

0113As used herein, the term "hybridizes under low stringency, medium stringency, high stringency, or very high stringency conditions" describes conditions for hybridization and washing. Guidance for performing hybridization reactions can be found in <nplcit id="ncit0007" npl-type="b"><text>Current Protocols in Molecular Biology, John Wiley &amp; Sons, N.Y. (1989), 6.3.1 - 6.3.6</text></nplcit>. Aqueous and nonaqueous methods are described in that reference and either method can be used. Specific hybridization conditions referred to herein are as follows: 1) low stringency hybridization conditions in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by two washes in 0.2X SSC, 0.1% SDS at least at 50°C (the temperature of the washes can be increased to 55°C for low stringency conditions); 2) medium stringency hybridization conditions in 6X SSC at about 45°C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 60°C; 3) high stringency hybridization conditions in 6X SSC at about 45°C, followed by one or more washes in 0.2.X SSC, 0.1% SDS at 65°C; and preferably 4) very high stringency hybridization conditions are 0.5M sodium phosphate, 7% SDS at 65°C, followed by one or more washes at 0.2X SSC, 1% SDS at 65°C. Very high stringency conditions (4) are the preferred conditions unless otherwise specified.

0114The term "isolated" as used herein with respect to nucleic acids, such as DNA or RNA, refers to molecules separated from other DNAs or RNAs, respectively, that are present in the natural source of the nucleic acid. Moreover, an "isolated nucleic acid" includes nucleic acid fragments, such as fragments that are not naturally occurring. The term "isolated" is also used herein to refer to polypeptides, which are isolated from other cellular proteins, and encompasses both purified endogenous polypeptides and recombinant polypeptides. The term "isolated" as used herein also refers to a nucleic acid or polypeptide that is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques. The term "isolated" as used herein also refers to a nucleic acid or polypeptide that is substantially free of chemical precursors or other chemicals when chemically synthesized.

0115As used herein, the "level of expression of a gene in a cell" refers to the level of mRNA, pre-mRNA nascent transcript(s), transcript processing intermediates, mature mRNA(s), and/or degradation products encoded by the gene in the cell.

0116As used herein, the term "microorganism" means 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 term "microbial cell", as used herein, means a cell from a microorganism.

0117As used herein, the term "nucleic acid" refers to polynucleotides, such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA). The term also includes analogs of either RNA or DNA made from nucleotide analogs, and, as applicable to the embodiment being described, single (sense or antisense) and double-stranded polynucleotides, ESTs, chromosomes, cDNAs, mRNAs, and rRNAs.

0118As used herein, the term "operably linked" means that a selected nucleotide sequence (<i>e.g</i>., encoding a polypeptide described herein) is in proximity with a promoter to allow the promoter to regulate expression of the selected nucleotide sequence. In addition, the promoter is located upstream of the selected nucleotide sequence in terms of the direction of transcription and translation. By "operably linked" is meant that a nucleotide sequence and a regulatory sequence(s) are connected in such a way as to permit gene expression when the appropriate molecules (<i>e.g</i>., transcriptional activator proteins) are bound to the regulatory sequence(s).

0119The term "or" is used herein to mean, and is used interchangeably with, the term "and/or," unless context clearly indicates otherwise.

0120As used herein, "overexpress" means to express or cause to be expressed a nucleic acid, polypeptide, or hydrocarbon in a cell at a greater concentration than is normally expressed in a corresponding wild-type cell. For example, a polypeptide can be "overexpressed" in a recombinant host cell when the polypeptide is present in a greater concentration in the recombinant host cell compared to its concentration in a non-recombinant host cell of the same species.

0121As used herein, "partition coefficient" or "P," is defined as the equilibrium concentration of a compound in an organic phase divided by the concentration at equilibrium in an aqueous phase (<i>e.g</i>., fermentation broth). In one embodiment of a bi-phasic system described herein, the organic phase is formed by the aldehyde or alkane during the production process. However, in some examples, an organic phase can be provided, such as by providing a layer of octane, to facilitate product separation. When describing a two phase system, the partition characteristics of a compound can be described as logP. For example, a compound with a logP of 1 would partition 10:1 to the organic phase. A compound with a logP of -1 would partition 1:10 to the organic phase. By choosing an appropriate fermentation broth and organic phase, an aldehyde or alkane with a high logP value can separate into the organic phase even at very low concentrations in the fermentation vessel.

0122As used herein, the term "purify," "purified," or "purification" means the removal or isolation of a molecule from its environment by, for example, isolation or separation. "Substantially purified" molecules are at least about 60% free, preferably at least about 75% free, and more preferably at least about 90% free from other components with which they are associated. As used herein, these terms also refer to the removal of contaminants from a sample. For example, the removal of contaminants can result in an increase in the percentage of aldehydes or alkanes in a sample. For example, when aldehydes or alkanes are produced in a host cell, the aldehydes or alkanes can be purified by the removal of host cell proteins. After purification, the percentage of aldehydes or alkanes in the sample is increased.

0123The terms "purify," "purified," and "purification" do not require absolute purity. They are relative terms. Thus, for example, when aldehydes or alkanes are produced in host cells, a purified aldehyde or purified alkane is one that is substantially separated from other cellular components (<i>e.g</i>., nucleic acids, polypeptides, lipids, carbohydrates, or other hydrocarbons). In another example, a purified aldehyde or purified alkane preparation is one in which the aldehyde or alkane is substantially free from contaminants, such as those that might be present following fermentation. In some embodiments, an aldehyde or an alkane is purified when at least about 50% by weight of a sample is composed of the aldehyde or alkane. In other embodiments, an aldehyde or an alkane is purified when at least about 60%, 70%, 80%, 85%, 90%, 92%, 95%, 98%, or 99% or more by weight of a sample is composed of the aldehyde or alkane.

0124As used herein, the term "recombinant polypeptide" refers to a polypeptide that is produced by recombinant DNA techniques, wherein generally DNA encoding the expressed polypeptide or RNA is inserted into a suitable expression vector and that is in turn used to transform a host cell to produce the polypeptide or RNA.

0125As used herein, the term "substantially identical" (or "substantially homologous") is used to refer to a first amino acid or nucleotide sequence that contains a sufficient number of identical or equivalent (<i>e.g</i>., with a similar side chain, <i>e.g</i>., conserved amino acid substitutions) amino acid residues or nucleotides to a second amino acid or nucleotide sequence such that the first and second amino acid or nucleotide sequences have similar activities.

0126As used herein, the term "synthase" means an enzyme which catalyzes a synthesis process. As used herein, the term synthase includes synthases, synthetases, and ligases.

0127As used herein, the term "transfection" means the introduction of a nucleic acid (<i>e.g</i>., via an expression vector) into a recipient cell by nucleic acid-mediated gene transfer.

0128As used herein, "transformation" refers to a process in which a cell's genotype is changed as a result of the cellular uptake of exogenous nucleic acid. This may result in the transformed cell expressing a recombinant form of an RNA or polypeptide. In the case of antisense expression from the transferred gene, the expression of a naturally-occurring form of the polypeptide is disrupted.

0129As used herein, a "transport protein" is a polypeptide that facilitates the movement of one or more compounds in and/or out of a cellular organelle and/or a cell.

0130As used herein, a "variant" of polypeptide X refers to a polypeptide having the amino acid sequence of polypeptide X in which one or more amino acid residues is altered. The variant may have conservative changes or nonconservative changes. Guidance in determining which amino acid residues may be substituted, inserted, or deleted without affecting biological activity may be found using computer programs well known in the art, for example, LASERGENE software (DNASTAR).

0131The term "variant," when used in the context of a polynucleotide sequence, may encompass a polynucleotide sequence related to that of a gene or the coding sequence thereof. This definition may also include, for example, "allelic," "splice," "species," or "polymorphic" variants. A splice variant may have significant identity to a reference polynucleotide, but will generally have a greater or fewer number of polynucleotides due to alternative splicing of exons during mRNA processing. The corresponding polypeptide may possess additional functional domains or an absence of domains. Species variants are polynucleotide sequences that vary from one species to another. The resulting polypeptides generally will have significant amino acid identity relative to each other. A polymorphic variant is a variation in the polynucleotide sequence of a particular gene between individuals of a given species.

0132As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of useful vector is an episome (<i>i.e</i>., a nucleic acid capable of extra-chromosomal replication). Useful vectors are those capable of autonomous replication and/or expression of nucleic acids to which they are linked. Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as "expression vectors". In general, expression vectors of utility in recombinant DNA techniques are often in the form of "plasmids," which refer generally to circular double stranded DNA loops that, in their vector form, are not bound to the chromosome. In the present specification, "plasmid" and "vector" are used interchangeably, as the plasmid is the most commonly used form of vector. However, also included are such other forms of expression vectors that serve equivalent functions and that become known in the art subsequently hereto.

0133Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

0134Other features and advantages of the invention will be apparent from the following detailed description and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

0135<ul id="ul0001" list-style="none" compact="compact"><li><figref idref="f0001">FIG. 1A</figref> is a GC/MS trace of hydrocarbons produced by <i>Prochlorococcus marinus</i> CCMP1986 cells. <figref idref="f0001">FIG. 1B</figref> is a mass fragmentation pattern of the peak at 7.55 min of <figref idref="f0001">FIG. 1A</figref>.</li><li><figref idref="f0001">FIG. 2A</figref> is a GC/MS trace of hydrocarbons produced by <i>Nostoc punctiforme</i> PCC73102 cells. <figref idref="f0001">FIG. 2B</figref> is a mass fragmentation pattern of the peak at 8.73 min of <figref idref="f0001">FIG. 2A</figref>.</li><li><figref idref="f0002">FIG. 3A</figref> is a GC/MS trace of hydrocarbons produced by <i>Gloeobaceter violaceus</i> ATCC29082 cells. <figref idref="f0002">FIG. 3B</figref> is a mass fragmentation pattern of the peak at 8.72 min of <figref idref="f0002">FIG. 3A. FIG. 4A</figref> is a GC/MS trace of hydrocarbons produced by <i>Synechocystic sp.</i> PCC6803 cells. <figref idref="f0002">FIG. 4B</figref> is a mass fragmentation pattern of the peak at 7.36 min of <figref idref="f0002">FIG. 4A</figref>.</li><li><figref idref="f0003">FIG. 5A</figref> is a GC/MS trace of hydrocarbons produced by <i>Synechocystis sp.</i> PCC6803 wild type cells. <figref idref="f0003">FIG. 5B</figref> is a GC/MS trace of hydrocarbons produced by <i>Synechocystis sp.</i> PCC6803 cells with a deletion of the sll0208 and sll0209 genes.</li><li><figref idref="f0004">FIG. 6A</figref> is a GC/MS trace of hydrocarbons produced by <i>E. coli</i> MG1655 wild type cells. <figref idref="f0004">FIG. 6B</figref> is a GC/MS trace of hydrocarbons produced by <i>E. coli</i> MG1655 cells expressing <i>Synechococcus elongatus</i> PCC7942 YP_400611 (Synpcc7942_1594) (SEQ ID NO:65).</li><li><figref idref="f0005">FIG. 7</figref> is a GC/MS trace of hydrocarbons produced by <i>E. coli</i> cells expressing <i>Cyanothece</i> sp. ATCC51142 cce_1430 (YP_001802846) (SEQ ID NO:69).</li><li><figref idref="f0006">FIG. 8A</figref> is a GC/MS trace of hydrocarbons produced by <i>E. coli</i> cells expressing <i>Synechococcus elongatus</i> PCC7942 YP_400611 (Synpcc7942_1594) (SEQ ID NO:65) and <i>Synechococcus elongatus</i> PCC7942 YP_400610 (Synpcc7942_1593) (SEQ ID NO:1). <figref idref="f0006">FIG. 8B</figref> depicts mass fragmentation patterns of the peak at 6.98 min of <figref idref="f0006">FIG. 8A</figref> and of pentadecane. <figref idref="f0006">FIG. 8C</figref> depicts mass fragmentation patterns of the peak at 8.12 min of <figref idref="f0006">FIG. 8A</figref> and of 8-heptadecene.</li><li><figref idref="f0007">FIG. 9</figref> is a GC/MS trace of hydrocarbons produced by <i>E. coli</i> MG1655 cells expressing <i>Synechococcus elongatus</i> PCC7942 YP_400611 (Synpcc7942_1594) (SEQ ID NO:65) and <i>Nostoc punctiforme</i> PCC73102 Npun02004178 (ZP_00108838) (SEQ ID NO:5).</li><li><figref idref="f0007">FIG. 10</figref> is a GC/MS trace of hydrocarbons produced by <i>E. coli</i> MG1655 cells expressing <i>Synechococcus elongatus</i> PCC7942 YP_400611 (Synpcc7942_1594) (SEQ ID NO:65) and <i>Synechocystis</i> sp. PCC6803 sll0208 (NP_442147) (SEQ ID NO:3).</li><li><figref idref="f0008">FIG. 11</figref> is a GC/MS trace of hydrocarbons produced by <i>E. coli</i> MG1655 cells expressing <i>Synechococcus elongatus</i> PCC7942 YP_400611 (Synpcc7942_1594) (SEQ ID NO:65) and <i>Nostoc</i> sp. PCC7210 alr5283 (NP_489323) (SEQ ID NO:7).</li><li><figref idref="f0008">FIG. 12</figref> is a GC/MS trace of hydrocarbons produced by <i>E. coli</i> MG1655 cells expressing <i>Synechococcus elongatus</i> PCC7942 YP_400611 (Synpcc7942_1594) (SEQ ID NO:65) and codon-optimized <i>Acaryochloris marina</i> MBIC11017 AM1_4041 (YP_001518340) (SEQ ID NO:46).</li><li><figref idref="f0009">FIG. 13</figref> is a GC/MS trace of hydrocarbons produced by <i>E. coli</i> MG1655 cells expressing <i>Synechococcus elongatus</i> PCC7942 YP_400611 (Synpcc7942_1594) (SEQ ID NO:65) and codon-optimized <i>Thermosynechococcus elongatus</i> BP-1 tll1313 (NP_682103) (SEQ ID NO:47).</li><li><figref idref="f0009">FIG. 14</figref> is a GC/MS trace of hydrocarbons produced by <i>E. coli</i> MG1655 cells expressing <i>Synechococcus elongatus</i> PCC7942 YP_400611 (Synpcc7942_1594) (SEQ ID NO:65) and codon-optimized <i>Synechococcus sp.</i> JA-3-3Ab CYA_0415 (YP_473897) (SEQ ID NO:48).</li><li><figref idref="f0010">FIG. 15</figref> is a GC/MS trace of hydrocarbons produced by <i>E. coli</i> MG1655 cells expressing <i>Synechococcus elongatus</i> PCC7942 YP_400611 (Synpcc7942_1594) (SEQ ID NO:65) and <i>Gloeobacter violaceus</i> PCC7421 gll3146 (NP_926092) (SEQ ID NO:15).</li><li><figref idref="f0010">FIG. 16</figref> is a GC/MS trace of hydrocarbons produced by <i>E. coli</i> MG1655 cells expressing <i>Synechococcus elongatus</i> PCC7942 YP_400611 (Synpcc7942_1594) (SEQ ID NO:65) and codon-optimized <i>Prochlorococcus marinus</i> MIT9313 PMT1231 (NP_895059) (SEQ ID NO:49).</li><li><figref idref="f0011">FIG. 17</figref> is a GC/MS trace of hydrocarbons produced by <i>E. coli</i> MG1655 cells expressing <i>Synechococcus elongatus</i> PCC7942 YP_400611 (Synpcc7942_1594) (SEQ ID NO:65) and <i>Prochlorococcus marinus</i> CCMP1986 PMM0532 (NP_892650) (SEQ ID NO:19).</li><li><figref idref="f0011">FIG. 18</figref> is a GC/MS trace of hydrocarbons produced by <i>E. coli</i> MG1655 cells expressing <i>Synechococcus elongatus</i> PCC7942 YP_400611 (Synpcc7942_1594) (SEQ ID NO:65) and codon-optimized <i>Prochlorococcus mariunus</i> NATL2A PMN2A_1863 (YP_293054) (SEQ ID NO:51).</li><li><figref idref="f0012">FIG. 19</figref> is a GC/MS trace of hydrocarbons produced by <i>E. coli</i> MG1655 cells expressing <i>Synechococcus elongatus</i> PCC7942 YP_400611 (Synpcc7942_1594) (SEQ ID NO:65) and codon-optimized <i>Synechococcus</i> sp. RS9917 RS9917_09941 (ZP_01079772) (SEQ ID NO:52).</li><li><figref idref="f0012">FIG. 20</figref> is a GC/MS trace of hydrocarbons produced by <i>E. coli</i> MG1655 cells expressing <i>Synechococcus elongatus</i> PCC7942 YP_400611 (Synpcc7942_1594) (SEQ ID NO:65) and codon-optimized <i>Synechococcus</i> sp. RS9917 RS9917_12945 (ZP_01080370) (SEQ ID NO:53).</li><li><figref idref="f0013">FIG. 21</figref> is a GC/MS trace of hydrocarbons produced by <i>E. coli</i> MG1655 cells expressing <i>Synechococcus elongatus</i> PCC7942 YP_400611 (Synpcc7942_1594) (SEQ ID NO:65) and <i>Cyanothece</i> sp. ATCC51142 cce_0778 (YP_001802195) (SEQ ID NO:27).</li><li><figref idref="f0013">FIG. 22</figref> is a GC/MS trace of hydrocarbons produced by <i>E. coli</i> MG1655 cells expressing <i>Synechococcus elongatus</i> PCC7942 YP_400611 (Synpcc7942_1594) (SEQ ID NO:65) and <i>Cyanothece</i> sp. PCC7425 Cyan7425_0398 (YP_002481151) (SEQ ID NO:29).</li><li><figref idref="f0014">FIG. 23</figref> is a GC/MS trace of hydrocarbons produced by <i>E. coli</i> MG1655 cells expressing <i>Synechococcus elongatus</i> PCC7942 YP_400611 (Synpcc7942_1594) (SEQ ID NO:65) and <i>Cyanothece</i> sp. PCC7425 Cyan7425_2986 (YP_002483683) (SEQ ID NO:31).</li><li><figref idref="f0014">FIG. 24A</figref> is a GC/MS trace of hydrocarbons produced by <i>E. coli</i> MG1655 cells expressing <i>Prochlorococcus marinus</i> CCMP1986 PMM0533 (NP_892651) (SEQ ID NO:71). <figref idref="f0014">FIG. 24B</figref> is a GC/MS trace of hydrocarbons produced by <i>E. coli</i> MG1655 cells expressing <i>Prochlorococcus marinus</i> CCMP1986 PMM0533 (NP_892651) (SEQ ID NO:71) and <i>Prochlorococcus mariunus</i> CCMP1986 PMM0532 (NP_892650) (SEQ ID NO:19).</li><li><figref idref="f0015">FIG. 25A</figref> is a GC/MS trace of hydrocarbons produced by <i>E. coli</i> MG1655 Δ<i>fadE lacZ</i>::<i>P_trc</i> '<i>tesA-fadD</i> cells. <figref idref="f0015">FIG. 25B</figref> is a GC/MS trace of hydrocarbons produced by <i>E. coli</i> MG1655 Δ<i>fadE lacZ</i>::<i>P_trc</i> '<i>tesA-fadD</i> cells expressing <i>Synechococcus elongatus</i> PCC7942 YP_400611 (Synpcc7942_1594) (SEQ ID NO:65) and <i>Acaryochloris marina</i> MBIC11017 AM1_4041 (YP_001518340) (SEQ ID NO:9).</li><li><figref idref="f0016">FIG. 26A</figref> is a GC/MS trace of hydrocarbons produced by <i>E. coli</i> MG1655 Δ<i>fadE lacZ</i>::<i>P_trc</i> '<i>tesA-fadD</i> cells expressing <i>Synechocystis</i> sp. PCC6803 sll0209 (NP_442146) (SEQ ID NO:67). <figref idref="f0016">FIG. 26B</figref> is a GC/MS trace of hydrocarbons produced by <i>E. coli</i> MG1655 Δ<i>fadE lacZ</i>::<i>P_trc</i> '<i>tesA-fadD</i> cells expressing <i>Synechocystis</i> sp. PCC6803 sll0209 (NP_442146) (SEQ ID NO:67) and <i>Synechocystis</i> sp. PCC6803 sll0208 (NP_442147) (SEQ ID NO:3).</li><li><figref idref="f0017">FIG. 27A</figref> is a GC/MS trace of hydrocarbons produced by <i>E. coli</i> MG1655 Δ<i>fadD lacZ</i>::<i>P_trc</i>-'<i>tesA</i> cells expressing <i>M. smegmatis</i> strain MC2 155 MSMEG_5739 (YP_889972) (SEQ ID NO:85). <figref idref="f0017">FIG. 27B</figref> is a GC/MS trace of hydrocarbons produced by <i>E. coli</i> MG1655 Δ<i>fadD lacZ</i>::<i>P_trc</i>-'<i>tesA</i> cells expressing <i>M. smegmatis</i> strain MC2 155 MSMEG_5739 (YP_889972) (SEQ ID NO:85) and <i>Nostoc punctiforme</i> PCC73102 Npun02004178 (ZP_00108838) (SEQ ID NO:5).</li><li><figref idref="f0018">FIG. 28</figref> is a graphic representation of hydrocarbons produced by <i>E. coli</i> MG1655 Δ<i>fadD lacZ</i>::<i>P_trc</i>-'<i>tesA</i> cells expressing <i>M. smegmatis</i> strain MC2 155 MSMEG_5739 (YP_889972) (SEQ ID NO:85) either alone or in combination with <i>Nostoc</i> sp. PCC7120 alr5283 (SEQ ID NO:7), <i>Nostoc punctiforme</i> PCC73102 Npun02004178 (SEQ ID NO:5), <i>P. mariunus</i> CCMP1986 PMM0532 (SEQ ID NO:19), <i>G. violaceus</i> PCC7421 gll3146 (SEQ ID NO:15), <i>Synechococcus</i> sp. RS9917_09941 (SEQ ID NO:23), <i>Synechococcus</i> sp. RS9917_12945 (SEQ ID NO:25), or <i>A. marina</i> MBIC11017 AM1_4041 (SEQ ID NO:9).</li><li><figref idref="f0019">FIG. 29A</figref> is a representation of the three-dimensional structure of a class I ribonuclease reductase subunit β protein, RNRβ. <figref idref="f0019">FIG. 29B</figref> is a representation of the three-dimensional structure of <i>Prochlorococcus marinus</i> MIT9313 PMT1231 (NP_895059) (SEQ ID NO:17). <figref idref="f0019">FIG. 29C</figref> is a representation of the three-dimensional structure of the active site of <i>Prochlorococcus marinus</i> MIT9313 PMT1231 (NP_895059) (SEQ ID NO:17).</li><li><figref idref="f0020">FIG. 30A</figref> is a GC/MS trace of hydrocarbons produced by <i>E. coli</i> MG1655 cells expressing <i>Nostoc punctiforme</i> PCC73102 Npun02004178 (ZP_00108838) (SEQ ID NO:5). <figref idref="f0020">FIG. 30B</figref> is a GC/MS trace of hydrocarbons produced by <i>E. coli</i> MG1655 cells expressing <i>Nostoc punctiforme</i> PCC73102 Npun02004178 (ZP_00108838) Y123F variant. <figref idref="f0020">FIG. 30C</figref> is a GC/MS trace of hydrocarbons produced by <i>E. coli</i> MG1655 cells expressing <i>Nostoc punctiforme</i> PCC73102 Npun02004178 (ZP_00108838) Y126F variant.</li><li><figref idref="f0021">FIG. 31</figref> depicts GC/MS traces of hydrocarbons produced <i>in vitro</i> using <i>Nostoc punctiforme</i> PCC73102 Npun02004178 (ZP_00108838) (SEQ ID NO:6) and octadecanal (A); Npun02004178 (ZP_00108838) (SEQ ID NO:6), octadecanal, spinach ferredoxin reductase, and NADPH (B); octadecanal, spinach ferredoxin, spinach ferredoxin reductase, and NADPH (C); or Npun02004178 (ZP_00108838) (SEQ ID NO:6), spinach ferredoxin, and spinach ferredoxin (D).</li><li><figref idref="f0022">FIG. 32</figref> depicts GC/MS traces of hydrocarbons produced <i>in vitro</i> using <i>Nostoc punctiforme</i> PCC73102 Npun02004178 (ZP_00108838) (SEQ ID NO:6), NADPH, octadecanal, and either (A) spinach ferredoxin and spinach ferredoxin reductase; (B) <i>N. punctiforme</i> PCC73102 Npun02003626 (ZP_00109192) (SEQ ID NO:88) and <i>N. punctiforme</i> PCC73102 Npun02001001 (ZP_00111633) (SEQ ID NO:90); (C) Npun02003626 (ZP_00109192) (SEQ ID NO:88) and <i>N. punctiforme</i> PCC73102 Npun02003530 (ZP_00109422) (SEQ ID NO:92); or (D) Npun02003626 (ZP_00109192) (SEQ ID NO:88) and <i>N. punctiforme</i> PCC73102 Npun02003123 (ZP_00109501) (SEQ ID NO:94).</li><li><figref idref="f0022">FIG. 33A</figref> is a GC/MS trace of hydrocarbons produced <i>in vitro</i> using octadecanoyl-CoA, <i>Synechococcus elongatus</i> PCC7942 YP_400611 (Synpcc7942_1594) (SEQ ID NO:66), NADH, and Mg²⁺. <figref idref="f0022">FIG. 33B</figref> is a GC/MS trace of hydrocarbons produced <i>in vitro</i> using octadecanoyl-CoA, <i>Synechococcus elongatus</i> PCC7942 YP_400611 (Synpcc7942_1594) (SEQ ID NO:66), NADPH, and Mg²⁺. <figref idref="f0022">FIG. 33C</figref> is a GC/MS trace of hydrocarbons produced <i>in vitro</i> using octadecanoyl-CoA, <i>Synechococcus elongatus</i> PCC7942 YP_400611 (Synpcc7942_1594) (SEQ ID NO:66) and NADPH.</li><li><figref idref="f0023">FIG. 34A</figref> is a GC/MS trace of hydrocarbons produced <i>in vitro</i> using octadecanoyl-CoA, labeled NADPH, <i>Synechococcus elongatus</i> PCC7942 YP_400611 (Synpcc7942_1594) (SEQ ID NO:66), and unlabeled NADPH. <figref idref="f0023">FIG. 34B</figref> is a GC/MS trace of hydrocarbons produced <i>in vitro</i> using octadecanoyl-CoA, labeled NADPH, <i>Synechococcus elongatus</i> PCC7942 YP_400611 (Synpcc7942_1594) (SEQ ID NO:66), and S-(4-²H)NADPH. <figref idref="f0023">FIG. 34C</figref> is a GC/MS trace of hydrocarbons produced <i>in vitro</i> using octadecanoyl-CoA, labeled NADPH, <i>Synechococcus elongatus</i> PCC7942 YP_400611 (Synpcc7942_1594) (SEQ ID NO:66), and R-(4-²H)NADPH.</li><li><figref idref="f0024">FIG. 35</figref> is a GC/MS trace of hydrocarbons in the cell-free supernatant produced by <i>E. coli</i> MG1655 Δ<i>fadE</i> cells in Che-9 media expressing <i>Synechococcus elongatus</i> PCC7942 YP_400611 (Synpcc7942_1594) (SEQ ID NO:65).</li><li><figref idref="f0024">FIG. 36</figref> is a GC/MS trace of hydrocarbons in the cell-free supernatant produced by <i>E. coli</i> MG1655 Δ<i>fadE</i> cells in Che-9 media expressing <i>Synechococcus elongatus</i> PCC7942 YP_400611 (Synpcc7942_1594) (SEQ ID NO:65) and <i>Nostoc punctiforme</i> PCC73102 Npun02004178 (ZP_00108838) (SEQ ID NO:5).</li><li><figref idref="f0025">FIG. 37</figref> is a GC/MS trace of hydrocarbons produced by <i>E. coli</i> MG1655 cells expressing <i>Nostoc</i> sp. PCC7120 alr5283 (NP_489323) (SEQ ID NO:7) and <i>Nostoc</i> sp. PCC7120 alr5284 (NP_489324) (SEQ ID NO:81).</li><li><figref idref="f0026 f0027 f0028 f0029">FIG. 38</figref> is a list of examples of homologs of <i>Synechococcus elongatus</i> PCC7942 YP_400610 (Synpcc7942_1593) (SEQ ID NO:1) from a metagenomic database.</li><li><figref idref="f0030 f0031 f0032 f0033 f0034 f0035 f0036">FIG. 39</figref> is a list of examples of homologs of <i>Synechococcus elongatus</i> PCC7942 YP_400611 (Synpcc7942_1594) (SEQ ID NO:65) from a metagenomic database.</li><li><figref idref="f0037 f0038 f0039 f0040 f0041 f0042 f0043 f0044 f0045 f0046 f0047 f0048 f0049 f0050 f0051 f0052 f0053 f0054 f0055 f0056 f0057 f0058 f0059">FIG. 40</figref> is a table identifying various genes that can be expressed, overexpressed, or attenuated to increase production of particular substrates.</li></ul>

