ES2446565T3 - CO2 fixing microorganisms obtained by genetic engineering that produce carbon-based products of interest - Google Patents

Abstract

A genetically modified photosynthetic microbial host cell that produces ethanol, where the host cell is genetically modified to comprise an exogenous genetically engineered nucleic acid encoding an NADPH-dependent alcohol dehydrogenase activity.

Description

CO2-fixing microorganisms obtained by genetic engineering that produce carbon-based products of interest 5

Cross reference to related requests

This application claims the priority benefit of U.S. Provisional Application No. 61 / 033,411 filed on March 3, 2008; U.S. Provisional Application No. 61 / 033,402, filed March 3, 2008; U.S. Provisional Application No. 61 / 044,419 filed on April 11, 2008; U.S. Provisional Application No. 61 / 106,543 filed on October 17, 2008; and U.S. Provisional Application No. 61 / 121,532 filed on December 10, 2008.

Countryside

15 The present disclosure relates to mechanisms to confer production of carbon-based products to a photoautotrophic organism so that it effectively converts carbon dioxide and light into various carbon-based products, and in particular the use of such organisms for commercial production of various carbon based products of interest.

Background

Photosynthesis is a procedure whereby biological entities use sunlight and CO2 to produce sugars for energy. Photosynthesis, as it has evolved naturally, is an extremely system

25 complex with numerous and poorly understood feedback loops, control mechanisms and inefficiencies of the process. This complicated system presents probably insurmountable obstacles to one-time or global optimization approaches [Nedbal et al., Photosynth Res., 93 (1-3): 223-34 (2007); Salvucci et al., Physiol Plant. 120 (2): 179-186 (2004); Greene et al., Biochem J., 404 (3): 517-24 (2007)].

Existing photoautotrophic organisms (ie plants, algae and photosynthetic bacteria) are poorly suited for industrial bioprocessing and therefore have not demonstrated commercial viability for this purpose. Such organisms have slow doubling time (3-72 hours) compared to industrialized heterotrophic organisms such as Escherichia coli (20 minutes), which reflects low total productivity. In addition, techniques for genetic manipulation (cancellation, overexpression of transgenes by integration or propagation

35 episomic plasmid) are ineffective, time consuming, laborious or non-existent.

Summary

Pathways and mechanisms to confer production capacity of direct carbon-based products to photoautotrophic organisms are described herein. The resulting photoautotrophs of resulting genetically engineered carbon-based products allow exceptionally efficient production of carbon-based products directly from carbon dioxide and light, eliminating the long and expensive processing steps currently required to generate biofuels and products biochemicals from biomass sources including corn, sugarcane, miscanthus, cellulose and others. The invention

45 provides a genetically modified photosynthetic microbial host cell that produces ethanol, where the host cell is genetically modified to comprise an exogenous genetically engineered nucleic acid encoding an NADPH-dependent alcohol dehydrogenase activity. The invention also provides use of the cell of the invention to produce ethanol and a method of producing ethanol, which comprises culturing the cell of the invention and then isolating the ethanol from said cell or culture medium.

In some aspects, ethanol production is optimized by channeling carbon away from glycogen and into pyruvate, etc. during its exposure to light. Normally glycogen is formed in the light and consumed to reduce energy in the dark. In one embodiment, glycogen synthesis genes are attenuated or nullified and in other embodiments, the glycolytic genes become constitutive. In other aspects, certain routes of

Fermentation, such as those leading to acetate, lactate, succinate, etc., if present.

In other aspects, if a light-dark cycle must be implemented, glycogen production is optimized during exposure to light (as opposed to biomass, etc.) and the dry weight% of cells that can be increased is increased. glycogen (that is, the cell is engineered to counteract any limitation that prevents cells from swelling full of glycogen). Then, during the dark, ethanol synthesis from accumulated glycogen is allowed to be performed, the other fermentation routes having been attenuated or canceled. In addition, it is disclosed to use a light-dark cycle that coincides with glycogen synthesis / catabolism rates so that the minimum time is wasted (glycogen is not depleted and cells are maintained nonproductively in darkness, or there is too much glycogen to consume completely during the period of

65 darkness).