DETAILED DESCRIPTION

0136The invention relates to compositions and methods of producing aldehydes, fatty alcohols, and hydrocarbons (such as alkanes, alkenes, and alkynes) from substrates, for example, an acyl-ACP, a fatty acid, an acyl-CoA, a fatty aldehyde, or a fatty alcohol substrate (<i>e.g</i>., as described in <patcit id="pcit0002" dnum="US08058788W"><text>PCT/US08/058788</text></patcit>). Such aldehydes, alkanes, and alkenes are useful as biofuels (<i>e.g</i>., substitutes for gasoline, diesel, jet fuel, <i>etc</i>.), specialty chemicals (<i>e.g</i>., lubricants, fuel additive, <i>etc</i>.), or feedstock for further chemical conversion (<i>e.g</i>., fuels, polymers, plastics, textiles, solvents, adhesives, <i>etc</i>.). The invention is based, in part, on the identification of genes that are involved in aldehyde, alkane, and alkene biosynthesis.

0137Such alkane and alkene biosynthetic genes include, for example, <i>Synechococcus elongatus</i> PCC7942 Synpcc7942_1593 (SEQ ID NO:1), <i>Synechocystis</i> sp. PCC6803 sll0208 (SEQ ID NO:3), <i>Nostoc punctiforme</i> PCC 73102 Npun02004178 (SEQ ID NO:5), <i>Nostoc</i> sp. PCC 7120 alr5283 (SEQ ID NO:7), <i>Acaryochloris marina</i> MBIC11017 AM1_4041 (SEQ ID NO:9), <i>Thermosynechococcus elongatus</i> BP-1 tll1313 (SEQ ID NO:11), <i>Synechococcus</i> sp. JA-3-3A CYA_0415 (SEQ ID NO:13), <i>Gloeobacter violaceus</i> PCC 7421 gll3146 (SEQ ID NO:15), <i>Prochlorococcus marinus</i> MIT9313 PM123 (SEQ ID NO:17), <i>Prochlorococcus marinus</i> subsp. pastoris str. CCMP1986 PMM0532 (SEQ ID NO:19), <i>Prochlorococcus marinus</i> str. NATL2A PMN2A_1863 (SEQ ID NO:21), <i>Synechococcus</i> sp. RS9917 RS9917_09941 (SEQ ID NO:23), <i>Synechococcus</i> sp. RS9917 RS9917_12945 (SEQ ID NO:25), <i>Cyanothece</i> sp. ATCC51142 cce_0778 (SEQ ID NO:27), <i>Cyanothece</i> sp. PCC7245 Cyan7425DRAFT_1220 (SEQ ID NO:29), <i>Cyanothece</i> sp. PCC7245 cce_0778 (SEQ ID NO:31), <i>Anabaena variabilis</i> ATCC29413 YP_323043 (Ava_2533) (SEQ ID NO:33), and <i>Synechococcus elongatus</i> PCC6301 YP_170760 (syc0050_d) (SEQ ID NO:35). Other alkane and alkene biosynthetic genes are listed in Table 1 and <figref idref="f0026 f0027 f0028 f0029">Figure 38</figref>.

0138Aldehyde biosynthetic genes include, for example, <i>Synechococcus elongatus</i> PCC7942 Synpcc7942_1594 (SEQ ID NO:65), <i>Synechocystis</i> sp. PCC6803 sll0209 (SEQ ID NO:67), <i>Cyanothece</i> sp. ATCC51142 cce_1430 (SEQ ID NO:69), <i>Prochlorococcus marinus</i> subsp. pastoris str. CCMP1986 PMM0533 (SEQ ID NO:71), <i>Gloeobacter violaceus</i> PCC7421 NP_96091 (gll3145) (SEQ ID NO:73), <i>Nostoc punctiforme</i> PCC73102 ZP_00108837 (Npun02004176) (SEQ ID NO:75), <i>Anabaena variabilis</i> ATCC29413 YP_323044 (Ava_2534) (SEQ ID NO:77), <i>Synechococcus elongatus</i> PCC6301 YP_170761 (syc0051_d) (SEQ ID NO:79), and <i>Nostoc</i> sp. PCC 7120 alr5284 (SEQ ID NO:81). Other aldehyde biosynthetic genes are listed in Table 1 and <figref idref="f0030 f0031 f0032 f0033 f0034 f0035 f0036">Figure 39</figref>.

0139Using the methods described herein, aldehydes, fatty alcohols, alkanes, and alkenes can be prepared using one or more aldehyde, alkane, and/or alkene biosynthetic genes or polypeptides described herein, or variants thereof, utilizing host cells or cell-free methods. <tables id="tabl0001" num="0001"><img file="EP2288694B1_D0001.tif" /></tables>

Aldehyde, Alkane, and Alkene Biosynthetic Genes and Variants

0140The methods and compositions described herein include, for example, alkane or alkene biosynthetic genes having the nucleotide sequence of SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, or 35, as well as polynucleotide variants thereof. In some instances, the alkane or alkene biosynthetic gene encodes one or more of the amino acid motifs described herein. For example, the alkane or alkene biosynthetic gene can encode a polypeptide comprising SEQ ID NO:37, 38, 39, 41, 42, 43, or 44. The alkane or alkene biosynthetic gene can also include a polypeptide comprising SEQ ID NO:40 and also any one of SEQ ID NO:37, 38, or 39.

0141The methods and compositions described herein also include, for example, aldehyde biosynthetic genes having the nucleotide sequence of SEQ ID NO:65, 67, 69, 71, 73, 75, 77, 79, or 81, as well as polynucleotide variants thereof. In some instances, the aldehyde biosynthetic gene encodes one or more of the amino acid motifs described herein. For example, the aldehyde biosynthetic gene can encode a polypeptide comprising SEQ ID NO:54, 55, 56, 57, 58, 59, 60, 61, 62, 63, or 64.

0142The variants can be naturally occurring or created <i>in vitro.</i> In particular, such variants can be created using genetic engineering techniques, such as site directed mutagenesis, random chemical mutagenesis, Exonuclease III deletion procedures, and standard cloning techniques. Alternatively, such variants, fragments, analogs, or derivatives can be created using chemical synthesis or modification procedures.

0143Methods of making variants are well known in the art. These include procedures in which nucleic acid sequences obtained from natural isolates are modified to generate nucleic acids that encode polypeptides having characteristics that enhance their value in industrial or laboratory applications. In such procedures, a large number of variant sequences having one or more nucleotide differences with respect to the sequence obtained from the natural isolate are generated and characterized. Typically, these nucleotide differences result in amino acid changes with respect to the polypeptides encoded by the nucleic acids from the natural isolates.

0144For example, variants can be created using error prone PCR (see, <i>e.g.,</i><nplcit id="ncit0008" npl-type="s"><text>Leung et al., Technique 1:11-15, 1989</text></nplcit>; and <nplcit id="ncit0009" npl-type="s"><text>Caldwell et al., PCR Methods Applic. 2:28-33, 1992</text></nplcit>). In error prone PCR, PCR is performed 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. Briefly, in such procedures, nucleic acids to be mutagenized (<i>e.g</i>., an aldehyde or alkane biosynthetic polynucleotide sequence), are mixed with PCR primers, reaction buffer, MgCl₂, MnCl₂, Taq polymerase, and an appropriate concentration of dNTPs for achieving a high rate of point mutation along the entire length of the PCR product. For example, the reaction can be performed using 20 fmoles of nucleic acid to be mutagenized (<i>e.g</i>., an aldehyde or alkane biosynthetic polynucleotide sequence), 30 pmole of each PCR primer, a reaction buffer comprising 50 mM KCl, 10 mM Tris HCl (pH 8.3), and 0.01% gelatin, 7 mM MgCl₂, 0.5 mM MnCl₂, 5 units of Taq polymerase, 0.2 mM dGTP, 0.2 mM dATP, 1 mM dCTP, and 1 mM dTTP. PCR can be performed for 30 cycles of 94°C for 1 min, 45°C for 1 min, and 72°C for 1 min. However, it will be appreciated that these parameters can be varied as appropriate. The mutagenized nucleic acids are then cloned into an appropriate vector and the activities of the polypeptides encoded by the mutagenized nucleic acids are evaluated.

0145Variants can also be created using oligonucleotide directed mutagenesis to generate site-specific mutations in any cloned DNA of interest. Oligonucleotide mutagenesis is described in, for example, <nplcit id="ncit0010" npl-type="s"><text>Reidhaar-Olson et al., Science 241:53-57, 1988</text></nplcit>. Briefly, in such procedures a plurality of double stranded oligonucleotides bearing one or more mutations to be introduced into the cloned DNA are synthesized and inserted into the cloned DNA to be mutagenized (<i>e.g</i>., an aldehyde or alkane biosynthetic polynucleotide sequence). Clones containing the mutagenized DNA are recovered, and the activities of the polypeptides they encode are assessed.

0146Another method for generating variants is assembly PCR. Assembly PCR involves the assembly of a PCR product from a mixture of small DNA fragments. A large number of different PCR reactions occur in parallel in the same vial, with the products of one reaction priming the products of another reaction. Assembly PCR is described in, for example, <patcit id="pcit0003" dnum="US5965408A"><text>U.S. Pat. No. 5,965,408</text></patcit>.

0147Still another method of generating variants is sexual PCR mutagenesis. In sexual PCR mutagenesis, forced homologous recombination occurs between DNA molecules of different, but highly related, DNA sequence <i>in vitro</i> as a result of random fragmentation of the DNA molecule based on sequence homology. This is followed by fixation of the crossover by primer extension in a PCR reaction. Sexual PCR mutagenesis is described in, for example, <nplcit id="ncit0011" npl-type="s"><text>Stemmer, PNAS, USA 91:10747-10751, 1994</text></nplcit>.

0148Variants can also be created by <i>in vivo</i> mutagenesis. In some embodiments, random mutations in a nucleic acid sequence are generated by propagating the sequence in a bacterial strain, such as an <i>E. coli</i> strain, which carries mutations in one or more of the DNA repair pathways. Such "mutator" strains have a higher random mutation rate than that of a wild-type strain. Propagating a DNA sequence (<i>e.g</i>., an aldehyde or alkane biosynthetic polynucleotide sequence) in one of these strains will eventually generate random mutations within the DNA. Mutator strains suitable for use for <i>in vivo</i> mutagenesis are described in, for example, <patcit id="pcit0004" dnum="WO9116427A"><text>PCT Publication No. WO 91/16427</text></patcit>.

0149Variants can also be generated using cassette mutagenesis. In cassette mutagenesis, a small region of a double stranded DNA molecule is replaced with a synthetic oligonucleotide "cassette" that differs from the native sequence. The oligonucleotide often contains a completely and/or partially randomized native sequence.

0150Recursive ensemble mutagenesis can also be used to generate variants. Recursive ensemble mutagenesis is an algorithm for protein engineering (<i>i.e.,</i> protein mutagenesis) developed to produce diverse populations of phenotypically related mutants whose members differ in amino acid sequence. This method uses a feedback mechanism to control successive rounds of combinatorial cassette mutagenesis. Recursive ensemble mutagenesis is described in, for example, <nplcit id="ncit0012" npl-type="s"><text>Arkin et al., PNAS, USA 89:7811-7815, 1992</text></nplcit>.

0151In some embodiments, variants are created using exponential ensemble mutagenesis. Exponential ensemble mutagenesis is a process for generating combinatorial libraries with a high percentage of unique and functional mutants, wherein small groups of residues are randomized in parallel to identify, at each altered position, amino acids which lead to functional proteins. Exponential ensemble mutagenesis is described in, for example, <nplcit id="ncit0013" npl-type="s"><text>Delegrave et al., Biotech. Res. 11:1548-1552, 1993</text></nplcit>. Random and site-directed mutagenesis are described in, for example, <nplcit id="ncit0014" npl-type="s"><text>Arnold, Curr. Opin. Biotech. 4:450-455, 1993</text></nplcit>.

0152In some embodiments, variants are created using shuffling procedures wherein portions of a plurality of nucleic acids that encode distinct polypeptides are fused together to create chimeric nucleic acid sequences that encode chimeric polypeptides as described in, for example, <patcit id="pcit0005" dnum="US5965408A"><text>U.S. Pat. Nos. 5,965,408 </text></patcit>and <patcit id="pcit0006" dnum="US5939250A"><text>5,939,250</text></patcit>.

0153Polynucleotide variants also include nucleic acid analogs. Nucleic acid analogs can be modified at the base moiety, sugar moiety, or phosphate backbone to improve, for example, stability, hybridization, or solubility of the nucleic acid. Modifications at the base moiety include deoxyuridine for deoxythymidine and 5-methyl-2'-deoxycytidine or 5-bromo-2'-doxycytidine for deoxycytidine. Modifications of the sugar moiety include modification of the 2' hydroxyl of the ribose sugar to form 2'-O-methyl or 2'-O-allyl sugars. The deoxyribose phosphate backbone can be modified to produce morpholino nucleic acids, in which each base moiety is linked to a six-membered, morpholino ring, or peptide nucleic acids, in which the deoxyphosphate backbone is replaced by a pseudopeptide backbone and the four bases are retained. (See, <i>e.g.,</i><nplcit id="ncit0015" npl-type="s"><text>Summerton et al., Antisense Nucleic Acid Drug Dev. (1997) 7:187-195</text></nplcit>; and <nplcit id="ncit0016" npl-type="s"><text>Hyrup et al., Bioorgan. Med. Chem. (1996) 4:5-23</text></nplcit>.) In addition, the deoxyphosphate backbone can be replaced with, for example, a phosphorothioate or phosphorodithioate backbone, a phosphoroamidite, or an alkyl phosphotriester backbone.

0154The aldehyde and alkane biosynthetic polypeptides Synpcc7942_1594 (SEQ ID NO:66) and Synpcc7942_1593 (SEQ ID NO:2) have homologs in other cyanobacteria (nonlimiting examples are depicted in Table 1). Thus, any polynucleotide sequence encoding a homolog listed in Table 1, or a variant thereof, can be used as an aldehyde or alkane biosynthetic polynucleotide in the methods described herein. Each cyanobacterium listed in Table 1 has copies of both genes. The level of sequence identity of the gene products ranges from 61% to 73% for Synpcc7942_1594 (SEQ ID NO:66) and from 43% to 78% for Synpcc7942_1593 (SEQ ID NO:2).

0155Further homologs of the aldehyde biosynthetic polypeptide Synpcc7942_1594 (SEQ ID NO:66) are listed in <figref idref="f0030 f0031 f0032 f0033 f0034 f0035 f0036">Figure 39</figref>, and any polynucleotide sequence encoding a homolog listed in <figref idref="f0030 f0031 f0032 f0033 f0034 f0035 f0036">Figure 39</figref>, or a variant thereof, can be used as an aldehyde biosynthetic polynucleotide in the methods described herein. Further homologs of the alkane biosynthetic polypeptide Synpcc7942_1593 (SEQ ID NO:2) are listed in <figref idref="f0026 f0027 f0028 f0029">Figure 38</figref>, and any polynucleotide sequence encoding a homolog listed in <figref idref="f0026 f0027 f0028 f0029">Figure 38</figref>, or a variant thereof, can be used as an alkane biosynthetic polynucleotide in the methods described herein.

0156In certain instances, an aldehyde, alkane, and/or alkene biosynthetic gene is codon optimized for expression in a particular host cell. For example, for expression in <i>E. coli</i>, one or more codons can be optimized as described in, <i>e.g.,</i><nplcit id="ncit0017" npl-type="s"><text>Grosjean et al., Gene 18:199-209 (1982</text></nplcit>).

Aldehyde, Alkane, and Alkene Biosynthetic Polypeptides and Variants

0157The methods and compositions described herein also include alkane or alkene biosynthetic polypeptides having the amino acid sequence of SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36, as well as polypeptide variants thereof. In some instances, an alkane or alkene biosynthetic polypeptide is one that includes one or more of the amino acid motifs described herein. For example, the alkane or alkene biosynthetic polypeptide can include the amino acid sequence of SEQ ID NO:37, 38, 39, 41, 42, 43, or 44. The alkane or alkene biosynthetic polypeptide can also include the amino acid sequence of SEQ ID NO:40 and also any one of SEQ ID NO:37, 38, or 39.

0158The methods and compositions described herein also include aldehyde biosynthetic polypeptides having the amino acid sequence of SEQ ID NO:66, 68, 70, 72, 74, 76, 78, 80, or 82, as well as polypeptide variants thereof. In some instances, an aldehyde biosynthetic polypeptide is one that includes one or more of the amino acid motifs described herein. For example, the aldehyde biosynthetic polypeptide can include the amino acid sequence of SEQ ID NO:54, 55, 56, 57, 58, 59, 60, 61, 62, 63, or 64.

0159Aldehyde, alkane, and alkene biosynthetic polypeptide variants can be variants in which one or more amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue). Such substituted amino acid residue may or may not be one encoded by the genetic code.

0160Conservative substitutions are those that substitute a given amino acid in a polypeptide by another amino acid of similar characteristics. Typical conservative substitutions are the following replacements: replacement of an aliphatic amino acid, such as alanine, valine, leucine, and isoleucine, with another aliphatic amino acid; replacement of a serine with a threonine or vice versa; replacement of an acidic residue, such as aspartic acid and glutamic acid, with another acidic residue; replacement of a residue bearing an amide group, such as asparagine and glutamine, with another residue bearing an amide group; exchange of a basic residue, such as lysine and arginine, with another basic residue; and replacement of an aromatic residue, such as phenylalanine and tyrosine, with another aromatic residue.

0161Other polypeptide variants are those in which one or more amino acid residues include a substituent group. Still other polypeptide variants are those in which the polypeptide is associated with another compound, such as a compound to increase the half-life of the polypeptide (<i>e.g</i>., polyethylene glycol).

0162Additional polypeptide variants are those in which additional amino acids are fused to the polypeptide, such as a leader sequence, a secretory sequence, a proprotein sequence, or a sequence which facilitates purification, enrichment, or stabilization of the polypeptide.

0163In some instances, an alkane or alkene biosynthetic polypeptide variant retains the same biological function as a polypeptide having the amino acid sequence of SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36 (<i>e.g.,</i> retains alkane or alkene biosynthetic activity) and has an amino acid sequence substantially identical thereto.

0164In other instances, the alkane or alkene biosynthetic polypeptide variants have at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or more than about 95% homology to the amino acid sequence of SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36. In another embodiment, the polypeptide variants include a fragment comprising at least about 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150 consecutive amino acids thereof.

0165In some instances, an aldehyde biosynthetic polypeptide variant retains the same biological function as a polypeptide having the amino acid sequence of SEQ ID NO:66, 68, 70, 72, 74, 76, 78, 80, or 82 (<i>e.g</i>., retains aldehyde biosynthetic activity) and has an amino acid sequence substantially identical thereto.

0166In yet other instances, the aldehyde biosynthetic polypeptide variants have at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or more than about 95% homology to the amino acid sequence of SEQ ID NO:66, 68, 70, 72, 74, 76, 78, 80, or 82. In another embodiment, the polypeptide variants include a fragment comprising at least about 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150 consecutive amino acids thereof.

0167The polypeptide variants or fragments thereof can be obtained by isolating nucleic acids encoding them using techniques described herein or by expressing synthetic nucleic acids encoding them. Alternatively, polypeptide variants or fragments thereof can be obtained through biochemical enrichment or purification procedures. The sequence of polypeptide variants or fragments can be determined by proteolytic digestion, gel electrophoresis, and/or microsequencing. The sequence of the alkane or alkene biosynthetic polypeptide variants or fragments can then be compared to the amino acid sequence of SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36 using any of the programs described herein. The sequence of the aldehyde biosynthetic polypeptide variants or fragments can be compared to the amino acid sequence of SEQ ID NO:66, 68, 70, 72, 74, 76, 78, 80, or 82 using any of the programs described herein.

0168The polypeptide variants and fragments thereof can be assayed for aldehyde-, fatty alcohol-, alkane-, and/or alkene-producing activity using routine methods. For example, the polypeptide variants or fragment can be contacted with a substrate (<i>e.g</i>., a fatty acid derivative substrate or other substrate described herein) under conditions that allow the polypeptide variant to function. A decrease in the level of the substrate or an increase in the level of an aldehyde, alkane, or alkene can be measured to determine aldehyde-, fatty alcohol-, alkane-, or alkene-producing activity, respectively.

Anti-Aldehyde, Anti-Fatty Alcohol, Anti-Alkane, and Anti-Alkene Biosynthetic Polypeptide Antibodies

0169The aldehyde, fatty alcohol, alkane, and alkene biosynthetic polypeptides described herein can also be used to produce antibodies directed against aldehyde, fatty alcohol, alkane, and alkene biosynthetic polypeptides. Such antibodies can be used, for example, to detect the expression of an aldehyde, fatty alcohol, alkane, or alkene biosynthetic polypeptide using methods known in the art. The antibody can be, <i>e.g.,</i> a polyclonal antibody; a monoclonal antibody or antigen binding fragment thereof; a modified antibody such as a chimeric antibody, reshaped antibody, humanized antibody, or fragment thereof (<i>e.g</i>., Fab', Fab, F(ab')₂); or a biosynthetic antibody, <i>e.g</i>., a single chain antibody, single domain antibody (DAB), Fv, single chain Fv (scFv), or the like.

0170Methods of making and using polyclonal and monoclonal antibodies are described, <i>e.g.,</i> in<nplcit id="ncit0018" npl-type="b"><text> Harlow et al., Using Antibodies: A Laboratory Manual: Portable Protocol I. Cold Spring Harbor Laboratory (December 1, 1998</text></nplcit>). Methods for making modified antibodies and antibody fragments (<i>e.g</i>., chimeric antibodies, reshaped antibodies, humanized antibodies, or fragments thereof, <i>e.g.,</i> Fab', Fab, F(ab')₂ fragments); or biosynthetic antibodies (<i>e.g</i>., single chain antibodies, single domain antibodies (DABs), Fv, single chain Fv (scFv), and the like), are known in the art and can be found, <i>e.g.,</i> in <nplcit id="ncit0019" npl-type="b"><text>Zola, Monoclonal Antibodies: Preparation and Use of Monoclonal Antibodies and Engineered Antibody Derivatives, Springer Verlag (December 15, 2000; 1st editi</text></nplcit>on).

Substrates

0171The compositions and methods described herein can be used to produce aldehydes, fatty alcohols, alkanes, and/or alkenes from an appropriate substrate. While not wishing to be bound by a particular theory, it is believed that the alkane or alkene biosynthetic polypeptides described herein produce alkanes or alkenes from substrates <i>via</i> a decarbonylation mechanism. In some instances, the substrate is a fatty acid derivative, <i>e.g</i>., a fatty aldehyde, and an alkane having particular branching patterns and carbon chain length can be produced from a fatty acid derivative, <i>e.g</i>., a fatty aldehyde, having those particular characteristics. In other instances, the substrate is an unsaturated fatty acid derivative, <i>e.g</i>., an unsaturated fatty aldehyde, and an alkene having particular branching patterns and carbon chain length can be produced from an unsaturated fatty acid derivative, <i>e.g</i>., an unsaturated fatty aldehyde, having those particular characteristics.

0172While not wishing to be bound by a particular theory, it is believed that the aldehyde biosynthetic polypeptides described herein produce aldehydes from substrates <i>via</i> a reduction mechanism. In certain instances, the substrate is an acyl-ACP.

0173While not wishing to be bound by a particular theory, it is believed that the fatty alcohols described herein are produced from substrates <i>via</i> a reduction mechanism. In certain instances, the substrate is a fatty aldehyde.

0174Accordingly, each step within a biosynthetic pathway that leads to the production of these substrates can be modified to produce or overproduce the substrate of interest. For example, known genes involved in the fatty acid biosynthetic pathway, the fatty aldehyde pathway, and the fatty alcohol pathway can be expressed, overexpressed, or attenuated in host cells to produce a desired substrate (see, <i>e.g.</i>, <patcit id="pcit0007" dnum="US08058788W"><text>PCT/US08/058788</text></patcit>). Exemplary genes are provided in <figref idref="f0037 f0038 f0039 f0040 f0041 f0042 f0043 f0044 f0045 f0046 f0047 f0048 f0049 f0050 f0051 f0052 f0053 f0054 f0055 f0056 f0057 f0058 f0059">Figure 40</figref>.

Synthesis of Substrates

0175Fatty acid synthase (FAS) is a group of polypeptides that catalyze the initiation and elongation of acyl chains (<nplcit id="ncit0020" npl-type="s"><text>Marrakchi et al., Biochemical Society, 30:1050-1055, 2002</text></nplcit>). The acyl carrier protein (ACP) along with the enzymes in the FAS pathway control the length, degree of saturation, and branching of the fatty acid derivatives produced. The fatty acid biosynthetic pathway involves the precursors acetyl-CoA and malonyl-CoA. The steps in this pathway are catalyzed by enzymes of the fatty acid biosynthesis (<i>fab</i>) and acetyl-CoA carboxylase (<i>acc</i>) gene families (see, <i>e.g.,</i><nplcit id="ncit0021" npl-type="s"><text>Heath et al., Prog. Lipid Res. 40(6):467-97 (2001</text></nplcit>)).

0176Host cells can be engineered to express fatty acid derivative substrates by recombinantly expressing or overexpressing acetyl-CoA and/or malonyl-CoA synthase genes. For example, to increase acetyl-CoA production, one or more of the following genes can be expressed in a host cell: <i>pdh, panK, aceEF</i> (encoding the E1p dehydrogenase component and the E2p dihydrolipoamide acyltransferase component of the pyruvate and 2-oxoglutarate dehydrogenase complexes), <i>fabH</i>, <i>fabD, fabG, acpP,</i> and <i>fabF.</i> Exemplary GenBank accession numbers for these genes are: <i>pdh</i> (BAB34380, AAC73227, AAC73226), <i>panK</i> (also known as coaA, AAC76952), <i>aceEF</i> (AAC73227, AAC73226), <i>fabH</i> (AAC74175), <i>fabD</i> (AAC74176), <i>fabG</i> (AAC74177), <i>acpP</i> (AAC74178), <i>fabF</i> (AAC74179). Additionally, the expression levels of <i>fadE, gpsA, ldhA, pflb, adhE, pta, poxB, ackA,</i> and/or <i>ackB</i> can be attenuated or knocked-out in an engineered host cell by transformation with conditionally replicative or non-replicative plasmids containing null or deletion mutations of the corresponding genes or by substituting promoter or enhancer sequences. Exemplary GenBank accession numbers for these genes are: <i>fadE</i> (AAC73325), <i>gspA</i> (AAC76632), <i>ldhA</i> (AAC74462), <i>pflb</i> (AAC73989), <i>adhE</i> (AAC74323), <i>pta</i> (AAC75357), <i>poxB</i> (AAC73958), <i>ackA</i> (AAC75356), and <i>ackB</i> (BAB81430). The resulting host cells will have increased acetyl-CoA production levels when grown in an appropriate environment.

0177Malonyl-CoA overexpression can be effected by introducing <i>accABCD</i> (<i>e.g</i>., accession number AAC73296, EC 6.4.1.2) into a host cell. Fatty acids can be further overexpressed in host cells by introducing into the host cell a DNA sequence encoding a lipase (<i>e.g</i>., accession numbers CAA89087, CAA98876).

0178In addition, inhibiting PlsB can lead to an increase in the levels of long chain acyl-ACP, which will inhibit early steps in the pathway (<i>e.g</i>., accABCD, fabH, and fabI). The <i>plsB</i> (<i>e.g.,</i> accession number AAC77011) D311E mutation can be used to increase the amount of available acyl-CoA.

0179In addition, a host cell can be engineered to overexpress a <i>sfa</i> gene (suppressor of fabA, <i>e.g</i>., accession number AAN79592) to increase production of monounsaturated fatty acids (<nplcit id="ncit0022" npl-type="s"><text>Rock et al., J. Bacteriology 178:5382-5387, 1996</text></nplcit>).

0180In some instances, host cells can be engineered to express, overexpress, or attenuate expression of a thioesterase to increase fatty acid substrate production. The chain length of a fatty acid substrate is controlled by thioesterase. In some instances, a <i>tes</i> or <i>fat</i> gene can be overexpressed. In other instances, C₁₀ fatty acids can be produced by attenuating thioesterase C₁₈ (<i>e.g</i>., accession numbers AAC73596 and P0ADA1), which uses C_18:1-ACP, and expressing thioesterase C₁₀ (<i>e.g.,</i> accession number Q39513), which uses C₁₀-ACP. This results in a relatively homogeneous population of fatty acids that have a carbon chain length of 10. In yet other instances, C₁₄ fatty acids can be produced by attenuating endogenous thioesterases that produce non-C₁₄ fatty acids and expressing the thioesterases, that use C₁₄-ACP (for example, accession number Q39473). In some situations, C₁₂ fatty acids can be produced by expressing thioesterases that use C₁₂-ACP (for example, accession number Q41635) and attenuating thioesterases that produce non-C₁₂ fatty acids. Acetyl-CoA, malonyl-CoA, and fatty acid overproduction can be verified using methods known in the art, for example, by using radioactive precursors, HPLC, and GC-MS subsequent to cell lysis. Non-limiting examples of thioesterases that can be used in the methods described herein are listed in Table 2. <tables id="tabl0002" num="0002"><table frame="all"><title><b>Table 2: Thioesterases</b></title><tgroup cols="4"><colspec colnum="1" colname="col1" colwidth="35mm" /><colspec colnum="2" colname="col2" colwidth="42mm" /><colspec colnum="3" colname="col3" colwidth="45mm" /><colspec colnum="4" colname="col4" colwidth="45mm" /><thead><row><entry valign="top"><u>Accession Number</u></entry><entry valign="top"><u>Source Organism</u></entry><entry valign="top"><u>Gene</u></entry><entry valign="top"><u>Preferential product produced</u></entry></row></thead><tbody><row><entry><u>AAC73596</u></entry><entry><i><u>E. coli</u></i></entry><entry><u><i>tesA</i> without leader sequence</u></entry><entry><u>C_18:1</u></entry></row><row><entry><u>AAC73555</u></entry><entry><i><u>E. coli</u></i></entry><entry><i><u>tesB</u></i></entry><entry /></row><row><entry><u>Q41635, AAA34215</u></entry><entry><i><u>Umbellularia california</u></i></entry><entry><i><u>fatB</u></i></entry><entry><u>C_12:0</u></entry></row><row><entry><u>Q39513; AAC49269</u></entry><entry><i><u>Cuphea hookeriana</u></i></entry><entry><i><u>fatB2</u></i></entry><entry><u>C_8:0 - C_10:0</u></entry></row><row><entry><u>AAC49269; AAC72881</u></entry><entry><i><u>Cuphea hookeriana</u></i></entry><entry><i><u>fatB3</u></i></entry><entry><u>C_14:0 - C_16:0</u></entry></row><row><entry><u>Q39473, AAC49151</u></entry><entry><i><u>Cinnamonum camphorum</u></i></entry><entry><i><u>fatB</u></i></entry><entry><u>C_14:0</u></entry></row><row><entry><u>CAA85388</u></entry><entry><i><u>Arabidopsis thaliana</u></i></entry><entry><u><i>fatB</i> [M141T]*</u></entry><entry><u>C_16:1</u></entry></row><row><entry><u>NP 189147: NP 193041</u></entry><entry><i><u>Arabidopsis thaliana</u></i></entry><entry><i><u>fatA</u></i></entry><entry><u>C_18:1</u></entry></row><row><entry><u>CAC39106</u></entry><entry><i><u>Bradyrhiizobium japonicum</u></i></entry><entry><i><u>fatA</u></i></entry><entry><u>C_18:1</u></entry></row><row><entry><u>AAC72883</u></entry><entry><i><u>Cuphea hookeriana</u></i></entry><entry><i><u>fatA</u></i></entry><entry><u>C_18:1</u></entry></row><row><entry><u>AAL79361</u></entry><entry><i><u>Helianthus annus</u></i></entry><entry><i><u>fatAl</u></i></entry><entry /></row></tbody></tgroup><tgroup cols="4" rowsep="0"><colspec colnum="1" colname="col1" colwidth="35mm" /><colspec colnum="2" colname="col2" colwidth="42mm" /><colspec colnum="3" colname="col3" colwidth="45mm" /><colspec colnum="4" colname="col4" colwidth="45mm" /><tbody><row><entry namest="col1" nameend="col4" align="justify">* <nplcit id="ncit0023" npl-type="s"><text>Mayer et al., BMC Plant Biology 7:1-11, 2007</text></nplcit></entry></row></tbody></tgroup></table></tables>

Formation of Branched Aldehydes, Fatty Alcohols, Alkanes, and Alkenes

0181Aldehydes, fatty alcohols, alkanes, an alkenes can be produced that contain branch points by using branched fatty acid derivatives as substrates. For example, although <i>E. coli</i> naturally produces straight chain fatty acid derivatives (sFAs), <i>E. coli</i> can be engineered to produce branched chain fatty acid derivatives (brFAs) by introducing and expressing or overexpressing genes that provide branched precursors in the <i>E. coli</i> (<i>e.g., bkd, ilv, icm,</i> and <i>fab</i> gene families). Additionally, a host cell can be engineered to express or overexpress genes encoding proteins for the elongation of brFAs (<i>e.g</i>., ACP, FabF, <i>etc</i>.) and/or to delete or attenuate the corresponding host cell genes that normally lead to sFAs.

0182The first step in forming brFAs is the production of the corresponding α-keto acids by a branched-chain amino acid aminotransferase. Host cells may endogenously include genes encoding such enzymes or such genes can be recombinantly introduced. <i>E. coli,</i> for example, endogenously expresses such an enzyme, IlvE (EC 2.6.1.42; GenBank accession YP_026247). In some host cells, a heterologous branched-chain amino acid aminotransferase may not be expressed. However, <i>E. coli</i> IlvE or any other branched-chain amino acid aminotransferase (<i>e.g.,</i> IlvE from <i>Lactococcus lactis</i> (GenBank accession AAF34406), IlvE from <i>Pseudomonas putida</i> (GenBank accession NP_745648), or IlvE from <i>Streptomyces coelicolor</i> (GenBank accession NP_629657)), if not endogenous, can be introduced and recombinantly expressed.

0183The second step is the oxidative decarboxylation of the α-ketoacids to the corresponding branched-chain acyl-CoA. This reaction can be catalyzed by a branched-chain α-keto acid dehydrogenase complex (<i>bkd</i>; EC 1.2.4.4.) (<nplcit id="ncit0024" npl-type="s"><text>Denoya et al., J. Bacteriol. 177:3504, 1995</text></nplcit>), which consists of E1α/β (decarboxylase), E2 (dihydrolipoyl transacylase), and E3 (dihydrolipoyl dehydrogenase) subunits. These branched-chain α-keto acid dehydrogenase complexes are similar to pyruvate and α-ketoglutarate dehydrogenase complexes. Any microorganism that possesses brFAs and/or grows on branched-chain amino acids can be used as a source to isolate <i>bkd</i> genes for expression in host cells, for example, <i>E. coli.</i> Furthermore, <i>E. coli</i> has the E3 component as part of its pyruvate dehydrogenase complex (<i>lpd,</i> EC 1.8.1.4, GenBank accession NP_414658). Thus, it can be sufficient to express only the <i>E1 α</i>/<i>β</i> and <i>E2 bkd</i> genes. Table 3 lists non-limiting examples of <i>bkd</i> genes from several microorganisms that can be recombinantly introduced and expressed in a host cell to provide branched-chain acyl-CoA precursors. <tables id="tabl0003" num="0003"><table frame="all"><title><b>Table 3: <i>Bkd</i> genes from selected microorganisms</b></title><tgroup cols="3"><colspec colnum="1" colname="col1" colwidth="39mm" /><colspec colnum="2" colname="col2" colwidth="23mm" /><colspec colnum="3" colname="col3" colwidth="36mm" /><thead><row><entry valign="top">Organism</entry><entry valign="top">Gene</entry><entry valign="top">GenBank Accession #</entry></row></thead><tbody><row rowsep="0"><entry morerows="2" rowsep="1"><i>Streptomyces coelicolor</i></entry><entry><i>bkdA1</i> (E1α)</entry><entry>NP_628006</entry></row><row rowsep="0"><entry><i>bkdB1</i> (E1β)</entry><entry>NP_628005</entry></row><row><entry><i>bkdC1</i> (E2)</entry><entry>NP_638004</entry></row><row rowsep="0"><entry morerows="2" rowsep="1"><i>Streptomyces coelicolor</i></entry><entry><i>bkdA2</i> (E1α)</entry><entry>NP_733618</entry></row><row rowsep="0"><entry><i>bkdB2</i> (E1β)</entry><entry>NP_628019</entry></row><row><entry><i>bkdC2</i> (E2)</entry><entry>NP_628018</entry></row><row rowsep="0"><entry morerows="2" rowsep="1"><i>Streptomyces avermitilis</i></entry><entry><i>bkdA</i> (E1a)</entry><entry>BAC72074</entry></row><row rowsep="0"><entry><i>bkdB</i> (E1b)</entry><entry>BAC72075</entry></row><row><entry><i>bkdC</i> (E2)</entry><entry>BAC72076</entry></row><row rowsep="0"><entry morerows="2" rowsep="1"><i>Streptomyces avermitilis</i></entry><entry><i>bkdF</i> (E1α)</entry><entry>BAC72088</entry></row><row rowsep="0"><entry><i>bkdG</i> (E1β)</entry><entry>BAC72089</entry></row><row><entry><i>bkdH</i> (E2)</entry><entry>BAC72090</entry></row><row rowsep="0"><entry morerows="2" rowsep="1"><i>Bacillus subtilis</i></entry><entry><i>bkdAA</i> (E1α)</entry><entry>NP_390288</entry></row><row rowsep="0"><entry><i>bkdAB</i> (E1β)</entry><entry>NP_390288</entry></row><row><entry><i>bkdB</i> (E2)</entry><entry>NP_390288</entry></row><row rowsep="0"><entry morerows="2" rowsep="1"><i>Pseudomonas putida</i></entry><entry><i>bkdA1</i> (E1α)</entry><entry>AAA65614</entry></row><row rowsep="0"><entry><i>bkdA2</i> (E1β)</entry><entry>AAA65615</entry></row><row><entry><i>bkdC</i> (E2)</entry><entry>AAA65617</entry></row></tbody></tgroup></table></tables>

0184In another example, isobutyryl-CoA can be made in a host cell, for example in <i>E. coli,</i> through the coexpression of a crotonyl-CoA reductase (Ccr, EC 1.6.5.5, 1.1.1.1) and isobutyryl-CoA mutase (large subunit IcmA, EC 5.4.99.2; small subunit IcmB, EC 5.4.99.2) (<nplcit id="ncit0025" npl-type="s"><text>Han and Reynolds, J. Bacteriol. 179:5157, 1997</text></nplcit>). Crotonyl-CoA is an intermediate in fatty acid biosynthesis in <i>E. coli</i> and other microorganisms. Non-limiting examples of <i>ccr</i> and <i>icm</i> genes from selected microorganisms are listed in Table 4. <tables id="tabl0004" num="0004"><table frame="all"><title><b>Table 4: <i>Ccr</i> and <i>icm</i> genes from selected microorganisms</b></title><tgroup cols="3"><colspec colnum="1" colname="col1" colwidth="46mm" /><colspec colnum="2" colname="col2" colwidth="24mm" /><colspec colnum="3" colname="col3" colwidth="36mm" /><thead><row><entry valign="top"><u>Organism</u></entry><entry valign="top"><u>Gene</u></entry><entry valign="top"><u>GenBank Accession #</u></entry></row></thead><tbody><row rowsep="0"><entry morerows="2" rowsep="1"><i><u>Streptomyces coelicolor</u></i></entry><entry><i><u>Ccr</u></i></entry><entry><u>NP_</u><u>630556</u></entry></row><row rowsep="0"><entry><i><u>icmA</u></i></entry><entry><u>NP_</u><u>629554</u></entry></row><row><entry><i><u>icmB</u></i></entry><entry><u>NP_</u><u>630904</u></entry></row><row rowsep="0"><entry morerows="2" rowsep="1"><i><u>Streptomyces</u><u>cinnamonensis</u></i></entry><entry><i><u>ccr</u></i></entry><entry><u>AAD53915</u></entry></row><row rowsep="0"><entry><i><u>icmA</u></i></entry><entry><u>AAC08713</u></entry></row><row><entry><i><u>icmB</u></i></entry><entry><u>AJ246005</u></entry></row></tbody></tgroup></table></tables>

0185In addition to expression of the <i>bkd</i> genes, the initiation of brFA biosynthesis utilizes β-ketoacyl-acyl-carrier-protein synthase III (FabH, EC 2.3.1.41) with specificity for branched chain acyl-CoAs (<nplcit id="ncit0026" npl-type="s"><text>Li et al., J. Bacteriol. 187:3795-3799, 2005</text></nplcit>). Non-limiting examples of such FabH enzymes are listed in Table 5. <i>fabH</i> genes that are involved in fatty acid biosynthesis of any brFA-containing microorganism can be expressed in a host cell. The Bkd and FabH enzymes from host cells that do not naturally make brFA may not support brFA production. Therefore, <i>bkd</i> and <i>fabH</i> can be expressed recombinantly. Vectors containing the <i>bkd</i> and <i>fabH</i> genes can be inserted into such a host cell. Similarly, the endogenous level of Bkd and FabH production may not be sufficient to produce brFA. In this case, they can be overexpressed. Additionally, other components of the fatty acid biosynthesis pathway can be expressed or overexpressed, such as acyl carrier proteins (ACPs) and β-ketoacyl-acyl-carrier-protein synthase II (<i>fabF</i>, EC 2.3.1.41) (non-limiting examples of candidates are listed in Table 5). In addition to expressing these genes, some genes in the endogenous fatty acid biosynthesis pathway can be attenuated in the host cell <i>(e.g.,</i> the <i>E. coli</i> genes <i>fabH</i> (GenBank accession # NP_415609) and/or <i>fabF</i> (GenBank accession # NP_415613)). <tables id="tabl0005" num="0005"><table frame="all"><title><b>Table 5: <i>FabH, ACP and fabF</i> genes from selected microorganisms with brFAs</b></title><tgroup cols="3"><colspec colnum="1" colname="col1" colwidth="48mm" /><colspec colnum="2" colname="col2" colwidth="43mm" /><colspec colnum="3" colname="col3" colwidth="36mm" /><thead><row><entry valign="top"><u>Organism</u></entry><entry valign="top"><u>Gene</u></entry><entry valign="top"><u>GenBank Accession #</u></entry></row></thead><tbody><row rowsep="0"><entry morerows="2" rowsep="1"><i><u>Streptomyces coelicolor</u></i></entry><entry><i><u>fabH1</u></i></entry><entry><u>NP_626634</u></entry></row><row rowsep="0"><entry><i><u>ACP</u></i></entry><entry><u>NP_626635</u></entry></row><row><entry><i><u>fabF</u></i></entry><entry><u>NP_626636</u></entry></row><row rowsep="0"><entry morerows="2" rowsep="1"><i><u>Streptomyces avermitilis</u></i></entry><entry><i><u>fabH3</u></i></entry><entry><u>NP_823466</u></entry></row><row rowsep="0"><entry><i><u>fabC3 (ACP)</u></i></entry><entry><u>NP_823467</u></entry></row><row><entry><i><u>fabF</u></i></entry><entry><u>NP_823468</u></entry></row><row rowsep="0"><entry morerows="3" rowsep="1"><u><i>Bacillus subtilis</i></u></entry><entry><i><u><b>f</b>abH_A</u></i></entry><entry><u>NP_389015</u></entry></row><row rowsep="0"><entry><u><i>fabH_ B</i></u></entry><entry><u>NP_388898</u></entry></row><row rowsep="0"><entry><i><u>ACP</u></i></entry><entry><u>NP_389474</u></entry></row><row><entry><i><u>fabF</u></i></entry><entry><u>NP_389016</u></entry></row><row rowsep="0"><entry morerows="2" rowsep="1"><u><i>Stenotrophomonas maltophilia</i></u></entry><entry><u>SmalDRAFT_0818 (<i>FabH</i>)</u></entry><entry><u>ZP_01643059</u></entry></row><row rowsep="0"><entry><u>SmalDRAFT_0821 (<i>ACP</i>)</u></entry><entry><u>ZP_01643063</u></entry></row><row><entry><u>SmalDRAFT_0822 (<i>FabF</i>)</u></entry><entry><u>ZP_01643064</u></entry></row><row rowsep="0"><entry morerows="2" rowsep="1"><i><u>Legionella pneumophila</u></i></entry><entry><i><u>FabH</u></i></entry><entry><u>YP_123672</u></entry></row><row rowsep="0"><entry><i><u>ACP</u></i></entry><entry><u>YP_123675</u></entry></row><row><entry><i><u>fabF</u></i></entry><entry><u>YP_123676</u></entry></row></tbody></tgroup></table></tables>