The genetically engineered nucleic acid comprised by the cell of the invention encodes an NADPH-dependent alcohol dehydrogenase activity. In one embodiment, the NADPH-dependent alcohol dehydrogenase activity is adhA of Moorella sp. HUC22-1. The coding sequence of NADPH-dependent alcohol dehydrogenase may be at least 77.1% identical to SEQ ID NO. 1. Alcohol-dependent dehydrogenase

5 of NADPH may be at least 72% identical to SEQ ID NO. 2.

In another embodiment, the cell of the invention also encodes a pyruvate decarboxylase activity. In a related embodiment, the pyruvate decarboxylase activity is selected from the pdc activity of Z. palmae and Z. mobilis The NADPH-dependent alcohol dehydrogenase coding sequence and / or the decarboxylene pyruvate coding sequence may be under the control of an inducible promoter.

In certain embodiments, the cell of the invention, in culture, is capable of producing ethanol in a yield of at least about 249 mg / l of culture medium in 72 hours. In certain other embodiments, the yield is at least about 296 mg / l of ethanol for 72 hours. In yet other embodiments, the performance of

15 ethanol is 2.5 to 5 g / l of culture medium per hour. In other embodiments, the level of acetaldehyde in said culture after 72 hours is less than about 14 mg / l. In other embodiments, the cultured cell produces at least about 36 mg / l of ethanol per OD, or at least about 47 mg / l of ethanol per OD.