Formation of Cyclic Aldehydes, Fatty Alcohols, Alkanes, and Alkenes

0186Cyclic aldehydes, fatty alcohols, alkanes, and alkenes can be produced by using cyclic fatty acid derivatives as substrates. To produce cyclic fatty acid derivative substrates, genes that provide cyclic precursors <i>(e.g.,</i> the <i>ans</i>, <i>chc,</i> and <i>plm</i> gene families) can be introduced into the host cell and expressed to allow initiation of fatty acid biosynthesis from cyclic precursors. For example, to convert a host cell, such as <i>E. coli,</i> into one capable of synthesizing ω-cyclic fatty acid derivatives (cyFA), a gene that provides the cyclic precursor cyclohexylcarbonyl-CoA (CHC-CoA) (<nplcit id="ncit0027" npl-type="s"><text>Cropp et al., Nature Biotech. 18:980-983, 2000</text></nplcit>) can be introduced and expressed in the host cell. Non-limiting examples of genes that provide CHC-CoA in <i>E. coli</i> include: <i>ansJ</i>, <i>ansK</i>, <i>ansL</i>, <i>chcA</i>, and <i>ansM</i> from the ansatrienin gene cluster of <i>Streptomyces collinus</i> (<nplcit id="ncit0028" npl-type="s"><text>Chen et al., Eur. J. Biochem. 261: 98-107, 1999</text></nplcit>) or <i>plmJ, plmK, plmL, chcA,</i> and <i>plmM</i> from the phoslactomycin B gene cluster of <i>Streptomyces</i> sp. HK803 (<nplcit id="ncit0029" npl-type="s"><text>Palaniappan et al., J. Biol. Chem. 278:35552-35557, 2003</text></nplcit>) together with the <i>chcB</i> gene (<nplcit id="ncit0030" npl-type="s"><text>Patton et al., Biochem. 39:7595-7604, 2000</text></nplcit>) from <i>S</i>. <i>collinus, S. avermitilis,</i> or <i>S. coelicolor</i> (<i>see</i> Table 6). The genes listed in Table 5 can then be expressed to allow initiation and elongation of ω-cyclic fatty acids. Alternatively, the homologous genes can be isolated from microorganisms that make cyFA and expressed in a host cell (<i>e.g., E. coli</i>)<i>.</i><tables id="tabl0006" num="0006"><table frame="all"><title><b>Table 6: Genes for the synthesis of CHC-CoA</b></title><tgroup cols="3"><colspec colnum="1" colname="col1" colwidth="63mm" /><colspec colnum="2" colname="col2" colwidth="44mm" /><colspec colnum="3" colname="col3" colwidth="59mm" /><thead><row><entry valign="top"><u>Organism</u></entry><entry valign="top"><u>Gene</u></entry><entry valign="top"><u>GenBank Accession #</u></entry></row></thead><tbody><row rowsep="0"><entry morerows="4" rowsep="1"><i><u>Streptomyces collinus</u></i></entry><entry><i><u>ansJK</u></i></entry><entry><u>U72144*</u></entry></row><row rowsep="0"><entry><i><u>ansL</u></i></entry><entry /></row><row rowsep="0"><entry><i><u>chcA</u></i></entry><entry /></row><row rowsep="0"><entry><i><u>ansM</u></i></entry><entry /></row><row><entry><i><u>chcB</u></i></entry><entry><u>AF268489</u></entry></row><row rowsep="0"><entry morerows="3" rowsep="1"><u><i>Streptomyces</i> sp. HK803</u></entry><entry><i><u>pmlJK</u></i></entry><entry><u>AAQ84158</u></entry></row><row rowsep="0"><entry><i><u>pmlL</u></i></entry><entry><u>AAQ84159</u></entry></row><row rowsep="0"><entry><i><u>chcA</u></i></entry><entry><u>AAQ84160</u></entry></row><row><entry><i><u>pmlM</u></i></entry><entry><u>AAQ84161</u></entry></row><row><entry><i><u>Streptomyces coelicolor</u></i></entry><entry><u><i>chcB</i>/<i>caiD</i></u></entry><entry><u>NP_629292</u></entry></row><row><entry><u><i>Streptomyces avermitilis</i></u></entry><entry><u><i>chcB</i>/<i>caiD</i></u></entry><entry><u>NP_629292</u></entry></row></tbody></tgroup><tgroup cols="3" rowsep="0"><colspec colnum="1" colname="col1" colwidth="63mm" /><colspec colnum="2" colname="col2" colwidth="44mm" /><colspec colnum="3" colname="col3" colwidth="59mm" /><tbody><row><entry namest="col1" nameend="col3" align="justify">*Only <i>chcA</i> is annotated in GenBank entry U72144, <i>ansJKLM</i> are according to <nplcit id="ncit0031" npl-type="s"><text>Chen et al. (Eur. J. Biochem. 261:98-107, 1999</text></nplcit>).</entry></row></tbody></tgroup></table></tables>

0187The genes listed in Table 5 (<i>fabH, ACP,</i> and <i>fabF</i>) allow initiation and elongation of ω-cyclic fatty acid derivatives because they have broad substrate specificity. If the coexpression of any of these genes with the genes listed in Table 6 does not yield cyFA, then <i>fabH</i>, <i>ACP,</i> and/or <i>fabF</i> homologs from microorganisms that make cyFAs <i>(e.g.,</i> those listed in Table 7) can be isolated <i>(e.g.,</i> by using degenerate PCR primers or heterologous DNA sequence probes) and coexpressed. <tables id="tabl0007" num="0007"><table frame="all"><title><b>Table 7: Non-limiting examples of microorganisms that contain ω-cyclic fatty acids</b></title><tgroup cols="2"><colspec colnum="1" colname="col1" colwidth="79mm" /><colspec colnum="2" colname="col2" colwidth="74mm" /><thead><row><entry valign="top">Organism</entry><entry valign="top">Reference</entry></row></thead><tbody><row><entry><i>Curtobacterium pusillum</i></entry><entry>ATCC19096</entry></row><row><entry><i>Alicyclobacillus acidoterrestris</i></entry><entry>ATCC49025</entry></row><row><entry><i>Alicyclobacillus acidocaldarius</i></entry><entry>ATCC27009</entry></row><row><entry><i>Alicyclobacillus cycloheptanicus</i> *</entry><entry><nplcit id="ncit0032" npl-type="s"><text>Moore, J. Org. Chem. 62:pp. 2173, 1997</text></nplcit>.</entry></row></tbody></tgroup><tgroup cols="2" rowsep="0"><colspec colnum="1" colname="col1" colwidth="79mm" /><colspec colnum="2" colname="col2" colwidth="74mm" /><tbody><row><entry namest="col1" nameend="col2" align="justify">*Uses cycloheptylcarbonyl-CoA and not cyclohexylcarbonyl-CoA as precursor for cyFA biosynthesis.</entry></row></tbody></tgroup></table></tables>

Aldehyde, Fatty Alcohol, and Alkene Saturation Levels

0188The degree of saturation in fatty acid derivatives can be controlled by regulating the degree of saturation of fatty acid derivative intermediates. The <i>sfa</i>, <i>gns,</i> and <i>fab</i> families of genes can be expressed or overexpressed to control the saturation of fatty acids. <figref idref="f0037 f0038 f0039 f0040 f0041 f0042 f0043 f0044 f0045 f0046 f0047 f0048 f0049 f0050 f0051 f0052 f0053 f0054 f0055 f0056 f0057 f0058 f0059">Figure 40</figref> lists non-limiting examples of genes in these gene families that may be used in the methods and host cells described herein.

0189Host cells can be engineered to produce unsaturated fatty acids by engineering the host cell to overexpress <i>fabB</i> or by growing the host cell at low temperatures (e.g., less than 37 °C). FabB has preference to cis-δ3decenoyl-ACP and results in unsaturated fatty acid production in <i>E. coli.</i> Overexpression of <i>fabB</i> results in the production of a significant percentage of unsaturated fatty acids (<nplcit id="ncit0033" npl-type="s"><text>de Mendoza et al., J. Biol. Chem. 258:2098-2101, 1983</text></nplcit>). The gene <i>fabB</i> may be inserted into and expressed in host cells not naturally having the gene. These unsaturated fatty acid derivatives can then be used as intermediates in host cells that are engineered to produce fatty acid derivatives, such as fatty aldehydes, fatty alcohols, or alkenes.

0190In other instances, a repressor of fatty acid biosynthesis, for example, <i>fabR</i> (GenBank accession NP_418398), can be deleted, which will also result in increased unsaturated fatty acid production in <i>E. coli</i> (<nplcit id="ncit0034" npl-type="s"><text>Zhang et al., J. Biol. Chem. 277:15558, 2002</text></nplcit>). Similar deletions may be made in other host cells. A further increase in unsaturated fatty acid derivatives may be achieved, for example, by overexpressing <i>fabM</i> (trans-2, cis-3-decenoyl-ACP isomerase, GenBank accession DAA05501) and controlled expression of <i>fabK</i> (trans-2-enoyl-ACP reductase II, GenBank accession NP_357969) from <i>Streptococcus pneumoniae</i> (<nplcit id="ncit0035" npl-type="s"><text>Marrakchi et al., J. Biol. Chem. 277: 44809, 2002</text></nplcit>), while deleting <i>E. colifabl</i> (trans-2-enoyl-ACP reductase, GenBank accession NP_415804). In some examples, the endogenous <i>fabF</i> gene can be attenuated, thus increasing the percentage of palmitoleate (C16:1) produced.

Other Substrates

0191Other substrates that can be used to produce aldehydes, fatty alcohols, alkanes, and alkenes in the methods described herein are acyl-ACP, acyl-CoA, a fatty aldehyde, or a fatty alcohol, which are described in, for example, <patcit id="pcit0008" dnum="US08058788W"><text>PCT/US08/058788</text></patcit>. Exemplary genes that can be altered to express or overexpress these substrates in host cells are listed in <figref idref="f0037 f0038 f0039 f0040 f0041 f0042 f0043 f0044 f0045 f0046 f0047 f0048 f0049 f0050 f0051 f0052 f0053 f0054 f0055 f0056 f0057 f0058 f0059">Figure 40</figref>. Other exemplary genes are described in <patcit id="pcit0009" dnum="US08058788W"><text>PCT/US08/058788</text></patcit>.

Genetic Engineering of Host Cells to Produce Aldehydes, Fatty Alcohols, Alkanes, and Alkenes

0192Various host cells can be used to produce aldehydes, fatty alcohols, alkanes, and/or alkenes, as described herein. A host cell can be any prokaryotic or eukaryotic cell. For example, a polypeptide described herein can be expressed in bacterial cells (such as <i>E. coli),</i> insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) cells, COS cells, VERO cells, BHK cells, HeLa cells, Cv1 cells, MDCK cells, 293 cells, 3T3 cells, or PC12 cells). Other exemplary host cells include cells from the members of the genus <i>Escherichia, Bacillus, Lactobacillus, Rhodococcus, Pseudomonas, Aspergillus, Trichoderma</i>, <i>Neurospora, Fusarium, Humicola, Rhizomucor, Kluyveromyces, Pichia, Mucor, Myceliophtora</i>, <i>Penicillium, Phanerochaete, Pleurotus</i>, <i>Trametes, Chrysosporium, Saccharomyces, Schizosaccharomyces, Yarrowia,</i> or <i>Streptomyces.</i> Yet other exemplary host cells can be a <i>Bacillus lentus</i> cell, a <i>Bacillus brevis</i> cell, a <i>Bacillus stearothermophilus</i> cell, a <i>Bacillus licheniformis</i> cell, a <i>Bacillus alkalophilus</i> cell, a <i>Bacillus coagulans</i> cell, a <i>Bacillus circulans</i> cell, a <i>Bacillus pumilis</i> cell, a <i>Bacillus thuringiensis</i> cell, a <i>Bacillus clausii</i> cell, a <i>Bacillus megaterium</i> cell, a <i>Bacillus subtilis</i> cell, a <i>Bacillus amyloliquefaciens</i> cell, a <i>Trichoderma koningii</i> cell, a <i>Trichoderma viride</i> cell, a <i>Trichoderma reesei</i> cell, a <i>Trichoderma longibrachiatum</i> cell, an <i>Aspergillus awamori</i> cell, an <i>Aspergillus fumigates</i> cell, an <i>Aspergillus foetidus</i> cell, an <i>Aspergillus nidulans</i> cell, an <i>Aspergillus niger</i> cell, an <i>Aspergillus oryzae</i> cell, a <i>Humicola insolens</i> cell, a <i>Humicola lanuginose</i> cell, a <i>Rhizomucor miehei</i> cell, a <i>Mucor michei</i> cell, a <i>Streptomyces lividans</i> cell, a <i>Streptomyces murinus</i> cell, or an <i>Actinomycetes</i> cell.

0193Other nonlimiting examples of host cells are those listed in Table 1.

0194In a preferred embodiment, the host cell is an <i>E. coli</i> cell. In a more preferred embodiment, the host cell is from <i>E. coli</i> strains B, C, K, or W. Other suitable host cells are known to those skilled in the art.

0195Various methods well known in the art can be used to genetically engineer host cells to produce aldehydes, fatty alcohols, alkanes and/or alkenes. The methods include the use of vectors, preferably expression vectors, containing a nucleic acid encoding an aldehyde, fatty alcohol, alkane, and/or alkene biosynthetic polypeptide described herein, or a polypeptide variant or fragment thereof. As used herein, the term "vector" 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 can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (<i>e.g</i>., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell and are thereby replicated along with the host genome. Moreover, certain vectors, such as expression vectors, are capable of directing the expression of genes to which they are operatively linked. In general, expression vectors used in recombinant DNA techniques are often in the form of plasmids. However, other forms of expression vectors, such as viral vectors (<i>e.g.,</i> replication defective retroviruses, adenoviruses, and adeno-associated viruses), can also be used.

0196The recombinant expression vectors described herein include a nucleic acid described herein in a form suitable for expression of the nucleic acid in a host cell. The recombinant expression vectors can include one or more control sequences, selected on the basis of the host cell to be used for expression. The control sequence is operably linked to the nucleic acid sequence to be expressed. Such control sequences are described, for example, in <nplcit id="ncit0036" npl-type="b"><text>Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990</text></nplcit>). Control sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cells and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, <i>etc.</i> The expression vectors described herein can be introduced into host cells to produce polypeptides, including fusion polypeptides, encoded by the nucleic acids as described herein.

0197Recombinant expression vectors can be designed for expression of an aldehyde, fatty alcohol, alkane, and/or alkene biosynthetic polypeptide or variant in prokaryotic or eukaryotic cells <i>(e.g.,</i> bacterial cells, such as <i>E. coli,</i> insect cells (using baculovirus expression vectors), yeast cells, or mammalian cells). Suitable host cells are discussed further in <nplcit id="ncit0037" npl-type="b"><text>Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990</text></nplcit>). Alternatively, the recombinant expression vector can be transcribed and translated <i>in vitro,</i> for example, by using T7 promoter regulatory sequences and T7 polymerase.

0198Expression of polypeptides in prokaryotes, for example, <i>E. coli,</i> is most often carried out with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion polypeptides. Fusion vectors add a number of amino acids to a polypeptide encoded therein, usually to the amino terminus of the recombinant polypeptide. Such fusion vectors typically serve three purposes: (1) to increase expression of the recombinant polypeptide; (2) to increase the solubility of the recombinant polypeptide; and (3) to aid in the purification of the recombinant polypeptide by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant polypeptide. This enables separation of the recombinant polypeptide from the fusion moiety after purification of the fusion polypeptide. Examples of such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin, and enterokinase. Exemplary fusion expression vectors include pGEX (Pharmacia Biotech Inc; <nplcit id="ncit0038" npl-type="s"><text>Smith et al., Gene (1988) 67:31-40</text></nplcit>), pMAL (New England Biolabs, Beverly, Mass.), and pRITS (Pharmacia, Piscataway, N.J.), which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant polypeptide.

0199Examples of inducible, non-fusion <i>E. coli</i> expression vectors include pTrc (<nplcit id="ncit0039" npl-type="s"><text>Amann et al., Gene (1988) 69:301-315</text></nplcit>) and pET 11d (<nplcit id="ncit0040" npl-type="b"><text>Studier et al., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990) 60-89</text></nplcit>). Target gene expression from the pTrc vector relies on host RNA polymerase transcription from a hybrid trp-lac fusion promoter. Target gene expression from the pET 11d vector relies on transcription from a T7 gn10-lac fusion promoter mediated by a coexpressed viral RNA polymerase (T7 gn1). This viral polymerase is supplied by host strains BL21(DE3) or HMS174(DE3) from a resident λ prophage harboring a T7 gnl gene under the transcriptional control of the lacUV 5 promoter.

0200One strategy to maximize recombinant polypeptide expression is to express the polypeptide in a host cell with an impaired capacity to proteolytically cleave the recombinant polypeptide (see <nplcit id="ncit0041" npl-type="b"><text>Gottesman, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990) 119-128</text></nplcit>). Another strategy is to alter the nucleic acid sequence to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in the host cell (<nplcit id="ncit0042" npl-type="s"><text>Wada et al., Nucleic Acids Res. (1992) 20:2111-2118</text></nplcit>). Such alteration of nucleic acid sequences can be carried out by standard DNA synthesis techniques.

0201In another embodiment, the host cell is a yeast cell. In this embodiment, the expression vector is a yeast expression vector. Examples of vectors for expression in yeast <i>S. cerevisiae</i> include pYepSec1 (<nplcit id="ncit0043" npl-type="s"><text>Baldari et al., EMBO J. (1987) 6:229-234</text></nplcit>), pMFa (<nplcit id="ncit0044" npl-type="s"><text>Kurjan et al., Cell (1982) 30:933-943</text></nplcit>), pJRY88 (<nplcit id="ncit0045" npl-type="s"><text>Schultz et al., Gene (1987) 54:113-123</text></nplcit>), pYES2 (Invitrogen Corporation, San Diego, Calif.), and picZ (Invitrogen Corp, San Diego, Calif.).

0202Alternatively, a polypeptide described herein can be expressed in insect cells using baculovirus expression vectors. Baculovirus vectors available for expression of proteins in cultured insect cells <i>(e.g.,</i> Sf 9 cells) include, for example, the pAc series (<nplcit id="ncit0046" npl-type="s"><text>Smith et al., Mol. Cell Biol. (1983) 3:2156-2165</text></nplcit>) and the pVL series (<nplcit id="ncit0047" npl-type="s"><text>Lucklow et al., Virology (1989) 170:31-39</text></nplcit>).

0203In yet another embodiment, the nucleic acids described herein can be expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (<nplcit id="ncit0048" npl-type="s"><text>Seed, Nature (1987) 329:840</text></nplcit>) and pMT2PC (<nplcit id="ncit0049" npl-type="s"><text>Kaufman et al., EMBO J. (1987) 6:187-195</text></nplcit>). When used in mammalian cells, the expression vector's control functions can be provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40. Other suitable expression systems for both prokaryotic and eukaryotic cells are described in chapters 16 and 17 of <nplcit id="ncit0050" npl-type="b"><text>Sambrook et al., eds., Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989</text></nplcit>.

0204Vectors can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection" refer to a variety of art-recognized techniques for introducing foreign nucleic acid <i>(e.g.,</i> DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in, for example, Sambrook <i>et al.</i> (supra).

0205For stable transformation of bacterial cells, it is known that, depending upon the expression vector and transformation technique used, only a small fraction of cells will take-up and replicate the expression vector. In order to identify and select these transformants, a gene that encodes a selectable marker (<i>e.g.</i>, resistance to antibiotics) can be introduced into the host cells along with the gene of interest. Selectable markers include those that confer resistance to drugs, such as ampacillin, kanamycin, chloramphenicol, or tetracycline. Nucleic acids encoding a selectable marker can be introduced into a host cell on the same vector as that encoding a polypeptide described herein or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).

0206For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a gene that encodes a selectable marker (e.g., resistance to antibiotics) can be introduced into the host cells along with the gene of interest. Preferred selectable markers include those which confer resistance to drugs, such as G418, hygromycin, and methotrexate. Nucleic acids encoding a selectable marker can be introduced into a host cell on the same vector as that encoding a polypeptide described herein or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (<i>e.g.</i>, cells that have incorporated the selectable marker gene will survive, while the other cells die).

0207In certain methods, an aldehyde biosynthetic polypeptide and an alkane or alkene biosynthetic polypeptide are co-expressed in a single host cell. In alternate methods, an aldehyde biosynthetic polypeptide and an alcohol dehydrogenase polypeptide are co-expressed in a single host cell.

Transport Proteins

0208Transport proteins can export polypeptides and hydrocarbons (<i>e.g.,</i> aldehydes, alkanes, and/or alkenes) out of a host cell. Many transport and efflux proteins serve to excrete a wide variety of compounds and can be naturally modified to be selective for particular types of hydrocarbons.

0209Non-limiting examples of suitable transport proteins are ATP-Binding Cassette (ABC) transport proteins, efflux proteins, and fatty acid transporter proteins (FATP). Additional non-limiting examples of suitable transport proteins include the ABC transport proteins from organisms such as <i>Caenorhabditis elegans, Arabidopsis thalania, Alkaligenes eutrophus,</i> and <i>Rhodococcus erythropolis.</i> Exemplary ABC transport proteins that can be used are listed in <figref idref="f0037 f0038 f0039 f0040 f0041 f0042 f0043 f0044 f0045 f0046 f0047 f0048 f0049 f0050 f0051 f0052 f0053 f0054 f0055 f0056 f0057 f0058 f0059">Figure 40</figref> (<i>e.g.,</i> CER5, AtMRP5, AmiS2, and AtPGP1). Host cells can also be chosen for their endogenous ability to secrete hydrocarbons. The efficiency of hydrocarbon production and secretion into the host cell environment (e.g., culture medium, fermentation broth) can be expressed as a ratio of intracellular product to extracellular product. In some examples, the ratio can be about 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, or 1:5.

Fermentation

0210The production and isolation of aldehydes, fatty alcohols, alkanes and/or alkenes can be enhanced by employing beneficial fermentation techniques. One method for maximizing production while reducing costs is increasing the percentage of the carbon source that is converted to hydrocarbon products.

0211During normal cellular lifecycles, carbon is used in cellular functions, such as producing lipids, saccharides, proteins, organic acids, and nucleic acids. Reducing the amount of carbon necessary for growth-related activities can increase the efficiency of carbon source conversion to product. This can be achieved by, for example, first growing host cells to a desired density (for example, a density achieved at the peak of the log phase of growth). At such a point, replication checkpoint genes can be harnessed to stop the growth of cells. Specifically, quorum sensing mechanisms (reviewed in <nplcit id="ncit0051" npl-type="s"><text>Camilli et al., Science 311:1113, 2006</text></nplcit>; <nplcit id="ncit0052" npl-type="s"><text>Venturi FEMS Microbio. Rev. 30:274-291, 2006</text></nplcit>; and <nplcit id="ncit0053" npl-type="s"><text>Reading et al., FEMS Microbiol. Lett. 254:1-11, 2006</text></nplcit>) can be used to activate checkpoint genes, such as <i>p53, p21,</i> or other checkpoint genes.

0212Genes that can be activated to stop cell replication and growth in <i>E. coli</i> include <i>umuDC</i> genes. The overexpression of <i>umuDC</i> genes stops the progression from stationary phase to exponential growth (<nplcit id="ncit0054" npl-type="s"><text>Murli et al., J. of Bact. 182:1127, 2000</text></nplcit>). UmuC is a DNA polymerase that can carry out translesion synthesis over non-coding lesions - the mechanistic basis of most UV and chemical mutagenesis. The <i>umuDC</i> gene products are involved in the process of translesion synthesis and also serve as a DNA sequence damage checkpoint. The <i>umuDC</i> gene products include UmuC, UmuD, umuD', UmuD'₂C, UmuD'₂, and UmuD₂. Simultaneously, product-producing genes can be activated, thus minimizing the need for replication and maintenance pathways to be used while an aldehyde, alkane and/or alkene is being made. Host cells can also be engineered to express <i>umuC</i> and <i>umuD</i> from <i>E</i>. <i>coli</i> in pBAD24 under the <i>prpBCDE</i> promoter system through <i>de novo</i> synthesis of this gene with the appropriate end-product production genes.

0213The percentage of input carbons converted to aldehydes, fatty alcohols, alkanes and/or alkenes can be a cost driver. The more efficient the process is (<i>i.e.,</i> the higher the percentage of input carbons converted to aldehydes, fatty alcohols, alkanes and/or alkenes), the less expensive the process will be. For oxygen-containing carbon sources (e.g., glucose and other carbohydrate based sources), the oxygen must be released in the form of carbon dioxide. For every 2 oxygen atoms released, a carbon atom is also released leading to a maximal theoretical metabolic efficiency of approximately 34% (w/w) (for fatty acid derived products). This figure, however, changes for other hydrocarbon products and carbon sources. Typical efficiencies in the literature are approximately less than 5%. Host cells engineered to produce aldehydes, alkanes and/or alkenes can have greater than about 1, 3, 5, 10, 15, 20, 25, and 30% efficiency. In one example, host cells can exhibit an efficiency of about 10% to about 25%. In other examples, such host cells can exhibit an efficiency of about 25% to about 30%. In other examples, host cells can exhibit greater than 30% efficiency.

0214The host cell can be additionally engineered to express recombinant cellulosomes, such as those described in <patcit id="pcit0010" dnum="US2007003736W"><text>PCT application number PCT/US2007/003736</text></patcit>. These cellulosomes can allow the host cell to use cellulosic material as a carbon source. For example, the host cell can be additionally engineered to express invertases (EC 3.2.1.26) so that sucrose can be used as a carbon source. Similarly, the host cell can be engineered using the teachings described in <patcit id="pcit0011" dnum="US5000000A"><text>U.S. Patent Nos. 5,000,000</text></patcit>; <patcit id="pcit0012" dnum="US5028539A"><text>5,028,539</text></patcit>; <patcit id="pcit0013" dnum="US5424202A"><text>5,424,202</text></patcit>; <patcit id="pcit0014" dnum="US5482846A"><text>5,482,846</text></patcit>; and <patcit id="pcit0015" dnum="US5602030A"><text>5,602,030</text></patcit>; so that the host cell can assimilate carbon efficiently and use cellulosic materials as carbon sources.

0215In one example, the fermentation chamber can enclose a fermentation that is undergoing a continuous reduction. In this instance, a stable reductive environment can be created. The electron balance can be maintained by the release of carbon dioxide (in gaseous form). Efforts to augment the NAD/H and NADP/H balance can also facilitate in stabilizing the electron balance. The availability of intracellular NADPH can also be enhanced by engineering the host cell to express an NADH:NADPH transhydrogenase. The expression of one or more NADH:NADPH transhydrogenases converts the NADH produced in glycolysis to NADPH, which can enhance the production of aldehydes, alkanes and/or alkenes.

0216For small scale production, the engineered host cells can be grown in batches of, for example, around 100 mL, 500 mL, 1 L, 2 L, 5 L, or 10 L; fermented; and induced to express desired aldehydes, fatty alcohols, alkanes and/or alkenes based on the specific genes encoded in the appropriate plasmids. For example, <i>E. coli</i> BL21(DE3) cells harboring pBAD24 (with ampicillin resistance and the aldehyde, fatty alcohol, alkane, or alkene synthesis pathway) as well as pUMVC1 (with kanamycin resistance and the acetyl CoA/malonyl CoA overexpression system) can be incubated overnight in 2 L flasks at 37°C shaken at > 200 rpm in 500 mL LB medium supplemented with 75 µg/mL ampicillin and 50 µg/mL kanamycin until cultures reach an OD₆₀₀ of > 0.8. Upon achieving an OD₆₀₀ of > 0.8, the cells can be supplemented with 25 mM sodium proprionate (pH 8.0) to activate the engineered gene systems for production and to stop cellular proliferation by activating UmuC and UmuD proteins. Induction can be performed for 6 hrs at 30°C. After incubation, the media can be examined for aldehydes, fatty alcohols, alkanes and/or alkenes using GC-MS.

0217For large scale production, the engineered host cells can be grown in batches of 10 L, 100 L, 1000 L ,or larger; fermented; and induced to express desired aldehydes, fatty alcohols, alkanes and/or alkenes based on the specific genes encoded in the appropriate plasmids. For example, <i>E. coli</i> BL21(DE3) cells harboring pBAD24 (with ampicillin resistance and the aldehyde and/or alkane synthesis pathway) as well as pUMVC1 (with kanamycin resistance and the acetyl-CoA/malonyl-CoA overexpression system) can be incubated from a 500 mL seed culture for 10 L fermentations (5 L for 100 L fermentations, <i>etc.</i>) in LB media (glycerol free) with 50 µg/mL kanamycin and 75 µg/mL ampicillin at 37°C, and shaken at > 200 rpm until cultures reach an OD₆₀₀ of > 0.8 (typically 16 hrs). Media can be continuously supplemented to maintain 25 mM sodium proprionate (pH 8.0) to activate the engineered gene systems for production and to stop cellular proliferation by activating umuC and umuD proteins. Media can be continuously supplemented with glucose to maintain a concentration 25 g/100 mL.

0218After the first hour of induction, aliquots of no more than 10% of the total cell volume can be removed each hour and allowed to sit without agitation to allow the aldehydes, alkanes and/or alkenes to rise to the surface and undergo a spontaneous phase separation. The aldehyde, fatty alcohols, alkane and/or alkene component can then be collected, and the aqueous phase returned to the reaction chamber. The reaction chamber can be operated continuously. When the OD₆₀₀ drops below 0.6, the cells can be replaced with a new batch grown from a seed culture.

Producing Aldehydes, Fatty Alcohols, Alkanes and Alkenes using Cell-free Methods

0219In some methods described herein, an aldehyde, fatty alcohols, alkane and/or alkene can be produced using a purified polypeptide described herein and a substrate described herein. For example, a host cell can be engineered to express aldehyde, fatty alcohols, alkane and/or alkene biosynthetic polypeptide or variant as described herein. The host cell can be cultured under conditions suitable to allow expression of the polypeptide. Cell free extracts can then be generated using known methods. For example, the host cells can be lysed using detergents or by sonication. The expressed polypeptides can be purified using known methods. After obtaining the cell free extracts, substrates described herein can be added to the cell free extracts and maintained under conditions to allow conversion of the substrates to aldehydes, fatty alcohols, alkanes and/or alkenes. The aldehydes, fatty alcohols, alkanes and/or alkenes can then be separated and purified using known techniques.

Post-Production Processing

0220The aldehydes, fatty alcohols, alkanes and/or alkenes produced during fermentation can be separated from the fermentation media. Any known technique for separating aldehydes, fatty alcohols, alkanes and/or alkenes from aqueous media can be used. One exemplary separation process is a two phase (bi-phasic) separation process. This process involves fermenting the genetically engineered host cells under conditions sufficient to produce an aldehyde, fatty alcohols, alkane and/or alkene, allowing the aldehyde, fatty alcohols, alkane and/or alkene to collect in an organic phase, and separating the organic phase from the aqueous fermentation broth. This method can be practiced in both a batch and continuous fermentation setting.

0221Bi-phasic separation uses the relative immiscibility of aldehydes, fatty alcohols, alkanes and/or alkenes to facilitate separation. Immiscible refers to the relative inability of a compound to dissolve in water and is defined by the compound's partition coefficient. One of ordinary skill in the art will appreciate that by choosing a fermentation broth and organic phase, such that the aldehyde, alkane and/or alkene being produced has a high logP value, the aldehyde, alkane and/or alkene can separate into the organic phase, even at very low concentrations, in the fermentation vessel.

0222The aldehydes, fatty alcohols, alkanes and/or alkenes produced by the methods described herein can be relatively immiscible in the fermentation broth, as well as in the cytoplasm. Therefore, the aldehyde, fatty alcohols, alkane and/or alkene can collect in an organic phase either intracellularly or extracellularly. The collection of the products in the organic phase can lessen the impact of the aldehyde, fatty alcohols, alkane and/or alkene on cellular function and can allow the host cell to produce more product.

0223The methods described herein can result in the production of homogeneous compounds wherein at least about 60%, 70%, 80%, 90%, or 95% of the aldehydes, fatty alcohols, alkanes and/or alkenes produced will have carbon chain lengths that vary by less than about 6 carbons, less than about 4 carbons, or less than about 2 carbons. These compounds can also be produced with a relatively uniform degree of saturation. These compounds can be used directly as fuels, fuel additives, specialty chemicals, starting materials for production of other chemical compounds <i>(e.g.,</i> polymers, surfactants, plastics, textiles, solvents, adhesives, <i>etc.</i>)<i>,</i> or personal care product additives. These compounds can also be used as feedstock for subsequent reactions, for example, hydrogenation, catalytic cracking (<i>via</i> hydrogenation, pyrolisis, or both), to make other products.

0224In some embodiments, the aldehydes, fatty alcohols, alkanes and/or alkenes produced using methods described herein can contain between about 50% and about 90% carbon; or between about 5% and about 25% hydrogen. In other embodiments, the aldehydes, fatty alcohols, alkanes and/or alkenes produced using methods described herein can contain between about 65% and about 85% carbon; or between about 10% and about 15% hydrogen.

Fuel Compositions and Specialty Chemical Compositions

0225The aldehydes, fatty alcohols, alkanes and/or alkenes described herein can be used as or converted into a fuel or as a specialty chemical. One of ordinary skill in the art will appreciate that, depending upon the intended purpose of the fuel or specialty chemical, different aldehydes, fatty alcohols, alkanes and/or alkenes can be produced and used. For example, a branched aldehyde, fatty alcohol, alkane and/or alkene may be desirable for automobile fuel that is intended to be used in cold climates. In addition, when the aldehydes, fatty alcohols, alkanes and/or alkenes described herein are used as a feedstock for fuel or specialty chemical production, one of ordinary skill in the art will appreciate that the characteristics of the aldehyde, fatty alcohol, alkane and/or alkene feedstock will affect the characteristics of the fuel or specialty chemical produced. Hence, the characteristics of the fuel or specialty chemical product can be selected for by producing particular aldehydes, fatty alcohols, alkanes and/or alkenes for use as a feedstock.

0226Using the methods described herein, biofuels having desired fuel qualities can be produced from aldehydes, fatty alcohols, alkanes and/or alkenes. Biologically produced aldehydes, fatty alcohols, alkanes and/or alkenes represent a new source of biofuels, which can be used as jet fuel, diesel, or gasoline. Some biofuels made using aldehydes, fatty alcohols, alkanes and/or alkenes have not been produced from renewable sources and are new compositions of matter. These new fuels or specialty chemicals can be distinguished from fuels or specialty chemicals derived from petrochemical carbon on the basis of dual carbon-isotopic fingerprinting. Additionally, the specific source of biosourced carbon (e.g., glucose vs. glycerol) can be determined by dual carbon-isotopic fingerprinting (<i>see, e.g.,</i><patcit id="pcit0016" dnum="US7169588B"><text>U.S. Patent No. 7,169,588</text></patcit>, in particular col. 4, line 31, to col. 6, line 8).

0227The aldehydes, fatty alcohols, alkanes and/or alkenes and the associated biofuels, specialty chemicals, and mixtures can be distinguished from their petrochemical derived counterparts on the basis of ¹⁴C (f_M) and dual carbon-isotopic fingerprinting. In some examples, the aldehyde, fatty alcohol, alkane and/or alkene in the biofuel composition can have a fraction of modern carbon (f_M¹⁴C) of, for example, at least about 1.003, 1.010, or 1.5.

0228In some examples, a biofuel composition can be made that includes aldehydes, fatty alcohols, alkanes and/or alkenes having δ¹³C of from about -15.4 to about -10.9, where the aldehydes, fatty alcohols, alkanes and/or alkenes account for at least about 85% of biosourced material (<i>i.e.,</i> derived from a renewable resource, such as biomass, cellulosic materials, and sugars) in the composition.

0229The ability to distinguish these biologically derived products is beneficial in tracking these materials in commerce. For example, fuels or specialty chemicals comprising both biologically derived and petroleum-based carbon isotope profiles can be distinguished from fuels and specialty chemicals made only of petroleum-based materials. Thus, the aldehydes, fatty alcohols, alkanes and/or alkenes described herein can be followed in commerce or identified in commerce as a biofuel on the basis of their unique profile. In addition, other competing materials can be identified as being biologically derived or derived from a petrochemical source.

0230Fuel additives 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/or flash point. In the United States, all fuel additives must be registered with Environmental Protection Agency. The names of fuel additives and the companies that sell the fuel additives are publicly available by contacting the EPA or by viewing the agency's website. One of ordinary skill in the art will appreciate that the aldehyde- and/or alkane-based biofuels described herein can be mixed with one or more fuel additives to impart a desired quality.

0231The aldehyde, fatty alcohols, alkane and/or alkene-based biofuels described herein can be mixed with other fuels, such as various alcohols, such as ethanol and butanol, and petroleum-derived products, such as gasoline, diesel, or jet fuel.

0232In some examples, the mixture can include at least about 10%, 15%, 20%, 30%, 40%, 50%, or 60% by weight of the aldehyde, fatty alcohols, alkane, or alkene. In other examples, a biofuel composition can be made that includes at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90% or 95% of an aldehyde, fatty alcohols, alkane, or alkene that includes a carbon chain that is 8, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 carbons in length. Such biofuel compositions can additionally include at least one additive selected from a cloud point lowering additive that can lower the cloud point to less than about 5°C, or 0°C; a surfactant; a microemulsion; at least about 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, or 95% diesel fuel from triglycerides; petroleum-derived gasoline; or diesel fuel from petroleum.

EXAMPLES

0233The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

Example 1. Detection and verification of alkane biosynthesis in selected cyanobacteria

0234Seven cyanobacteria, whose complete genome sequences are publicly available, were selected for verification and/or detection of alkane biosynthesis: <i>Synechococcus elongatus</i> PCC7942, <i>Synechococcus elongatus</i> PCC6301, <i>Anabaena variabilis</i> ATCC29413, <i>Synechocystis sp.</i> PCC6803, <i>Nostoc punctiforme</i> PCC73102, <i>Gloeobacter violaceus</i> ATCC 29082, and <i>Prochlorococcus marinus</i> CCMP1986. Only the first three cyanobacterial strains from this list had previously been reported to contain alkanes (<nplcit id="ncit0055" npl-type="s"><text>Han et al., J. Am. Chem. Soc. 91:5156-5159 (1969</text></nplcit>);<nplcit id="ncit0056" npl-type="s"><text> Fehler et al., Biochem. 9:418-422 (1970</text></nplcit>)). The strains were grown photoautotrophically in shake flasks in 100 mL of the appropriate media (listed in Table 8) for 3-7 days at 30°C at a light intensity of approximately 3,500 lux. Cells were extracted for alkane detection as follows: cells from 1 mL culture volume were centrifuged for 1 min at 13,000 rpm, the cell pellets were resuspended in methanol, vortexed for 1 min and then sonicated for 30 min. After centrifugation for 3 min at 13,000 rpm, the supernatants were transferred to fresh vials and analyzed by GC-MS. The samples were analyzed on either 30 m DP-5 capillary column (0.25 mm internal diameter) or a 30 m high temperature DP-5 capillary column (0.25mm internal diameter) using the following method.

0235After a 1 µL splitless injection (inlet temperature held at 300°C) onto the GC/MS column, the oven was held at 100°C for 3 mins. The temperature was ramped up to 320°C at a rate of 20°C/min. The oven was held at 320°C for an additional 5 min. The flow rate of the carrier gas helium was 1.3 mL/min. The MS quadrapole scanned from 50 to 550 m/z. Retention times and fragmentation patterns of product peaks were compared with authentic references to confirm peak identity.

0236Out of the seven strains, six produced mainly heptadecane and one produced pentadecane (<i>P. marinus</i> CCMP1986); one of these strains produced methyl-heptadecane in addition to heptadecane (<i>A</i>. <i>variabilis</i> ATCC29413) (see Table 8). Therefore, alkane biosynthesis in three previously reported cyanobacteria was verified, and alkane biosynthesis was detected in four cyanobacteria that were not previously known to produce alkanes: <i>P. marinus</i> CCMP1986 (see <figref idref="f0001">Figure 1</figref>), <i>N. punctiforme</i> PCC73102 (see <figref idref="f0001">Figure 2</figref>), G. <i>violaceus</i> ATCC 29082 (see <figref idref="f0002">Figure 3</figref>) and <i>Synechocystis sp.</i> PCC6803 (see <figref idref="f0002">Figure 4</figref>).

0237<figref idref="f0001">Figure 1A</figref> depicts the GC/MS trace of <i>Prochlorococcus marinus</i> CCMP1986 cells extracted with methanol. The peak at 7.55 min had the same retention time as pentadecane (Sigma). In <figref idref="f0001">Figure 1B</figref>, the mass fragmentation pattern of the pentadecane peak is shown. The 212 peak corresponds to the molecular weight of pentadecane.

0238<figref idref="f0001">Figure 2A</figref> depicts the GC/MS trace of <i>Nostoc punctiforme</i> PCC73102 cells extracted with methanol. The peak at 8.73 min has the same retention time as heptadecane (Sigma). In <figref idref="f0001">Figure 2B</figref>, the mass fragmentation pattern of the heptadecane peak is shown. The 240 peak corresponds to the molecular weight of heptadecane.

0239<figref idref="f0002">Figure 3A</figref> depicts the GC/MS trace of <i>Gloeobaceter violaceus</i> ATCC29082 cells extracted with methanol. The peak at 8.72 min has the same retention time as heptadecane (Sigma). In <figref idref="f0002">Figure 3B</figref>, the mass fragmentation pattern of the heptadecane peak is shown. The 240 peak corresponds to the molecular weight of heptadecane.

0240<figref idref="f0002">Figure 4A</figref> depicts the GC/MS trace of <i>Synechocystic sp.</i> PCC6803 cells extracted with methanol. The peak at 7.36 min has the same retention time as heptadecane (Sigma). In <figref idref="f0002">Figure 4B</figref>, the mass fragmentation pattern of the heptadecane peak is shown. The 240 peak corresponds to the molecular weight of heptadecane. <tables id="tabl0008" num="0008"><img file="EP2288694B1_D0002.tif" /></tables>

0241Genomic analysis yielded two genes that were present in the alkane-producing strains. The <i>Synechococcus elongatus</i> PCC7942 homologs of these genes are depicted in Table 9 and are Synpcc7942_1593 (SEQ ID NO:1) and Synpcc7942_1594 (SEQ ID NO:65). <tables id="tabl0009" num="0009"><img file="EP2288694B1_D0003.tif" /></tables>

Example 2. Deletion of the sll0208 and sll0209 genes in <i>Synechocystis</i> sp. PCC6803 leads to loss of Alkane Biosynthesis

0242The genes encoding the putative decarbonylase (sll0208; NP_442147) (SEQ ID NO:3) and aldehyde-generating enzyme (sll0209; NP_442146) (SEQ ID NO:67) of <i>Synechocystis</i> sp. PCC6803 were deleted as follows. Approximately 1 kb of upstream and downstream flanking DNA were amplified using primer sll0208/9-KO1 (CGCGGATCCCTTGATTCTACTGCGGCGAGT) with primer sll0208/9-KO2 (CACGCACCTAGGTTCACACTCCCATGGTATAACAGGGGCGTTGGACTCC TGTG) and primer sll0208/9-KO3 (GTTATACCATGGGAGTGTGAACCTAGGTGCGTGGCCGACAGGATAGGG-CGTGT) with primer sll0208/9-KO4 (CGCGGATCCAACGCATCCTCACTAGTCGGG), respectively. The PCR products were used in a cross-over PCR with primers sll0208/9-KO1 and sll0208/9-KO4 to amplify the approximately 2 kb sll0208/sll0209 deletion cassette, which was cloned into the <i>Bam</i>HI site of the cloning vector pUC19. A kanamycin resistance cassette (aph, KanR) was then amplified from plasmid pRL27 (<nplcit id="ncit0057" npl-type="s"><text>Larsen et al., Arch. Microbiol. 178:193 (2002</text></nplcit>)) using primers Kan-aph-F (CATGCCATGGAAAGCCACGTTGTGTCTCAAAATCTCTG) and Kan-aph-R (CTAGTCTAGAGCGCTGAGGTCTGCCTCGTGAA), which was then cut with <i>Ncol</i> and <i>Xba</i>I and cloned into the <i>Ncol</i> and <i>Avr</i>II sites of the sll0208/sll0209 deletion cassette, creating a sll0208/sll0209-deletion KanR-insertion cassette in pUC19. The cassette-containing vector, which does not replicate in cyanobacteria, was transformed into <i>Synechocystis</i> sp. PCC6803 (<nplcit id="ncit0058" npl-type="s"><text>Zang et al., 2007, J. Microbiol., vol. 45, pp. 241</text></nplcit>) and transformants (<i>e.g</i>., chromosomal integrants by double-homologous recombination) were selected on BG-11 agar plates containing 100 µg/mL Kanamycin in a light-equipped incubator at 30 °C. Kanamycin resistant colonies were restreaked once and then subjected to genotypic analysis using PCR with diagnostic primers.

0243Confirmed deletion-insertion mutants were cultivated in 12 mL of BG11 medium with 50 µg/mL Kanamycin for 4 days at 30°C in a light-equipped shaker-incubator. 1 mL of broth was then centrifuged (1 min at 13,000 g) and the cell pellets were extracted with 0.1 mL methanol. After extraction, the samples were again centrifuged and the supernatants were subjected to GC-MS analysis as described in Example 1.

0244As shown in <figref idref="f0003">Fig. 5</figref>, the <i>Synechocystis</i> sp. PCC6803 strains in which the sl10208 and sll0209 genes were deleted lost their ability to produce heptadecene and octadecenal. This result demonstrates that the sll0208 and sll0209 genes in <i>Synechocystis</i> sp. PCC6803 and the orthologous genes in other cyanobacteria (see Table 1) are responsible for alkane and fatty aldehyde biosynthesis in these organisms.

Example 3. Production of Fatty Aldehydes and Fatty Alcohols in <i>E. coli</i> through Heterologous Expression of <i>Synechococcus elongatus</i> PCC7942 orf1594

0245The genomic DNA encoding <i>Synechococcus elongatus</i> PCC7942 orf1594 (YP_400611; putative aldehyde-generating enzyme) (SEQ ID NO:65) was amplified and cloned into the <i>Ncol</i> and <i>Eco</i>RI sites of vector OP-80 (pCL1920 derivative) under the control of the P_trc promoter. The resulting construct ("OP80-PCC7942_1594") was transformed into <i>E. coli</i> MG1655 and the cells were grown at 37°C in M9 minimal media with 1% (w/v) glucose as carbon source and supplemented with 100 µg/mL spectinomycin. When the culture reached OD₆₀₀ of 0.8-1.0, it was induced with 1mM IPTG and cells were grown for an additional 18-20 h at 37°C. Cells from 0.5 mL of culture were extracted with 0.5 mL of ethyl acetate. After sonication for 60 min, the sample was centrifuged at 15,000 rpm for 5 min. The solvent layer was analyzed by GC-MS as described in Example 1.

0246As shown in <figref idref="f0004">Fig. 6</figref>, <i>E. coli</i> cells transformed with the <i>Synechococcus elongatus</i> PCC7942 orf1594 -bearing vector produced the following fatty aldehydes and fatty alcohols: hexadecanal, octadecenal, tetradecenol, hexadecenol, hexadecanol and octadecenol. This result indicates that PCC7942 orf1594 (i) generates aldehydes <i>in-vivo</i> as possible substrates for decarbonylation and (ii) may reduce acyl-ACPs as substrates, which are the most abundant form of activated fatty acids in wild type <i>E. coli</i> cells. Therefore, the enzyme was named Acyl-ACP reductase. <i>In-vivo,</i> the fatty aldehydes apparently are further reduced to the corresponding fatty alcohols by an endogenous <i>E. coli</i> aldehyde reductase activity.