In further embodiments, the genetically modified cell provided by the invention comprises a cell selected from algae, cyanobacteria, green sulfur bacteria, green non-sulfur bacteria, purple sulfur bacteria, purple non-sulfur bacteria, extremophiles, yeasts, fungi, organisms modified by genetic engineering of them and synthetic organisms. The cell is photosynthetic. The cell of the invention may be an algae and / or cyanobacterial organism selected from the group consisting of Acanthoceras, Acanthococcus, Acaryochloris, Achnanthes, Achnanthidium, Actinastrum, Actinochloris, Actinocyclus, Actinotaenium, Amphichrysis, 25 Amphidinium, Amphikrikos, Amphiple, Amphipra, Amphipra, Amphipra, Amphipra, Amphipra Amphithrix, Amphora, Anabaena, Anabaenopsis, Aneumastus, Ankistrodesmus, Ankyra, Anomoeoneis, Apatococcus, Aphanizomenon, Aphanocapsa, Aphanochaete, Aphanothece, Apiocystis. Bangia, Basichlamys, Batrachospermum, Binuclearia, Bitrichia, Blidingia, Botrdiopsis, Botrydium, Botryococcus, Botryosphaerella, Brachiomonas, Brachysira, Brachytrichia, Brebissonia, Bulbochaete, Bumilleria, Bumilleriopsis, Caloneis, Calipheria, Calothrix, Causcapus, Calipheria, Causcallumus, Calipheria, Causcallumus, Calipheria, Causcallumus, Calipheria, Causcapus, Calipheria, Causcapus, Calipheria, Causcallumus, Calipheria, Causcallus, Caliphrae, Capochnoca, Capochnoca Centronella, Ceratium, Chaetoceros, Chaetochloris, Chaeto morpha, Chaetonella, Chaetonema, Chaetopeltis, Chaetophora, Chaetosphaeridium, Chamaesiphon, chara, Characiochloris, Characiopsis, Characium, Charales, Chilomonas, Chlainomonas, Chlamydoblepharis, Chlamydocapsa, Chlamydomonas, Chlamydomonopsis, Chlamydomyxa, Chlamydonephris, Chlorangiella, Chlorangiopsis, Chlorella, 35 Chlorobotrys, Chlorobrachis , Chlorochytrium, Chlorococcum, Chlorogloea, Chlorogloeopsis, Chlorogonium, Chlorolobion, Chloromonas, Chlorophysema, Chlorophyta, Chlorosaccus, Chlorosarcina, Choricystis, Chromophyton, Chromulina, Chroococcidiopsis, Chroococcus, Chroodactylon, Chroomonas, Chroothece, Chrysamoeba, Chrysapsis, Chrysidiastrum, Chrysocapsa, Chrysocapsella, Chrysochaete , Chrysochromulina, Chrysococcus, Chrysocrinus, Chrysolepidomonas, Chrysolykos, Chrysonebula, Chrysophyta, Chrysopyxis, Chrysosaccus, Chrysophaerella, Chrysostephanosphaera, Clodophora, Clastidium, Closteriopsis, Closterioe Cochloe, Cocrometer, Cocrometeria Cochloe, Cocrometer, Cocrometerus s, Coenococcus, Coenocystis, Colacium, Coleochaete, Collodictyon, Compsogonopsis, Compsopogon, Conjugatophyta, Conochaete, Coronastrum, Cosmarium, Cosmioneis, Cosmocladium, Crateriportula, Craticula, Crinalium, Crucigenia, Crucigenononon, Cryicodonela, Crypto-cyanone, Crypodotopus Cyanophora, Cianophyta, Cyanothece, Cyanothomonas, 45 Cyclonexis, Cyclostephanos, Cyclotella, Cylindrocapsa, Cylindrocystis, Cylindrospermum, Cylindrotheca, Cymatopleura, Cymbella, Cymbellonitzschia, Cystodinium, Dactylococcopsis, Debarya, Denticula, Dermatochrysis, Dentiocarpa, Dermocarpella, Desmatractum, Desmidium, Desmococcus, Desmonema , Desmosiphon, Diacanthos, Diacronema, Diadesmis, Diatoma, Diatomella, Dicellula, Dichothrix, Dichotomococcus, Dicranochaete, Dictyochloris, Dictyococcus, Dictyosphaerium, Didymocystis, Didymogenes, Didymosphenia, Dilabifilum, Dimorphococcus, Dinobryon, Dinococcus, Diplochloris, Diploneis, Diplostauron, Distrionella, Docidium Draparnaldia , Dunaliella, Dysmorphococcus, Ecballocystis, Elakatothrix, Ellerbeckia, Encyonema, Enteromorpha, Entocladia, Entomoneis, Entophysalis, Epichrysis, Epipyxis, Epithemia, Eremosphaera, Euastropsis, Euastrum, Eremospharaptia, Ecaptus, Echinaptus, Ecaphastothaeugia, Ecaphastusia, Echiaptus, Echinaptusia , Fischerella, Fragilaria, Fragilariforma, Franceia, Frustulia, Curcilla, Geminella, Genicularia, Glaucocystis, Glaucophyta, Glenodiniopsis, 55 Glenodinium, Gloeocapsa, Gloeochaete, Gloeochrysis, Gloeococcus, Gloeocystis, Gloeodendron, Gloeomonas, Gloeoplax, Gloeothece, Gloeotila, andGloeotrichia, Gloiodictyon, Golenkinia, Golenkiniopsis, Gomontia, Gomphocymbella, Gomphonema, Gomphosphaeria, Gonatozygon, Gongrosia, Gongrosira, Goniochloris, Gonium, Gonyostomum, Granulochloris, Granulocystopsis, Groenbladia, Gymnodinium, Gymnozyga, Gyrosigma, Haematococcus, Hafniomonas, Hallassia, Hammatoidea, Hannaea, Hantzschia, Hapalosiphon, Haplotaenium, Haptophyta, Haslea, Hemidinium, Hemitoma, Herib audiella, Heteromastix, Heterothrix, Hibberdia, Hildenbrandia, Hillea, Holopedium, Homoeothrix, Hormanthonema, Hormotila, Hyalobrachion, Hyalocardium, Hyalodiscus, Hyalogonium, Hyalotheca, Hydrianum, Hydrococcus, Hydroconemoralronia, Hydroconemoralronia, Hydroconemoralronia, Hydrocoemoralumonia, Hydroconemoralumonia, Hydrocoemonium, Hydrocoemonium, Hydrocoemonium, Hydrocoemonium, Hydrocoemonium, Hydrocoemonium, Hydrocoemonium, Hydrocoemonium, Hydrocoemonium Johannesbaptistia, Juranyiella, Karayevia, Kathablepharis, Katodinium, Kephyrion, Keratococcus, Kirchneriella, Klebsormidium, Kolbesia, Koliella, Komarekia, Korshikoviella, 65 Kraskella, Lagerheimia, Lagynion, Lamprothamnium, Lemaneabom, Lepoismacous, Lymphocytole, Lymphocytic, Lymphaemia , Malleochloris, Mallomonas, Mantoniella, Marssoniella, Martyana, Mastigocoleus, Gastogloia, Melosira, Merismopedia, Mesostigma, Mesotaenium, Micractinium, Micrasterias, Microchaete, Microcoleus, Microcystis, Microglena, Micromonas, Microspora, Microthamcodium, Monopod, Monopod, Monopod , Monostroma, Mougeotia, Mougeotiopsis, Myochloris, Myrome cia, Myxosarcina, Naegeliella, Nannochloris, Nautococcus, Navicula, Neglectella, Neidium, Nephroclamys, Nephrocytium, 5 Nephrodiella, Nephroselmis, Netrium, Nitella, Nitellopsis, Nitzschia, Nodularia, Nostoc, Ochromonas, Oedogonium, Oligochaetophora, Onychonema, Oocardium, Oocystis, Opephora , Ophiocytium, Orthoseira, Oscillatoria, Oxyneis, Pachycladella, Palmella, Palmodictyon, Pnadorina, Pannus, Paralia, Pascherina, Paulschulzia, Pediastrum, Pedinella, Pedinomonas, Pedinopera, Pelagodictyon, Penium, Peranema, Peridiniumps, Phaeidiscus, Peridiniumps, Phaeidiscus , Phaeaster, Phaeodermatium, Phaeophyta, Phaeosphaera, Phaeothamnion, Phormidium, Phycopeltis, Phyllariochloris, Phyllocardium, Phyllomitas, Pinnularia, Pitophora, Placoneis, Planctonema, Planktosphaeria, Planothidium, Plectonema, Pleodorina, Pleurastrum, Pleurocapsa, Pleurocladia, Pleurodiscus, Pleurosigma, Pleurosira, Pleurotaenium , Pocillononas, Podohedra, Polyblepharides, Polychaetophora, Polyedriella, Polyedriops is, Polygoniochloris, Polyepidomonas, Polytaenia, Polytoma, Polytomella, Porphyridium, Posteriochromonas, Prasinochloris, Prasinocladus, Prasinophyta, Prasiola, Prochlorphyta, Prochlorothrix, 15 Protoderma, Protosiphon, Provasoliella, Pammdochanochadiadane, Piummidium, Syndrome, Prymidium, Syndrome , Pseudococcomyxa, Pseudodictyosphaerium, Pseudokephyrion, Pseudoncobyrsa, Pseudoquadrigula, Pseudosphaerocystis, Pseudostaurastrum, Pseudostaurosira, Pseudotetrastrum, Pteromonas, Punctastruata, Pyramichlamys, Pyramimonas, Pyrrophyta, Quadrichloris, Quadricoccus, Quadrigula, Radiococcus, Radiofilum, Raphidiopsis, Raphidocelis, Raphidonema, Raphidophyta, Peimeria, Rhabdoderma , Rhabdomonas, Rhizoclonium, Rhodomonas, Rhodophyta, Rhoicosphenia, Rhopalodia, Rivularia, Rosenvingiella, Rossithidium, Roya, Scenedesmus, Scherffelia, Schizochlamydella, Schizochlamys, Schizomeris, Schizothrix, Schroederia, Scolioneis, Scotchlois, Scotchlois, Scotchlois fìeldia, Scytonema, Selenastrum, Selenochloris, Sellaphora, Semiorbis, Siderocelis, Diderocystopsis, Dimonsenia, Siphononema, Sirocladium, Sirogonium, Skeletonerna, Sorastrum, Spermatozopsis, Sphaerellocystis, Sphaerellopsis, 25 Sphaerodinium, Sphaeroplea, Sphaerozosma, Spiniferomonas, Spirogyra, Spirotaenia, Spirulina, Spondylomorum , Spondylosium, Sporotetras, Spumella, Staurastrum, Stauerodesmus, Stauroneis, Staurosira, Staurosirella, Stenopterobia, Stephanocostis, Stephanodiscus, Stephanoporos, Stephanosphaera, Stichococcus, Stichogloea, Stigeoclonium, Stigonema, Stipitolololaphylolaphylolaphylaphylolaphylaemia, Stipitololaphylolaphylolaphylaemia, Styrofocalous, Styrophylolaphylolaphylaemia, Styrophylolaphylolaemia , Sykidion, Symploca, Synechococcus, Synechocystis, Synedra, Synochromonas, Synura, Tabellaria, Tabularia, Teilingia, Temnogametum, Tetmemorus, Tetrachlorella, Tetracyclus, Tetradesmus. Thorea, Tolypella, Tolypothrix, Trachelomonas, Trachydiscus, Trebouxia, Trentepholia, Treubaria, Tribonema, Trichodesmium, Trichodiscus, Trochiscia, Tryblionella, Ulothrix, Uroglena, Uronema, Urosolenia, Urospora, Uva, Vacuolaria, Vacuolaria, Vacuolaria, Vacuolaria Xanthidium, Xanthophyta, Xenococcus, Zygnema, Zygnemopsis and Zygonium. The cell provided by the invention may be derived from a cell of Chloroflexus, Chloronema, Oscillochloris, Heliothrix, Herpetosiphon, Roseiflexus, and Thermomicrobium; a green sulfur bacteria selected from: Chlorobium, Clathrochloris, and Prosthecochloris; a purple sulfur bacteria selected from: Allochromatium, Chromatium, Halochromatium, Isochromatium, Marichromatium, Rhodovulum, Thermochromatium, Thiocapsa, Thiorhodococcus, and Thiocystis; a purple non-sulfur bacteria selected from: Phaeospirillum, Rhodobaca, Rhodobacter, Rhodomicrobium, Rhodopila, Rhodopseudomonas, Rhodothalassium, Rhodospirillum, Rodovibrio and Roseospira; an aerobic chemolithotrophic bacterium selected from: nitrifying bacteria, Nitrobacteraceae sp., Nitrobacter sp., Nitrospina sp., Nitrococcus sp., Nitrospira sp., Nitrosomonas sp., Nitrosococcus sp., Nitrosaspira sp., Nitrosolobus sp., Nitrosovibrio sp .; colorless sulfur bacteria such as Thiovulum sp., Thiobacillus sp., Thiomicrospira sp., Thiosphaera sp., 45 Thermothrix sp; Compulsory chemilitotrophic hydrogen bacteria Hydrogenobacter sp., oxidizing bacteria and / or iron and manganese depositors, Siderococcus. sp, and magnetotactic bacteria, Aquaspirillum sp; an archeobacterium selected from: methanobacterium sp., Methanobacterium sp., Methanobrevibacter sp., Methanothermus sp., Methanococcus sp., Methanomicrobium sp., Methanospirillum sp., Methanogenium sp., Methanosarcina sp., Methanothbus spco., Methanothbus spco. ., Methanoplanus sp., Extremely thermophilic sulfur metabolizers such as Thermoproteus sp., Pyrodictium sp., Sulfolobus sp., Acidianus sp., Bacillus subtilis, Saccharomyces cerevisiae, Streptomyces sp., Ralstonia sp., Rhodococcus sp., Corynebacter , Brevibacteria sp., Mycobacteria sp., And oil yeast; and extremophiles selected from Pyrolobus fumarii; Synechococcus lividis, mesophiles, psychrophiles, Psychrobacter, insects, Deinococcus radiodurans, piezophiles, barophiles, hypergravity tolerant organisms, hypogravity tolerant organisms, organisms