Example 4. Production of Fatty Aldehydes and Fatty Alcohols in <i>E. coli</i> through Heterologous Expression of <i>Cyanothece</i> sp. ATCC51142 cce 1430

0247The genomic DNA encoding <i>Cyanothece</i> sp. ATCC51142 cce_1430 (YP_001802846; putative aldehyde-generating enzyme) (SEQ ID NO:69) was amplified and cloned into the <i>Ncol</i> and <i>Eco</i>RI sites of vector OP-80 (pCL1920 derivative) under the control of the P_trc promoter. The resulting construct was transformed into <i>E. coli</i> MG1655 and the cells were grown at 37°C in M9 minimal media with 1% (w/v) glucose as carbon source and supplemented with 100 µg/mL spectinomycin. The cells were cultured and extracted as in Example 3 and analyzed by GC-MS as described in Example 26.

0248As shown in <figref idref="f0005">Fig. 7</figref>, <i>E. coli</i> cells transformed with the <i>Cyanothece</i> sp. ATCC51142 cce_1430 -bearing vector produced the following fatty aldehydes and fatty alcohols: hexadecanal, octadecenal, tetradecenol, hexadecenol, hexadecanol and octadecenol. This result indicates that ATCC51142 cce_1430 (i) generates aldehydes <i>in-vivo</i> as possible substrates for decarbonylation and (ii) may reduce acyl-ACPs as substrates, which are the most abundant form of activated fatty acids in wild type <i>E. coli</i> cells. Therefore, this enzyme is also an Acyl-ACP reductase.

Example 5. Production of Alkanes and Alkenes in <i>E. coli</i> through Heterologous Expression of <i>Synechococcus elongatus</i> PCC7942 orf1594 and <i>Synechococcus elongatus</i> PCC7942 orf1593

0249The genomic DNA encoding <i>Synechococcus elongatus</i> PCC7942 orf1593 (YP_400610; putative decarbonylase) (SEQ ID NO:1) was amplified and cloned into the <i>Nde</i>I and <i>Xho</i>I sites of vector OP-183 (pACYC derivative) under the control of the P_trc promoter. The resulting construct was cotransformed with OP80-PCC7942_1594 into <i>E. coli</i> MG1655 and the cells were grown at 37°C in M9 minimal media supplemented with 100 µg/mL spectinomycin and 100 µg/mL carbenicillin. The cells were cultured and extracted as in Example 3 and analyzed by GC-MS as described in Example 1.

0250As shown in <figref idref="f0006">Fig. 8</figref>, <i>E. coli</i> cells cotransformed with the <i>S. elongatus</i> PCC7942_1594 and <i>S. elongatus</i> PCC7942_1593-bearing vectors produced the same fatty aldehydes and fatty alcohols as in Example 3, but also pentadecane and heptadecene. This result indicates that PCC7942_1593 in <i>E. coli</i> converts hexadecanal and octadecenal to pentadecane and heptadecene, respectively, and therefore is an active fatty aldehyde decarbonylase.

Example 6. Production of Alkanes and Alkenes in <i>E. coli</i> through Heterologous Expression of <i>Synechococcus elongatus</i> PCC7942 orf1594 and <i>Nostoc punctiforme</i> PCC73102 Npun02004178

0251The genomic DNA encoding <i>Nostoc punctiforme</i> PCC73102 Npun02004178 (ZP_00108838; putative decarbonylase) (SEQ ID NO:5) was amplified and cloned into the <i>Nde</i>I and <i>Xho</i>I sites of vector OP-183 (pACYC derivative) under the control of the P_trc promoter. The resulting construct was cotransformed with OP80-PCC7942_1594 into <i>E. coli</i> MG1655 and the cells were grown at 37°C in M9 minimal media supplemented with 100 µg/mL spectinomycin and 100 µg/mL carbenicillin. The cells were cultured and extracted as in Example 3 and analyzed by GC-MS as described in Example 1.

0252As shown in <figref idref="f0007">Fig. 9</figref>, <i>E. coli</i> cells cotransformed with the <i>S. elongatus</i> PCC7942_1594 and <i>N. punctiforme</i> PCC73102 Npun02004178-bearing vectors produced the same fatty aldehydes and fatty alcohols as in Example 3, but also tridecane, pentadecene, pentadecane and heptadecene. This result indicates that Npun02004178 in <i>E. coli</i> converts tetradecanal, hexadecenal, hexadecanal and octadecenal to tridecane, pentadecene, pentadecane and heptadecene, respectively, and therefore is an active fatty aldehyde decarbonylase.

Example 7. Production of Alkanes and Alkenes in <i>E. coli</i> through Heterologous Expression of <i>Synechococcus elongatus</i> PCC7942 orf1594 and <i>Synechocystis</i> sp. PCC6803 sll0208

0253The genomic DNA encoding <i>Synechocystis</i> sp. PCC6803 sll0208 (NP_442147; putative decarbonylase) (SEQ ID NO:3) was amplified and cloned into the <i>Nde</i>I and <i>Xho</i>I sites of vector OP-183 (pACYC derivative) under the control of the P_trc promoter. The resulting construct was cotransformed with OP80-PCC7942_1594 into <i>E. coli</i> MG1655 and the cells were grown at 37°C in M9 minimal media supplemented with 100 µg/mL spectinomycin and 100 µg/mL carbenicillin. The cells were cultured and extracted as in Example 3 and analyzed by GC-MS as described in Example 1.

0254As shown in <figref idref="f0007">Fig. 10</figref>, <i>E. coli</i> cells cotransformed with the <i>S. elongatus</i> PCC7942_1594 and <i>Synechocystis</i> sp. PCC6803 sll0208-bearing vectors produced the same fatty aldehydes and fatty alcohols as in Example 3, but also pentadecane and heptadecene. This result indicates that Npun02004178 in <i>E. coli</i> converts hexadecanal and octadecenal to pentadecane and heptadecene, respectively, and therefore is an active fatty aldehyde decarbonylase.

Example 8. Production of Alkanes and Alkenes in <i>E. coli</i> through Heterologous Expression of <i>Synechococcus elongatus</i> PCC7942 orf1594 and <i>Nostoc</i> sp. PCC7210 alr5283

0255The genomic DNA encoding <i>Nostoc</i> sp. PCC7210 alr5283 (NP_489323; putative decarbonylase) (SEQ ID NO:7) was amplified and cloned into the <i>Nde</i>I and <i>Xho</i>I sites of vector OP-183 (pACYC derivative) under the control of the P_trc promoter. The resulting construct was cotransformed with OP80-PCC7942_1594 into <i>E. coli</i> MG1655 and the cells were grown at 37°C in M9 minimal media supplemented with 100 µg/mL spectinomycin and 100 µg/mL carbenicillin. The cells were cultured and extracted as in Example 3 and analyzed by GC-MS as described in Example 1.

0256As shown in <figref idref="f0008">Fig. 11</figref>, <i>E. coli</i> cells cotransformed with the S. <i>elongatus</i> PCC7942_1594 and <i>Nostoc</i> sp. PCC7210 alr5283-bearing vectors produced the same fatty aldehydes and fatty alcohols as in Example 3, but also pentadecane and heptadecene. This result indicates that alr5283 in <i>E. coli</i> converts hexadecanal and octadecenal to pentadecane and heptadecene, respectively, and therefore is an active fatty aldehyde decarbonylase.

Example 9. Production of Alkanes and Alkenes in <i>E. coli</i> through Heterologous Expression of <i>Synechococcus elongatus</i> PCC7942 orf1594 and <i>Acaryochloris marina</i> MBIC11017 AMI 4041

0257The genomic DNA encoding <i>Acaryochloris marina</i> MBIC11017 AM1_4041 (YP_001518340; putative decarbonylase) (SEQ ID NO:9) was codon optimized for expression in <i>E. coli</i> (SEQ ID NO:46), synthesized, and cloned into the <i>Nde</i>I and <i>Xho</i>I sites of vector OP-183 (pACYC derivative) under the control of the P_trc promoter. The resulting construct was cotransformed with OP80-PCC7942_1594 into <i>E. coli</i> MG1655 and the cells were grown at 37°C in M9 minimal media supplemented with 100 µg/mL spectinomycin and 100 µg/mL carbenicillin. The cells were cultured and extracted as in Example 3 and analyzed by GC-MS as described in Example 26.

0258As shown in <figref idref="f0008">Fig. 12</figref>, <i>E. coli</i> cells cotransformed with the <i>S</i>. <i>elongatus</i> PCC7942_1594 and <i>A.marina</i> MBIC11017 AM1_4041-bearing vectors produced the same fatty aldehydes and fatty alcohols as in Example 3, but also tridecane, pentadecene, pentadecane and heptadecene. This result indicates that AM1_4041 in <i>E. coli</i> converts tetradecanal, hexadecenal, hexadecanal and octadecenal to tridecane, pentadecene, pentadecane and heptadecene, respectively, and therefore is an active fatty aldehyde decarbonylase.

Example 10. Production of Alkanes and Alkenes in <i>E. coli</i> through Heterologous Expression of <i>Svnechococcus elongatus</i> PCC7942 orf1594 and <i>Thermosynechococcus elongatus</i> BP-1 tll1313

0259The genomic DNA encoding <i>Thermosynechococcus elongatus</i> BP-1 tll1313 (NP_682103; putative decarbonylase) (SEQ ID NO:11) was codon optimized for expression in <i>E. coli</i> (SEQ ID NO:47), synthesized, and cloned into the <i>Nde</i>I and <i>Xho</i>I sites of vector OP-183 (pACYC derivative) under the control of the P_trc promoter. The resulting construct was cotransformed with OP80-PCC7942_1594 into <i>E. coli</i> MG1655 and the cells were grown at 37°C in M9 minimal media supplemented with 100 µg/mL spectinomycin and 100 µg/mL carbenicillin. The cells were cultured and extracted as in Example 3 and analyzed by GC-MS as described in Example 26.

0260As shown in <figref idref="f0009">Fig. 13</figref>, <i>E. coli</i> cells cotransformed with the <i>S</i>. <i>elongatus</i> PCC7942_1594 and <i>T. elongatus</i> BP-1 tll1313-bearing vectors produced the same fatty aldehydes and fatty alcohols as in Example 3, but also pentadecane and heptadecene. This result indicates that tll1313 in <i>E. coli</i> converts hexadecanal and octadecenal to pentadecane and heptadecene, respectively, and therefore is an active fatty aldehyde decarbonylase.

Example 11. Production of Alkanes and Alkenes in <i>E. coli</i> through Heterologous Expression of <i>Synechococcus elongatus</i> PCC7942 orf1594 and <i>Synechococcus sp.</i> JA-3-3Ab CYA 0415

0261The genomic DNA encoding <i>Synechococcus sp.</i> JA-3-3Ab CYA_0415 (YP_473897; putative decarbonylase) (SEQ ID NO:13) was codon optimized for expression in <i>E. coli</i> (SEQ ID NO:48), synthesized, and cloned into the <i>Nde</i>I and <i>Xho</i>I sites of vector OP-183 (pACYC derivative) under the control of the P_trc promoter. The resulting construct was cotransformed with OP80-PCC7942_1594 into <i>E. coli</i> MG1655 and the cells were grown at 37°C in M9 minimal media supplemented with 100 µg/mL spectinomycin and 100 µg/mL carbenicillin. The cells were cultured and extracted as in Example 3 and analyzed by GC-MS as described in Example 26.

0262As shown in <figref idref="f0009">Fig. 14</figref>, <i>E. coli</i> cells cotransformed with the S. <i>elongatus</i> PCC7942_1594 and <i>Synechococcus sp.</i> JA-3-3Ab CYA_0415-bearing vectors produced the same fatty aldehydes and fatty alcohols as in Example 3, but also pentadecane and heptadecene. This result indicates that Npun02004178 in <i>E. coli</i> converts hexadecanal and octadecenal to pentadecane and heptadecene, respectively, and therefore is an active fatty aldehyde decarbonylase.

Example 12. Production of Alkanes and Alkenes in <i>E. coli</i> through Heterologous Expression of <i>Synechococcus elongatus</i> PCC7942 orf1594 and <i>Gloeobacter violaceus</i> PCC7421 gll3146

0263The genomic DNA encoding <i>Gloeobacter violaceus</i> PCC7421 gll3146 (NP_926092; putative decarbonylase) (SEQ ID NO:15) was amplified and cloned into the <i>NdeI</i> and <i>Xho</i>I sites of vector OP-183 (pACYC derivative) under the control of the P_trc promoter. The resulting construct was cotransformed with OP80-PCC7942_1594 into <i>E. coli</i> MG1655 and the cells were grown at 37°C in M9 minimal media supplemented with 100 µg/mL spectinomycin and 100 µg/mL carbenicillin. The cells were cultured and extracted as in Example 3 and analyzed by GC-MS as described in Example 1.

0264As shown in <figref idref="f0010">Fig. 15</figref>, <i>E. coli</i> cells cotransformed with the S. <i>elongatus</i> PCC7942_1594 and G. <i>violaceus</i> PCC7421 gll3146-bearing vectors produced the same fatty aldehydes and fatty alcohols as in Example 3, but also pentadecane and heptadecene. This result indicates that gll3146 in <i>E. coli</i> converts hexadecanal and octadecenal to pentadecane and heptadecene, respectively, and therefore is an active fatty aldehyde decarbonylase.

Example 13. Production of Alkanes and Alkenes in <i>E. coli</i> through Heterologous Expression of <i>Synechococcus elongatus</i> PCC7942 orf1594 and <i>Prochlorococcus marinus</i> MIT9313 PMT1231

0265The genomic DNA encoding <i>Prochlorococcus marinus</i> MIT9313 PMT1231 (NP_895059; putative decarbonylase) (SEQ ID NO:17) was codon optimized for expression in <i>E. coli</i> (SEQ ID NO:49), synthesized, and cloned into the <i>Nde</i>I and <i>Xho</i>I sites of vector OP-183 (pACYC derivative) under the control of the P_trc promoter. The resulting construct was cotransformed with OP80-PCC7942_1594 into <i>E. coli</i> MG1655 and the cells were grown at 37°C in M9 minimal media supplemented with 100 µg/mL spectinomycin and 100 µg/mL carbenicillin. The cells were cultured and extracted as in Example 3 and analyzed by GC-MS as described in Example 26.

0266As shown in <figref idref="f0010">Fig. 16</figref>, <i>E. coli</i> cells cotransformed with the <i>S. elongatus</i> PCC7942_1594 and <i>P.marinus</i> MIT9313 PMT1231-bearing vectors produced the same fatty aldehydes and fatty alcohols as in Example 3, but also pentadecane and heptadecene. This result indicates that PMT1231 in <i>E. coli</i> converts hexadecanal and octadecenal to pentadecane and heptadecene, respectively, and therefore is an active fatty aldehyde decarbonylase.

Example 14. Production of Alkanes and Alkenes in <i>E. coli</i> through Heterologous Expression of <i>Synechococcus elongatus</i> PCC7942 orf1594 and <i>Prochlorococcus marinus</i> CCMP1986 PMM0532

0267The genomic DNA encoding <i>Prochlorococcus marinus</i> CCMP1986 PMM0532 (NP_892650; putative decarbonylase) (SEQ ID NO:19) was amplified and cloned into the <i>Nde</i>I and <i>Xho</i>I sites of vector OP-183 (pACYC derivative) under the control of the P_trc promoter. The resulting construct was cotransformed with OP80-PCC7942_1594 into <i>E. coli</i> MG1655 and the cells were grown at 37°C in M9 minimal media supplemented with 100 µg/mL spectinomycin and 100 µg/mL carbenicillin. The cells were cultured and extracted as in Example 3 and analyzed by GC-MS as described in Example 1.

0268As shown in <figref idref="f0011">Fig. 17</figref>, <i>E. coli</i> cells cotransformed with the <i>S. elongatus</i> PCC7942_1594 and <i>P.marinus</i> CCMP1986 PMM0532-bearing vectors produced the same fatty aldehydes and fatty alcohols as in Example 3, but also pentadecane and heptadecene. This result indicates that PMM0532 in <i>E. coli</i> converts hexadecanal and octadecenal to pentadecane and heptadecene, respectively, and therefore is an active fatty aldehyde decarbonylase.

Example 15. Production of Alkanes and Alkenes in <i>E. coli</i> through Heterologous Expression of <i>Synechococcus elongatus</i> PCC7942 orf1594 and <i>Prochlorococcus mariunus</i> NATL2A PMN2A 1863

0269The genomic DNA encoding <i>Prochlorococcus mariunus</i> NATL2A PMN2A_1863 (YP_293054; putative decarbonylase) (SEQ ID NO:21) was codon optimized for expression in <i>E. coli</i> (SEQ ID NO:51), synthesized, and cloned into the <i>Nde</i>I and <i>Xho</i>I sites of vector OP-183 (pACYC derivative) under the control of the P_trc promoter. The resulting construct was cotransformed with OP80-PCC7942_1594 into <i>E. coli</i> MG1655 and the cells were grown at 37°C in M9 minimal media supplemented with 100 µg/mL spectinomycin and 100 µg/mL carbenicillin. The cells were cultured and extracted as in Example 3 and analyzed by GC-MS as described in Example 26.

0270As shown in <figref idref="f0011">Fig. 18</figref>, <i>E. coli</i> cells cotransformed with the <i>S. elongatus</i> PCC7942_1594 and <i>P.mariunus</i> NATL2A PMN2A_1863-bearing vectors produced the same fatty aldehydes and fatty alcohols as in Example 3, but also pentadecane and heptadecene. This result indicates that PMN2A_1863 in <i>E. coli</i> converts hexadecanal and octadecenal to pentadecane and heptadecene, respectively, and therefore is an active fatty aldehyde decarbonylase.

Example 16. Production of Alkanes and Alkenes in <i>E. coli</i> through Heterologous Expression of <i>Synechococcus elongatus</i> PCC7942 orf1594 and <i>Synechococcus</i> sp. RS9917 RS9917 09941

0271The genomic DNA encoding <i>Synechococcus</i> sp. RS9917 RS9917_09941 (ZP_01079772; putative decarbonylase) (SEQ ID NO:23) was codon optimized for expression in <i>E. coli</i> (SEQ ID NO:52), synthesized, and cloned into the <i>Nde</i>I and <i>Xho</i>I sites of vector OP-183 (pACYC derivative) under the control of the P_trc promoter. The resulting construct was cotransformed with OP80-PCC7942_1594 into <i>E. coli</i> MG1655 and the cells were grown at 37°C in M9 minimal media supplemented with 100 µg/mL spectinomycin and 100 µg/mL carbenicillin. The cells were cultured and extracted as in Example 3 and analyzed by GC-MS as described in Example 26.

0272As shown in <figref idref="f0012">Fig. 19</figref>, <i>E. coli</i> cells cotransformed with the S. <i>elongatus</i> PCC7942_1594 and <i>Synechococcus</i> sp. RS9917 RS9917_09941-bearing vectors produced the same fatty aldehydes and fatty alcohols as in Example 3, but also pentadecane and heptadecene. This result indicates that RS9917_09941 in <i>E. coli</i> converts hexadecanal and octadecenal to pentadecane and heptadecene, respectively, and therefore is an active fatty aldehyde decarbonylase.

Example 17. Production of Alkanes and Alkenes in <i>E. coli</i> through Heterologous Expression of <i>Synechococcus elongatus</i> PCC7942 orf1594 and <i>Synechococcus</i> sp. RS9917 RS9917 12945

0273The genomic DNA encoding <i>Synechococcus</i> sp. RS9917 RS9917_12945 (ZP_01080370; putative decarbonylase) (SEQ ID NO:25) was codon optimized for expression in <i>E. coli</i> (SEQ ID NO:53), synthesized, and cloned into the <i>Nde</i>I and <i>Xho</i>I sites of vector OP-183 (pACYC derivative) under the control of the P_trc promoter. The resulting construct was cotransformed with OP80-PCC7942_1594 into <i>E. coli</i> MG1655 and the cells were grown at 37°C in M9 minimal media supplemented with 100 µg/mL spectinomycin and 100 µg/mL carbenicillin. The cells were cultured and extracted as in Example 3 and analyzed by GC-MS as described in Example 26.

0274As shown in <figref idref="f0012">Fig. 20</figref>, <i>E. coli</i> cells cotransformed with the <i>S. elongatus</i> PCC7942_1594 and <i>Synechococcus</i> sp. RS9917 RS9917_12945-bearing vectors produced the same fatty aldehydes and fatty alcohols as in Example 3, but also pentadecane and heptadecene. This result indicates that RS9917_12945 in <i>E. coli</i> converts hexadecanal and octadecenal to pentadecane and heptadecene, respectively, and therefore is an active fatty aldehyde decarbonylase.

Example 18. Production of Alkanes and Alkenes in <i>E. coli</i> through Heterologous Expression of <i>Synechococcus elongatus</i> PCC7942 orf1594 and <i>Cyanothece</i> sp. ATCC51142 cce 0778

0275The genomic DNA encoding <i>Cyanothece</i> sp. ATCC51142 cce_0778 (YP_001802195; putative decarbonylase) (SEQ ID NO:27) was synthesized and cloned into the <i>Nde</i>I and <i>Xho</i>I sites of vector OP-183 (pACYC derivative) under the control of the P_trc promoter. The resulting construct was cotransformed with OP80-PCC7942_1594 into <i>E. coli</i> MG1655 and the cells were grown at 37°C in M9 minimal media supplemented with 100 µg/mL spectinomycin and 100 µg/mL carbenicillin. The cells were cultured and extracted as in Example 3 and analyzed by GC-MS as described in Example 26.

0276As shown in <figref idref="f0013">Fig. 21</figref>, <i>E. coli</i> cells cotransformed with the <i>S</i>. <i>elongatus</i> PCC7942_1594 and <i>Cyanothece</i> sp. ATCC51142 cce_0778 -bearing vectors produced the same fatty aldehydes and fatty alcohols as in Example 3, but also tridecane, pentadecene, pentadecane and heptadecene. This result indicates that ATCC51142 cce_0778 in <i>E. coli</i> converts tetradecanal, hexadecenal, hexadecanal and octadecenal to tridecane, pentadecene, pentadecane and heptadecene, respectively, and therefore is an active fatty aldehyde decarbonylase.

Example 19. Production of Alkanes and Alkenes in <i>E. coli</i> through Heterologous Expression of <i>Synechococcus elongatus</i> PCC7942 orf1594 and <i>Cyanothece</i> sp. PCC7425 Cyan7425_0398

0277The genomic DNA encoding <i>Cyanothece</i> sp. PCC7425 Cyan7425_0398 (YP_002481151; putative decarbonylase) (SEQ ID NO:29) was synthesized and cloned into the <i>Nde</i>I and <i>Xho</i>I sites of vector OP-183 (pACYC derivative) under the control of the P_trc promoter. The resulting construct was cotransformed with OP80-PCC7942_1594 into <i>E. coli</i> MG1655 and the cells were grown at 37°C in M9 minimal media supplemented with 100 µg/mL spectinomycin and 100 µg/mL carbenicillin. The cells were cultured and extracted as in Example 3 and analyzed by GC-MS as described in Example 26.

0278As shown in <figref idref="f0013">Fig. 22</figref>, <i>E. coli</i> cells cotransformed with the <i>S</i>. <i>elongatus</i> PCC7942_1594 and <i>Cyanothece</i> sp. PCC7425 Cyan7425_0398 -bearing vectors produced the same fatty aldehydes and fatty alcohols as in Example 3, but also tridecane, pentadecene, pentadecane and heptadecene. This result indicates that Cyan7425_0398in <i>E. coli</i> converts tetradecanal, hexadecenal, hexadecanal and octadecenal to tridecane, pentadecene, pentadecane and heptadecene, respectively, and therefore is an active fatty aldehyde decarbonylase.

Example 20. Production of Alkanes and Alkenes in <i>E. coli</i> through Heterologous Expression of <i>Synechococcus elongatus</i> PCC7942 orf1594 and <i>Cyanothece</i> sp. PCC7425 Cyan7425 2986

0279The genomic DNA encoding <i>Cyanothece</i> sp. PCC7425 Cyan7425_2986 (YP_002483683; putative decarbonylase) (SEQ ID NO:31) was synthesized and cloned into the <i>Nde</i>I and <i>Xho</i>I sites of vector OP-183 (pACYC derivative) under the control of the P_trc promoter. The resulting construct was cotransformed with OP80-PCC7942_1594 into <i>E. coli</i> MG1655 and the cells were grown at 37°C in M9 minimal media supplemented with 100 µg/mL spectinomycin and 100 µg/mL carbenicillin. The cells were cultured and extracted as in Example 3 and analyzed by GC-MS as described in Example 26.

0280As shown in <figref idref="f0014">Fig. 23</figref>, <i>E. coli</i> cells cotransformed with the <i>S</i>. <i>elongatus</i> PCC7942_1594 and <i>Cyanothece</i> sp. PCC7425 Cyan7425_2986-bearing vectors produced the same fatty aldehydes and fatty alcohols as in Example 3, but also tridecane, pentadecene, pentadecane and heptadecene. This result indicates that Cyan7425_2986 in <i>E. coli</i> converts tetradecanal, hexadecenal, hexadecanal and octadecenal to tridecane, pentadecene, pentadecane and heptadecene, respectively, and therefore is an active fatty aldehyde decarbonylase.

Example 21. Production of Alkanes and Alkenes in <i>E. coli</i> through Heterologous Expression of <i>Prochlorococcus marinus</i> CCMP1986 PMM0533 and <i>Prochlorococcus mariunus</i> CCMP1986 PMM0532

0281The genomic DNA encoding <i>P.mariunus</i> CCMP1986 PMM0533 (NP_892651; putative aldehyde-generating enzyme) (SEQ ID NO:71) and <i>Prochlorococcus mariunus</i> CCMP1986 PMM0532 (NP_892650; putative decarbonylase) (SEQ ID NO: 19) were amplified and cloned into the <i>Ncol</i> and <i>Eco</i>RI sites of vector OP-80 and the <i>Nde</i>I and <i>Xho</i>I sites of vector OP-183, respectively. The resulting constructs were separately transformed and cotransformed into <i>E. coli</i> MG1655 and the cells were grown at 37°C in M9 minimal media supplemented with 100 µg/mL spectinomycin and 100 µg/mL carbenicillin. The cells were cultured and extracted as in Example 3 and analyzed by GC-MS as described in Example 26.

0282As shown in <figref idref="f0014">Fig. 24A</figref>, <i>E. coli</i> cells transformed with only the <i>P.mariunus</i> CCMP1986 PMM0533-bearing vector did not produce any fatty aldehydes or fatty alcohols. However, <i>E. coli</i> cells cotransformed with PMM0533 and PMM0532-bearing vectors produced hexadecanol, pentadecane and heptadecene (<figref idref="f0014">Fig. 24B</figref>). This result indicates that PMM0533 only provides fatty aldehyde substrates for the decarbonylation reaction when it interacts with a decarbonylase, such as PMM0532.

Example 22. Production of Alkanes and Alkenes in a Fatty Acyl-CoA-producing E. <i>coli</i> strain through Heterologous Expression of <i>Synechococcus elongatus</i> PCC7942 orf1594 and <i>Acarvochloris marina</i> MBIC11017 AMI 4041

0283The genomic DNA encoding <i>Acaryochloris marina</i> MBIC11017 AM1_4041 (YP_001518340; putative fatty aldehyde decarbonylase) (SEQ ID NO:9) was synthesized and cloned into the <i>Nde</i>I and <i>XhoI</i> sites of vector OP-183 (pACYC derivative) under the control of the P_trc promoter. The resulting construct was cotransformed with OP80-PCC7942_1594 into <i>E. coli</i> MG1655 Δ<i>fadE lacZ</i>::<i>P_trc 'tesA-fadD.</i> This strain expresses a cytoplasmic version of the <i>E. coli</i> thioesterase, 'TesA, and the <i>E. coli</i> acyl-CoA synthetase, FadD, under the control of the P_trc promoter, and therefore produces fatty acyl-CoAs. The cells were grown at 37°C in M9 minimal media supplemented with 100 µg/mL spectinomycin and 100 µg/mL carbenicillin. The cells were cultured and extracted as in Example 3 and analyzed by GC-MS as described in Example 1.

0284As shown in <figref idref="f0015">Fig. 25</figref>, these <i>E. coli</i> cells cotransformed with S. <i>elongatus</i> PCC7942_1594 and <i>A</i>. <i>marina</i> MBIC11017 AM1_4041 also produced alkanes and fatty alcohols. This result indicates that <i>S. elongatus</i> PCC7942_1594 is able to use acyl-CoA as a substrate to produce hexadecenal, hexadecanal and octadecenal, which is then converted into pentadecene, pentadecane and heptadecene, respectively, by <i>A</i>. <i>marina</i> MBIC11017 AM1_4041.

Example 23. Production of Alkanes and Alkenes in a Fatty Acyl-CoA-producing <i>E</i>. <i>coli</i> Strain through Heterologous Expression of <i>Synechocystis</i> sp. PCC6803 sll0209 and <i>Synechocystis</i> sp. PCC6803 sll0208

0285The genomic DNA encoding <i>Synechocystis</i> sp. PCC6803 sll0208 (NP_442147; putative fatty aldehyde decarbonylase) (SEQ ID NO:3) was synthesized and cloned into the <i>Nde</i>I and <i>Xho</i>I sites of vector OP-183 (pACYC derivative) under the control of the P_trc promoter. The genomic DNA encoding <i>Synechocystis</i> sp. PCC6803 sll0209 (NP_442146; acyl-ACP reductase) (SEQ ID NO:67) was synthesized and cloned into the <i>Ncol</i> and <i>EcoR</i>I sites of vector OP-183 (pACYC derivative) under the control of the P_trc promoter. The resulting constructs were cotransformed with into <i>E. coli</i> MG1655 Δ<i>fadE lacZ</i>::<i>P_trc 'tesA-fadD.</i> This strain expresses a cytoplasmic version of the <i>E. coli</i> thioesterase, 'TesA, and the <i>E</i>. <i>coli</i> acyl-CoA synthetase, FadD, under the control of the P_trc promoter, and therefore produces fatty acyl-CoAs. The cells were grown at 37°C in M9 minimal media supplemented with 100 µg/mL spectinomycin and 100 µg/mL carbenicillin. The cells were cultured and extracted as in Example 3 and analyzed by GC-MS as described in Example 26.

0286As shown in <figref idref="f0016">Fig. 26</figref>, these <i>E. coli</i> cells transformed with <i>Synechocystis</i> sp. PCC6803 sll0209 did not produce any fatty aldehydes or fatty alcohols. However, when cotransformed with <i>Synechocystis</i> sp. PCC6803 sll0208 and sll0209, they produced alkanes, fatty aldehydes and fatty alcohols. This result indicates that <i>Synechocystis</i> sp. PCC6803 sll0209 is able to use acyl-CoA as a substrate to produce fatty aldehydes such as tetradecanal, hexadecanal and octadecenal, but only when coexpressed with a fatty aldehyde decarbonylase. The fatty aldehydes apparently are further reduced to the corresponding fatty alcohols, tetradecanol, hexadecanol and octadecenol, by an endogenous <i>E. coli</i> aldehyde reductase activity. In this experiment, octadecenal was converted into heptadecene by <i>Synechocystis</i> sp. PCC6803 sll0208.

Example 24. Production of Alkanes and Alkenes in a Fatty Aldehyde-producing <i>E</i>. <i>coli</i> Strain through Heterologous Expression of <i>Nostoc punctiforme</i> PCC73102 Npun02004178

and Several of its Homologs

0287The genomic DNA encoding <i>Nostoc punctiforme</i> PCC73102 Npun02004178 (ZP_00108838; putative fatty aldehyde decarbonylase) (SEQ ID NO:5) was amplified and cloned into the <i>Nde</i>I and <i>Xho</i>I sites of vector OP-183 (pACYC derivative) under the control of the P_trc promoter. The genomic DNA encoding <i>Mycobacterium smegmatis</i> strain MC2 155 orf MSMEG_5739 (YP_889972, putative carboxylic acid reductase) (SEQ ID NO:85) was amplified and cloned into the <i>Ncol</i> and <i>EcoR</i>I sites of vector OP-180 (pCL1920 derivative) under the control of the P_trc promoter. The two resulting constructs were cotransformed into <i>E. coli</i> MG1655 Δ<i>fadD lacZ</i>::<i>P_trc-'tesA.</i> In this strain, fatty aldehydes were provided by MSMEG_5739, which reduces free fatty acids (formed by the action of 'TesA) to fatty aldehydes. The cells were grown at 37°C in M9 minimal media supplemented with 100 µg/mL spectinomycin and 100 µg/mL carbenicillin. The cells were cultured and extracted as in Example 3 and analyzed by GC-MS as described in Example 1.

0288As shown in <figref idref="f0017">Fig. 27</figref>, these <i>E. coli</i> cells cotransformed with the <i>N. punctiforme</i> PCC73102 Npun02004178 and <i>M. smegmatis</i> strain MC2 155 MSMEG_5739-bearing vectors produced tridecane, pentadecene and pentadecane. This result indicates that Npun02004178 in <i>E. coli</i> converts tetradecanal, hexadecenal and hexadecanal provided by the carboxylic acid reductase MSMEG_5739 to tridecane, pentadecene and pentadecane. As shown in <figref idref="f0018">Fig. 28</figref>, in the same experimental set-up, the following fatty aldehyde decarbonylases also converted fatty aldehydes provided by MSMEG_5739 to the corresponding alkanes when expressed in <i>E. coli</i> MG1655 Δ<i>fadD lacZ</i>::<i>P_trc-'tesA: Nostoc</i> sp. PCC7210 alr5283 (SEQ ID NO:7), <i>P. mariunus</i> CCMP1986 PMM0532 (SEQ ID NO:19), G. <i>violaceus</i> PCC7421 gll3146 (SEQ ID NO:15), <i>Synechococcus</i> sp. RS9917_09941 (SEQ ID NO:23), <i>Synechococcus</i> sp. RS9917_12945 (SEQ ID NO:25), and A. <i>marina</i> MBIC11017 AM1_4041 (SEQ ID NO:9).

Example 25: Cyanobacterial Fatty Aldehyde Decarbonylases belong to the class of Non-heme Diiron Proteins. Site-directed mutagenesis of conserved histidines to phenylalanines in <i>Nostoc punctiforme</i> PCC73102 Npun02004178

does not abolish its catalytic function

0289As discussed in Example 13, the hypothetical protein PMT1231 from <i>Prochlorococcus marinus</i> MIT9313 (SEQ ID NO: 18) is an active fatty aldehyde decarbonylase. Based on the three-dimensional structure of PMT1231, which is available at 1.8 Å resolution (pdb2OC5A) (see <figref idref="f0019">Fig. 29B</figref>), cyanobacterial fatty aldehyde decarbonylases have structural similarity with non-heme diiron proteins, in particular with class I ribonuclease reductase subunit β proteins, RNRβ (Stubbe and Riggs-Gelasco, TIBS 1998, vol. 23., pp. 438) (see <figref idref="f0019">Fig. 29A</figref>). Class Ia and Ib RNRβ contains a diferric tyrosyl radical that mediates the catalytic activity of RNRα (reduction of ribonucleotides to deoxyribonucleotides). In <i>E. coli</i> RNRβ, this tyrosine is in position 122 and is in close proximity to one of the active site's iron molecules. Structural alignment showed that PMT1231 contained a phenylalanine in the same position as RNRb tyr122, suggesting a different catalytic mechanism for cyanobacterial fatty aldehyde decarbonylases. However, an aligment of all decarbonylases showed that two tyrosine residues were completely conserved in all sequences, tyr135 and tyr138 with respect to PMT1231, with tyr135 being in close proximity (5.5 Å) to one of the active site iron molecules (see <figref idref="f0019">Fig. 29C</figref>). To examine whether either of the two conserved tyrosine residues is involved in the catalytic mechanism of cyanobacterial fatty aldehyde decarbonylases, these residues were replaced with phenylalanine in Npun02004178 (tyr 123 and tyr126) as follows.

0290The genomic DNA encoding <i>S</i>. <i>elongatus</i> PCC7942 ORF1594 (SEQ ID NO:65) was cloned into the NcoI and EcoRI sites of vector OP-80 (pCL1920 derivative) under the control of the P_trc promoter. The genomic DNA encoding <i>N</i>. <i>punctiforme</i> PCC73102 Npun02004178 (SEQ ID NO:5) was also cloned into the <i>Nde</i>I and <i>Xho</i>I sites of vector OP-183 (pACYC177 derivative) under the control of the P_trc promoter. The latter construct was used as a template to introduce a mutation at positions 123 and 126 of the decarbonylase protein, changing the tyrosines to phenylalanines using the primers gttttgcgatcgcagcatttaacatttacatccccgttgccgacg and gttttgcgatcgcagcatataacattttcatccccgttgccgacg, respectively. The resulting constructs were then transformed into <i>E. coli</i> MG1655. The cells were grown at 37°C in M9 minimal media supplemented with 1% glucose (w/v), and 100 µg/mL carbenicillin and spectinomycin. The cells were cultured and extracted as in Example 3.

0291As shown in <figref idref="f0020">Fig. 30</figref>, the two Npun02004178 Tyr to Phe protein variants were active and produced alkanes when coexpressed with <i>S</i>. <i>elongatus</i> PCC7942 ORF1594. This result indicates that in contrast to class Ia and Ib RNRβ proteins, the catalytic mechanism of fatty aldehyde decarbonylases does not involve a tyrosyl radical.

Example 26: Biochemical characterization of <i>Nostoc punctiforme</i> PCC73102 Npun02004178

0292The genomic DNA encoding <i>N. punctiforme</i> PCC73102 Npun02004178 (SEQ ID NO:5) was cloned into the <i>Nde</i>I and <i>Xho</i>I sites of vector pET-15b under the control of the T7 promoter. The resulting Npun02004178 protein contained an N-terminal His-tag. An <i>E. coli</i> BL21 strain (DE3) (Invitrogen) was transformed with the plasmid by routine chemical transformation techniques. Protein expression was carried out by first inoculating a colony of the <i>E. coli</i> strain in 5 mL of LB media supplemented with 100 mg/L of carbenicillin and shaken overnight at 37 °C to produce a starter culture. This starter cultures was used to inoculate 0.5 L of LB media supplemented with 100 mg/L of carbenecillin. The culture was shaken at 37 °C until an OD₆₀₀ value of 0.8 was reached, and then IPTG was added to a final concentration of 1 mM. The culture was then shaken at 37 °C for approximately 3 additional h. The culture was then centrifuged at 3,700 rpm for 20 min at 4 °C. The pellet was then resuspended in 10 mL of buffer containing 100 mM sodium phosphate buffer at pH 7.2 supplemented with Bacterial ProteaseArrest (GBiosciences). The cells were then sonicated at 12 W on ice for 9 s with 1.5 s of sonication followed by 1.5 s of rest. This procedure was repeated 5 times with one min intervals between each sonication cycle. The cell free extract was centrifuged at 10,000 rpm for 30 min at 4 °C. 5 mL of Ni-NTA (Qiagen) was added to the supernatant and the mixture was gently stirred at 4 °C. The slurry was passed over a column removing the resin from the lysate. The resin was then washed with 30 mL of buffer containing 100 mM sodium phosphate buffer at pH 7.2 plus 30 mM imidazole. Finally, the protein was eluted with 10 mL of 100 mM sodium phosphate buffer at pH 7.2 plus 250 mM imidazole. The protein solution was dialyzed with 200 volumes of 100 mM sodium phosphate buffer at pH 7.2 with 20% glycerol. Protein concentration was determined using the Bradford assay (Biorad). 5.6 mg/mL of Npun02004178 protein was obtained.

0293To synthesize octadecanal for the decarbonylase reaction, 500 mg of octadecanol (Sigma) was dissolved in 25 mL of dichloromethane. Next, 200 mg of pyridinium chlorochromate (TCI America) was added to the solution and stirred overnight. The reaction mixture was dried under vacuum to remove the dichloromethane. The remaining products were resuspended in hexane and filtered through Whatman filter paper. The filtrate was then dried under vacuum and resuspended in 5 mL of hexane and purified by silica flash chromatography. The mixture was loaded onto the gravity fed column in hexane and then washed with two column volumes of hexane. The octadecanal was then eluted with an 8:1 mixture of hexane and ethyl acetate. Fractions containing octadecanal were pooled and analyzed using the GC/MS methods described below. The final product was 95% pure as determined by this method.

0294To test Npun02004178 protein for decarbonylation activity, the following enzyme assays were set-up. 200 µL reactions were set up in 100 mM sodium phosphate buffer at pH 7.2 with the following components at their respective final concentrations: 30 µM of purified Npun02004178 protein, 200 µM octadecanal, 0.11 µg/mL spinach ferredoxin (Sigma), 0.05 units/mL spinach ferredoxin reductase (Sigma), and 1 mM NADPH (Sigma). Negative controls included the above reaction without Npun02004178, the above reaction without octadecanal, and the above reaction without spinach ferredoxin, ferredoxin reductase and NADPH. Each reaction was incubated at 37°C for 2 h before being extracted with 100 µL ethyl acetate. Samples were analyzed by GC/MS using the following parameters: run time: 13.13 min; column: HP-5-MS Part No. 19091S-433E (length of 30 meters; I.D.: 0.25 mm narrowbore; film: 0.251M); inject: 1 ìl Agilent 6850 inlet; inlet: 300 C splitless; carrier gas: helium; flow: 1.3 mL/min; oven temp: 75 °C hold 5 min, 320 at 40 °C/min, 320 hold 2 min; det: Agilent 5975B VL MSD; det. temp: 330 °C; scan: 50-550 M/Z. Heptadecane from Sigma was used as an authentic reference for determining compound retention time and fragmentation pattern.

0295As shown in <figref idref="f0021">Fig. 31</figref>, <i>in-vitro</i> conversion of octadecanal to heptadecane was observed in the presence of Npun02004178. The enzymatic decarbonylation of octadecanal by Npun02004178 was dependent on the addition of spinach ferredoxin reducatase, ferredoxin and NADPH.

0296Next, it was determined whether cyanobaterial ferredoxins and ferredoxin reductases can replace the spinach proteins in the <i>in-vitro</i> fatty aldehyde decarbonylase assay. The following four genes were cloned separately into the <i>Nde</i>I and <i>Xho</i>I sites of pET-15b: <i>N. punctiforme</i> PCC73102 Npun02003626 (ZP_00109192, ferredoxin oxidoreductase petH without the n-terminal allophycocyanin linker domain) (SEQ ID NO:87), <i>N. punctiforme</i> PCC73102 Npun02001001 (ZP_00111633, ferredoxin 1) (SEQ ID NO:89), <i>N. punctiforme</i> PCC73102 Npun02003530 (ZP_00109422, ferredoxin 2) (SEQ ID NO:91) and <i>N</i>. <i>punctiforme</i> PCC73102 Npun02003123 (ZP_00109501, ferredoxin 3) (SEQ ID NO:93). The four proteins were expressed and purified as described above. 1 mg/mL of each ferredoxin and 4 mg/mL of the ferredoxin oxidoreductase was obtained. The three cyanobacterial ferredoxins were tested with the cyanobacterial ferredoxin oxidoreductase using the enzymatic set-up described earlier with the following changes. The final concentration of the ferredoxin reductase was 60 µg/mL and the ferredoxins were at 50 µg/mL. The extracted enzymatic reactions were by GC/MS using the following parameters: run time: 6.33 min; column: J&amp;W 122-5711 DB-5ht (length of 15 meters; I.D.: 0.25 mm narrowbore; film: 0.10µM); inject: 1 µL Agilent 6850 inlet; inlet: 300 °C splitless; carrier gas: helium; flow: 1.3 mL/min; oven temp: 100 °C hold 0.5 min, 260 at 30 °C/min, 260 hold 0.5 min; det: Agilent 5975B VL MSD; det. temp: 230 °C; scan: 50-550 M/Z.

0297As shown in <figref idref="f0022">Fig. 32</figref>, Npun02004178-dependent <i>in-vitro</i> conversion of octadecanal to heptadecane was observed in the presence of NADPH and the cyanobacterial ferredoxin oxidoreductase and any of the three cyanobacterial ferredoxins.

Example 27. Biochemical characterization of <i>Synechococcus elongatus</i> PCC7942 orf1594

0298The genomic DNA encoding <i>S. elongatus</i> PCC7492 orf1594 (SEQ ID NO:65) was cloned into the <i>Ncol</i> and <i>Xho</i>I sites of vector pET-28b under the control of the T7 promoter. The resulting PCC7942_orf1594 protein contained a C-terminal His-tag. An <i>E. coli</i> BL21 strain (DE3) (Invitrogen) was transformed with the plasmid and PCC7942_orf1594 protein was expressed and purified as described in Example 22. The protein solution was stored in the following buffer: 50 mM sodium phosphate, pH 7.5, 100 mM NaCl, 1 mM THP, 10% glycerol. Protein concentration was determined using the Bradford assay (Biorad). 2 mg/mL of PCC7942_orf1594 protein was obtained.

0299To test PCC7942_orf1594 protein for acyl-ACP or acyl-CoA reductase activity, the following enzyme assays were set-up. 100 µL reactions were set-up in 50 mM Tris-HCl buffer at pH 7.5 with the following components at their respective final concentrations: 10 µM of purified PCC7942_orf1594 protein, 0.01-1 mM acyl-CoA or acyl-ACP, 2 mM MgCl₂, 0.2-2 mM NADPH. The reactions were incubated for 1 h at 37°C and where stopped by adding 100 µL ethyl acetate (containing 5 mg/l 1-octadecene as internal standard). Samples were vortexed for 15 min and centrifuged at max speed for 3 min for phase separation. 80 µL of the top layer were transferred into GC glass vials and analyzed by GC/MS as described in Example 26. The amount of aldehyde formed was calculated based on the internal standard.

0300As shown in <figref idref="f0022">Fig. 33</figref>, PCC7942_orf1594 was able to reduce octadecanoyl-CoA to octadecanal. Reductase activity required divalent cations such as Mg²⁺, Mn²⁺ or Fe²⁺ and NADPH as electron donor. NADH did not support reductase activity. PCC7942_orf1594 was also able to reduce octadecenoyl-CoA and octadecenoyl-ACP to octadecenal. The K_m values for the reduction of octadecanoyl-CoA, octadecenoyl-CoA and octadecenoyl-ACP in the presence of 2 mM NADPH were determined as 45 ± 20 µM, 82 ± 22 µM and 7.8 ± 2 µM, respectively. These results demonstrate that PCC7942_orf1594, <i>in vitro,</i> reduces both acyl-CoAs and acyl-ACPs and that the enzyme apparently has a higher affinity for acyl-ACPs as compared to acyl-CoAs. The K_m value for NADPH in the presence of 0.5 mM octadecanoyl-CoA for PCC7942_orf1594 was determined as 400 ± 80 µM.

0301Next, the stereospecific hydride transfer from NADPH to a fatty aldehyde catalyzed by PCC7942_orf1594 was examined. Deutero-NADPH was prepared according to the following protocol. 5 mg of NADP⁺ and 3.6 mg of D-glucose-1-d was added to 2.5 mL of 50 mM sodium phosphate buffer (pH 7.0). Enzymatic production of labeled NADPH was initiated by the addition of 5 units of glucose dehydrogenase from either <i>Bacillus megaterium</i> (USB Corporation) for the production of R-(4-²H)NADPH or <i>Thermoplasma acidophilum</i> (Sigma) for the production of S-(4-²H)NADPH. The reaction was incubated for 15 min at 37°C, centrifuge-filtered using a 10 KDa MWCO Amicon Ultra centrifuge filter (Millipore), flash frozen on dry ice, and stored at -80°C.

0302The <i>in vitro</i> assay reaction contained 50 mM Tris-HCl (pH 7.5), 10 µM of purified PCC7942_orf1594 protein, 1 mM octadecanoyl-CoA, 2 mM MgCl₂, and 50 µL deutero-NADPH (prepared as described above) in a total volume of 100 µL. After a 1 h incubation, the product of the enzymatic reaction was extracted and analyzed as described above. The resulting fatty aldehyde detected by GC/MS was octadecanal (see <figref idref="f0023">Fig. 34</figref>). Because hydride transfer from NADPH is stereospecific, both <i>R</i>-(4-²H)NADPH and <i>S</i>-(4-²H)NADPH were synthesized. Octadecanal with a plus one unit mass was observed using only the <i>S</i>-(4-²H)NADPH. The fact that the fatty aldehyde was labeled indicates that the deuterated hydrogen has been transferred from the labeled NADPH to the labeled fatty aldehyde. This demonstrates that NADPH is used in this enzymatic reaction and that the hydride transfer catalyzed by PCC7942_orf1594 is stereospecific.

Example 28. Intracellular and Extracellular Production of Fatty Aldehydes and Fatty Alcohols in <i>E. coli</i> through Heterologous Expression of <i>Svnechococcus elongatus</i> PCC7942 orf1594

0303The genomic DNA encoding <i>Synechococcus elongatus</i> PCC7942 orf1594 (YP_400611; acyl-ACP reductase) (SEQ ID NO:65) was amplified and cloned into the <i>Nco</i>I and <i>EcoR</i>I sites of vector OP-80 (pCL1920 derivative) under the control of the P_trc promoter. The resulting construct was cotransformed into <i>E</i>. <i>coli</i> MG1655 Δ<i>fadE</i> and the cells were grown at 37°C in 15 mL Che-9 minimal media with 3% (w/v) glucose as carbon source and supplemented with 100 µg/mL spectinomycin and carbenicillin, respectively. When the culture reached OD₆₀₀ of 0.8-1.0, it was induced with 1mM IPTG and cells were grown for an additional 24-48 h at 37°C. Che-9 minimal medium is defined as: 6g/L Na₂HPO₄, 3 g/L KH₂PO₄, 0.5 g/L NaCl, 2 g/L NH₄Cl, 0.25 g/L MgSO₄ x 7 H₂O, 11 mg/L CaCl₂, 27 mg/L Fe₃Cl x 6 H₂O, 2 mg/L ZnCl x 4 H₂O, 2 mg/L Na₂MoO₄ x 2 H₂O, 1.9 mg/L CuSO₄ x 5 H₂O, 0.5 mg/L H₃BO₃, 1 mg/L thiamine, 200 mM Bis-Tris (pH 7.25) and 0.1% (v/v) Triton-X100. When the culture reached OD₆₀₀ of 1.0-1.2, it was induced with 1 mM IPTG and cells were allowed to grow for an additional 40 hrs at 37°C. Cells from 0.5 mL of culture were extracted with 0.5 mL of ethyl acetate for total hydrocarbon production as described in Example 26. Additionally, cells and supernatant were separated by centrifugation (4,000 g at RT for 10 min) and extracted separately.

0304The culture produced 620 mg/L fatty aldehydes (tetradecanal, heptadecenal, heptadecanal and octadecenal) and 1670 mg/L fatty alcohols (dodecanol, tetradecenol, tetradecanol, heptadecenol, heptadecanol and octadecenol). <figref idref="f0024">Fig 35</figref> shows the chromatogram of the extracted supernatant. It was determined that 73 % of the fatty aldehydes and fatty alcohols were in the cell-free supernatant.

Example 29. Intracellular and Extracellular Production of Alkanes and Alkenes in <i>E. coli</i> through Heterologous Expression of <i>Synechococcus elongatus</i> PCC7942 orf1594 and <i>Nostoc punctiforme</i> PCC73102 Npun02004178

0305The genomic DNA encoding <i>Synechococcus elongatus</i> PCC7942 orf1594 (YP_400611; acyl-ACP reductase) (SEQ ID NO:65) was amplified and cloned into the <i>Ncol</i> and <i>Eco</i>RI sites of vector OP-80 (pCL1920 derivative) under the control of the P_trc promoter. The genomic DNA encoding <i>Nostoc punctiforme</i> PCC73102 Npun02004178 (ZP_00108838; fatty aldehyde decarbonylase) (SEQ ID NO:5) was amplified and cloned into the <i>Nde</i>I and <i>Xho</i>I sites of vector OP-183 (pACYC derivative) under the control of the P_trc promoter. The resulting constructs were cotransformed into <i>E. coli</i> MG1655 Δ<i>fadE</i> and the cells were grown at 37°C in 15 mL Che9 minimal media with 3% (w/v) glucose as carbon source and supplemented with 100 µg/mL spectinomycin and carbenicillin, respectively. The cells were grown, separated from the broth, extracted, and analyzed as described in Example 28.

0306The culture produced 323 mg/L alkanes and alkenes (tridecane, pentadecene, pentadecane and heptadecene), 367 mg/L fatty aldehydes (tetradecanal, heptadecenal, heptadecanal and octadecenal) and 819 mg/L fatty alcohols (tetradecanol, heptadecenol, heptadecanol and octadecenol). <figref idref="f0024">Fig. 36</figref> shows the chromatogram of the extracted supernatant. It was determined that 86% of the alkanes, alkenes, fatty aldehydes and fatty alcohols were in the cell-free supernatant.

Example 30. Production of Alkanes and Alkenes in <i>E. coli</i> through Heterologous Expression of <i>Nostoc</i> sp. PCC7210 alr5284 and <i>Nostoc</i> sp. PCC7210 alr5283

0307The genomic DNA encoding <i>Nostoc</i> sp. PCC7210 alr5284 (NP_489324; putative aldehyde-generating enzyme) (SEQ ID NO:81) was amplified and cloned into the <i>Ncol</i> and <i>EcoRI</i> sites of vector OP-80 (pCL1920 derivative) under the control of the P_trc promoter. The genomic DNA encoding <i>Nostoc</i> sp. PCC7210 alr5283 (NP_489323; putative decarbonylase) (SEQ ID NO:7) was amplified and cloned into the <i>Nde</i>I and <i>Xho</i>I sites of vector OP-183 (pACYC derivative) under the control of the P_trc promoter. The resulting constructs were cotransformed into <i>E</i>. <i>coli</i> MG1655 and the cells were grown at 37°C in 15 mL Che9 minimal media with 3% (w/v) glucose as carbon source and supplemented with 100 µg/mL spectinomycin and carbenicillin, respectively (as described in Example 28). Cells from 0.5 mL of culture were extracted and analyzed as described in Example 3 and analyzed by GC-MS as described in Example 26.

0308As shown in <figref idref="f0025">Fig. 37</figref>, <i>E. coli</i> cells cotransformed with the <i>Nostoc</i> sp. PCC7210 alr5284 and <i>Nostoc</i> sp. PCC7210 alr5283-bearing vectors produced tridecane, pentadecene, pentadecane, tetradecanol and hexadecanol. This result indicates that coexpression of <i>Nostoc</i> sp. PCC7210 alr5284 and alr5283 is sufficient for <i>E. coli</i> to produce fatty alcohols, alkanes and alkenes.

0309It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims.

SEQUENCE LISTING

0310<ul id="ul0002" list-style="none"><li><i>Synechococcus elongatus</i> PCC7942 Synpcc7942_1593 (YP_400610) nucleotide (SEQ ID NO:1) <img file="EP2288694B1_D0004.tif" /></li><li><i>Synechococcus elongatus</i> PCC7942 Synpcc7942_1593 (YP_400610) amino acid (SEQ ID NO:2) <img file="EP2288694B1_D0005.tif" /></li><li><i>Synechocystis</i> sp. PCC6803 sll0208 (NP_442147) nucleotide (SEQ ID NO:3) <img file="EP2288694B1_D0006.tif" /></li><li><i>Synechocystis</i> sp. PCC6803 sll0208 (NP_442147) amino acid (SEQ ID NO:4) <img file="EP2288694B1_D0007.tif" /></li><li><i>Nostoc punctiforme</i> PCC 73102 Npun02004178 (ZP_00108838) nucleotide (SEQ ID NO:5) <img file="EP2288694B1_D0008.tif" /><img file="EP2288694B1_D0009.tif" /></li><li><i>Nostoc punctiforme</i> PCC 73102 Npun02004178 (ZP_00108838) amino acid (SEQ ID NO:6) <img file="EP2288694B1_D0010.tif" /></li><li><i>Nostoc</i> sp. PCC 7120 alr5283 (NP_489323) nucleotide (SEQ ID NO:7) <img file="EP2288694B1_D0011.tif" /></li><li><i>Nostoc</i> sp. PCC 7120 alr5283 (NP_489323) amino acid (SEQ ID NO:8) <img file="EP2288694B1_D0012.tif" /></li><li><i>Acaryochloris marina</i> MBIC11017 AM1_4041 (YP_001518340) nucleotide (SEQ ID NO:9) <img file="EP2288694B1_D0013.tif" /></li><li><i>Acaryochloris marina</i> MBIC11017 AM1_4041 (YP_001518340) amino acid (SEQ ID NO:10) <img file="EP2288694B1_D0014.tif" /><img file="EP2288694B1_D0015.tif" /></li><li><i>Thermosynechococcus elongatus</i> BP-1 tll1313 (NP_682103) nucleotide (SEQ ID NO:11) <img file="EP2288694B1_D0016.tif" /></li><li><i>Thermosynechococcus elongatus</i> BP-1 tll1313 (NP_682103) amino acid (SEQ ID NO:12) <img file="EP2288694B1_D0017.tif" /></li><li><i>Synechococcus</i> sp. JA-3-3A CYA_0415 (YP_473897) nucleotide (SEQ ID NO:13) <img file="EP2288694B1_D0018.tif" /></li><li><i>Synechococcus</i> sp. JA-3-3A CYA_0415 (YP_473897) amino acid (SEQ ID NO:14) <img file="EP2288694B1_D0019.tif" /></li><li><i>Gloeobacter violaceus</i> PCC 7421 <u>gll3146</u> (NP_926092) nucleotide (SEQ ID NO:15) <img file="EP2288694B1_D0020.tif" /><img file="EP2288694B1_D0021.tif" /></li><li><i>Gloeobacter violaceus</i> PCC 7421 gll3146 (NP_926092) amino acid (SEQ ID NO:16) <img file="EP2288694B1_D0022.tif" /></li><li><i>Prochlorococcus marinus</i> MIT9313 PM123 (NP_895059) nucleotide (SEQ ID NO:17) <img file="EP2288694B1_D0023.tif" /></li><li><i>Prochlorococcus marinus</i> MIT9313 PM1231 (NP_895059) amino acid (SEQ ID NO:18) <img file="EP2288694B1_D0024.tif" /></li><li><i>Prochlorococcus marinus</i> subsp. pastoris str. CCMP1986 PMM0532 (NP_892650) nucleotide (SEQ ID NO:19) <img file="EP2288694B1_D0025.tif" /><img file="EP2288694B1_D0026.tif" /></li><li><i>Prochlorococcus marinus</i> subsp. pastoris str. CCMP1986 PMM0532 (NP_892650) amino acid (SEQ ID NO:20) <img file="EP2288694B1_D0027.tif" /></li><li><i>Prochlorococcus marinus</i> str. NATL2A PMN2A_1863 (YP_293054) nucleotide (SEQ ID NO:21) <img file="EP2288694B1_D0028.tif" /></li><li><i>Prochlorococcus marinus</i> str. NATL2A PMN2A_1863 (YP_293054) amino acid (SEQ ID NO:22) <img file="EP2288694B1_D0029.tif" /></li><li><i>Synechococcus</i> sp. RS9917 RS9917_09941 (ZP_01079772) nucleotide (SEQ ID NO:23) <img file="EP2288694B1_D0030.tif" /></li><li><i>Synechococcus</i> sp. RS9917 RS9917_09941 (ZP_01079772) amino acid (SEQ ID NO:24) <img file="EP2288694B1_D0031.tif" /></li><li><i>Synechococcus</i> sp. RS9917 RS9917_12945 (ZP_01080370) nucleotide (SEQ ID NO:25) <img file="EP2288694B1_D0032.tif" /></li><li><i>Synechococcus</i> sp. RS9917 RS9917_12945 (ZP_01080370) amino acid (SEQ ID NO:26) <img file="EP2288694B1_D0033.tif" /></li><li><i>Cyanothece</i> sp. ATCC51142 cce_0778 (YP_001802195) nucleotide (SEQ ID NO:27) <img file="EP2288694B1_D0034.tif" /></li><li><i>Cyanothece</i> sp. ATCC51142 cce_0778 (YP_001802195) amino acid (SEQ ID NO:28) <img file="EP2288694B1_D0035.tif" /></li><li><i>Cyanothece</i> sp. PCC7245 Cyan7425_0398 (YP_002481151) nucleotide (SEQ ID NO:29) <img file="EP2288694B1_D0036.tif" /><img file="EP2288694B1_D0037.tif" /></li><li><i>Cyanothece</i> sp. PCC7245 Cyan7425_0398 (YP_002481151) amino acid (SEQ ID NO:30) <img file="EP2288694B1_D0038.tif" /></li><li><i>Cyanothece</i> sp. PCC7245 Cyan7425_2986 (YP_002483683) nucleotide (SEQ ID NO:31) <img file="EP2288694B1_D0039.tif" /></li><li><i>Cyanothece</i> sp. PCC7245 Cyan7425_2986 (YP_002483683) amino acid (SEQ ID NO:32) <img file="EP2288694B1_D0040.tif" /></li><li><i>Anabaena variabilis</i> ATCC29413 YP_323043 (Ava_2533) nucleotide (SEQ ID NO:33) <img file="EP2288694B1_D0041.tif" /></li><li><i>Anabaena variabilis</i> ATCC29413 YP_323043 (Ava_2533) amino acid (SEQ ID NO:34) <img file="EP2288694B1_D0042.tif" /></li><li><i>Synechococcus elongatus</i> PCC6301 YP_170760 (syc0050_d) nucleotide (SEQ ID NO:35) <img file="EP2288694B1_D0043.tif" /><img file="EP2288694B1_D0044.tif" /></li><li><i>Synechococcus elongatus</i> PCC6301 YP_170760 (syc0050_d) amino acid (SEQ ID NO:36) <img file="EP2288694B1_D0045.tif" /></li><li>Motif 1: (SEQ ID NO:37) Y-X(2)-A-Y-X-R-X(4)-V-X(2)-G-E-X(2)-A</li><li>Motif 2: (SEQ ID NO:38) L-X(2)-M-E-X(2)-H-X(3)-F-X(2)-C</li><li>Motif 3: (SEQ ID NO:39) C-X(3)-Q-X(4)-E-X-F-A-X(2)-A-Y</li><li>Motif 4: (SEQ ID NO:40) T-X(2)-V-X(3)-E-X(2)-H-X(6)-W-L</li><li>Motif 5: (SEQ ID NO:41) Y-[KRQ]-X-A-Y-[SR]-R-[IV]-[DN]-[GA]-[VI]-V-[IL]-X-G-E-X(2)-A-X(2)-[NH]-[YF]</li><li>Motif 6: (SEQ ID NO:42) L-[ASG]-X-M-E-X-[RDK]-H-X(2)-[ASG]-F-X-[ASG]-C-[AG]-X-N-L-X-[VI]</li><li>Motif 7: (SEQ ID NO:43) C-[LMF]-[VL]-[IV]-Q-X-[LI]-[ILM]-[IV]-E-X-F-A-[IV]-[AS]-A-Y-[NHR]-X-Y-[IL]</li><li>Motif 8: (SEQ ID NO:44) <img file="EP2288694B1_D0046.tif" /></li><li><i>Nostoc punctiforme</i> PCC 73102 Npun02004178 (ZP_00108838) nucleotide, synthetic (SEQ ID NO:45) <img file="EP2288694B1_D0047.tif" /></li><li><i>Acaryochloris marina</i> MBIC11017 AM1_4041 (YP_001518340) nucleotide, synthetic (SEQ ID NO:46) <img file="EP2288694B1_D0048.tif" /></li><li><i>Thermosynechococcus elongatus</i> BP-1 tll1313 (NP_682103) nucleotide, synthetic (SEQ ID NO:47) <img file="EP2288694B1_D0049.tif" /></li><li><i>Synechococcus</i> sp. JA-3-3A CYA_0415 (YP_473897) nucleotide, synthetic (SEQ ID NO:48) <img file="EP2288694B1_D0050.tif" /><img file="EP2288694B1_D0051.tif" /></li><li><i>Prochlorococcus marinus</i> MIT9313 PM123 (NP_895059) nucleotide, synthetic (SEQ ID NO:49) <img file="EP2288694B1_D0052.tif" /></li><li><i>Prochlorococcus marinus</i> subsp. pastoris str. CCMP1986 PMM0532 (NP_892650) nucleotide, synthetic (SEQ ID NO:50) <img file="EP2288694B1_D0053.tif" /></li><li><i>Prochlorococcus marinus</i> str. NATL2A PMN2A_1863 (YP_293054) nucleotide, synthetic (SEQ ID NO:51) <img file="EP2288694B1_D0054.tif" /></li><li><i>Synechococcus</i> sp. RS9917 RS9917_09941 (ZP_01079772) nucleotide, synthetic (SEQ ID NO:52) <img file="EP2288694B1_D0055.tif" /><img file="EP2288694B1_D0056.tif" /></li><li><i>Synechococcus</i> sp. RS9917 RS9917_12945 (ZP_01080370) nucleotide, synthetic (SEQ ID NO:53) <img file="EP2288694B1_D0057.tif" /></li><li>Motif 9: (SEQ ID NO:54)</li><li>G-A-X-G-D-I-G-S-X(4)-W-X(12,13)-A-R</li><li>Motif 10: (SEQ ID NO:55) <img file="EP2288694B1_D0058.tif" /></li><li>Motif 11: (SEQ ID NO:56) {K}-L-X-[FLW]-X-R-F-T-T-G-N in which {K} corresponds to any amino acid but Lys</li><li>Motif 12: (SEQ ID NO:57) M-F-G-L-I-G-H-X(6)-A</li><li>Motif 13: (SEQ ID NO:58) L-[DE]-[FLVM]-W-[CS]-X-A-P-P-[QV]-X-[LV]-[DE]-X(3)-[VI]-X-S</li><li>Motif 14: (SEQ ID NO:59) S-X(2)-G-X(2)-I-X-G-X-Y-X(2)-S-X-F-X-PEML</li><li>Motif 15: (SEQ ID NO:60) K-X-A-X-R-K-X(3)-A-M-X(3)-Q-X(5)-I-X(2)-LGGF</li><li>Motif 16: (SEQ ID NO:61) A-[LVM]-X(2)-[AS]-[DN]-X-[VI]-[VI]-W-V-A-S-[LMI]</li><li>Motif 17: (SEQ ID NO:62) P-X(4)-D-G-G-Y-P-K-N</li><li>Motif 18: (SEQ ID NO:63) N-F-S-W-G-R-N-X-I-X(7)-I-G-X(2)S-X(2)-H-G</li><li>Motif 19: (SEQ IDNO:64) FTTGNTHTA</li><li><i>Synechococcus elongatus</i> PCC7942 Synpcc7942_1594 (YP_400611) nucleotide (SEQ ID NO:65) <img file="EP2288694B1_D0059.tif" /></li><li><i>Synechococcus elongatus</i> PCC7942 Synpcc7942_1594 (YP_400611) amino acid (SEQ ID NO:66) <img file="EP2288694B1_D0060.tif" /></li><li><i>Synechocystis</i> sp. PCC6803 sll0209 (NP 442146) nucleotide (SEQ ID NO:67) <img file="EP2288694B1_D0061.tif" /><img file="EP2288694B1_D0062.tif" /></li><li><i>Synechocystis</i> sp. PCC6803 sll0209 (NP_442146) amino acid (SEQ ID NO:68) <img file="EP2288694B1_D0063.tif" /></li><li><i>Cyanothece</i> sp. ATCC51142 cce_1430 (YP_001802846) nucleotide (SEQ ID NO:69) <img file="EP2288694B1_D0064.tif" /></li><li><i>Cyanothece</i> sp. ATCC51142 cce_1430 (YP 001802846) amino acid (SEQ ID NO:70) <img file="EP2288694B1_D0065.tif" /></li><li><i>Prochlorococcus marinus</i> subsp. pastoris str. CCMP1986 PMM0533 (NP_892651) nucleotide (SEQ ID NO:71) <img file="EP2288694B1_D0066.tif" /><img file="EP2288694B1_D0067.tif" /></li><li><i>Prochlorococcus marinus</i> subsp. pastoris str. CCMP1986 PMM0533 (NP_892651) amino acid (SEQ ID NO:72) <img file="EP2288694B1_D0068.tif" /></li><li><i>Gloeobacter violaceus</i> PCC7421 NP_96091 (gll3145) nucleotide (SEQ ID NO:73) <img file="EP2288694B1_D0069.tif" /></li><li><i>Gloeobacter violaceus</i> PCC7421 NP_96091 (gll3145) amino acid (SEQ ID NO:74) <img file="EP2288694B1_D0070.tif" /></li><li><i>Nostoc punctiforme</i> PCC73102 ZP_00108837 (Npun02004176) nucleotide (SEQ ID NO:75) <img file="EP2288694B1_D0071.tif" /><img file="EP2288694B1_D0072.tif" /></li><li><i>Nostoc punctiforme</i> PCC73102 ZP_00108837 (Npun02004176) amino acid (SEQ ID NO:76) <img file="EP2288694B1_D0073.tif" /></li><li><i>Anabaena variabilis</i> ATCC29413 YP_323044 (Ava_2534) nucleotide (SEQ ID NO:77) <img file="EP2288694B1_D0074.tif" /></li><li><i>Anabaena variabilis</i> ATCC29413 YP_323044 (Ava_2534) amino acid (SEQ ID NO:78) <img file="EP2288694B1_D0075.tif" /></li><li><i>Synechococcus elongatus</i> PCC6301 YP_170761 (syc0051_d) nucleotide (SEQ ID NO:79) <img file="EP2288694B1_D0076.tif" /><img file="EP2288694B1_D0077.tif" /></li><li><i>Synechococcus elongatus</i> PCC6301 YP_170761 (syc0051_d) amino acid (SEQ ID NO:80) <img file="EP2288694B1_D0078.tif" /></li><li><i>Nostoc</i> sp. PCC 7120 alr5284 (NP_489324) nucleotide (SEQ ID NO:81) <img file="EP2288694B1_D0079.tif" /></li><li><i>Nostoc</i> sp. PCC 7120 alr5284 (NP_489324) amino acid (SEQ ID NO:82) <img file="EP2288694B1_D0080.tif" /></li><li><i>Synechococcus elongatus</i> PCC7942 Synpcc7942_1594 (YP_400611) nucleotide, synthetic (SEQ ID NO:83) <img file="EP2288694B1_D0081.tif" /></li><li><i>Prochlorococcus marinus</i> subsp. pastoris str. CCMP1986 PMM0533 (NP_892651) nucleotide, synthetic (SEQ ID NO:84) <img file="EP2288694B1_D0082.tif" /></li><li><i>Mycobacterium smegmatis</i> strain MC2 155 orf MSMEG_5739 (YP_889972) nucleotide (SEQ ID NO:85) <img file="EP2288694B1_D0083.tif" /><img file="EP2288694B1_D0084.tif" /></li><li><i>Mycobacterium smegmatis</i> strain MC2 155 orf MSMEG_5739 (YP_889972) amino acid (SEQ ID NO:86) <img file="EP2288694B1_D0085.tif" /><img file="EP2288694B1_D0086.tif" /></li><li><i>Nostoc punctiforme</i> PCC73102 Npun02003626 (ZP_00109192) nucleotide (SEQ ID NO:87) <img file="EP2288694B1_D0087.tif" /></li><li><i>Nostoc punctiforme</i> PCC73102 Npun02003626 (ZP_00109192) amino acid (SEQ ID NO:88) <img file="EP2288694B1_D0088.tif" /></li><li><i>Nostoc punctiforme</i> PCC73102 Npun02001001 (ZP_00111633) nucleotide (SEQ ID NO:89) <img file="EP2288694B1_D0089.tif" /></li><li><i>Nostoc punctiforme</i> PCC73102 Npun02001001 (ZP_00111633) amino acid (SEQ ID NO:90) <img file="EP2288694B1_D0090.tif" /></li><li><i>Nostoc punctiforme</i> PCC73102 Npun02003530 (ZP_00109422) nucleotide (SEQ ID NO:91) <img file="EP2288694B1_D0091.tif" /></li><li><i>Nostoc punctiforme</i> PCC73102 Npun02003530 (ZP_00109422) amino acid (SEQ ID NO:92) <img file="EP2288694B1_D0092.tif" /></li><li><i>Nostoc punctiforme</i> PCC73102 Npun02003123 (ZP_00109501) nucleotide (SEQ ID NO:93) <img file="EP2288694B1_D0093.tif" /></li><li><i>Nostoc punctiforme</i> PCC73102 Npun02003123 (ZP_00109501) amino acid (SEQ ID NO:94) <img file="EP2288694B1_D0094.tif" /></li></ul>