55 vacuum tolerant, tardigrades, insects, microbe seeds, desiccation tolerant anhydrobial organisms; xerophilous, Artemia saline, nematodes, microbes, fungi, lichens, salinity-tolerant organisms, halophiles, halobacteriacea, Dunaliella saline, pH-tolerant organisms, alkalophils, Natronobacterium, Bacillus firmus OF4, Spirulina spp., acidophilus spider, Cryphoidium ., anaerobes, which cannot tolerate O2, Methanococcus jannaschii, microaerophils, which tolerate some O2, Clostridium, aerobes, which require O2, gas tolerant organisms, which tolerate pure CO2, Cyanidium caldarium, metal tolerant organisms, metallotorants, Ferroplasma acidarmanus Ralstonia sp. CH34.

In yet other embodiments, the genetically modified cell provided by the invention is derived from Botryococcus braunii, Chlamydomonas reinhardtii and Dunaliela salina (algae), Synechococcus sp. PCC 7002, 65 Synechococcus sp. PCC 7942, Synechocystis sp. PCC 6803 and Thermosynechococcus elongatus BP-1 (cyanobacteria), Chlorobium tepidum (green sulfur bacteria), Chloroflexus auranticus (non green bacteria

sulfur), Chromatium tepidum and Chromatium vinoum (purple sulfur bacteria), Rhodospirillum rubrum, Rhodobacter capsulatus and Rhodopseudomonas palusris (non-sulfur purple bacteria).

In yet other embodiments, the genetically modified cell provided by the invention is a cell of Clostridium ljungdahlii, Clostridium thermocellum, Penicillium chrysogenum, Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pseudomonas fluorescens or Zymomonas mobilis.

In the method of the invention, the cell can be cultured in a photobioreactor.

Brief description of the figures

Figure 1 (AO) provides several identified genes that can be expressed, regulated positively, attenuated or canceled in the genetic engineering of carbon dioxide fixing microorganisms in the production of carbon-based products of interest.

Figure 2 provides an example of routes to produce ethanol, succinate and other derivatives. Figure 3 shows the GC / FID chromatogram of the control Synechococcus 7002 strain. Figure 4 shows the GC / FID chromatogram of the recombinant strain 7002 / efe_rs. Figure 5 graphically illustrates the optical density of ethanol selected over time. Figure 6 graphically illustrates the ethanol concentrations of cultures in the supernatant over time. Figure 7 graphically illustrates the acetaldehyde concentrations of cultures in the supernatant over time. Figure 8 graphically illustrates the ethanol and acetaldehyde ratios of cultures in the supernatant over time.

Figure 9 graphically illustrates the concentration ratios of ethanol and OD (730 nm) of cultures in the supernatant over time.

Detailed description

Abbreviations and Terms

The following explanations of terms and methods are provided to better describe the present disclosure and to guide those skilled in the art in the practice of the present disclosure. As used herein, "comprising" means "including" and the singular forms "a" or "the" include plural references to

35 unless the context clearly dictates otherwise. For example, the reference to "comprising a cell" includes one or a plurality of said cells, and the reference to "comprising thioesterase" includes reference to one or more thioesterase peptides and equivalents thereof known to those of ordinary skill. in matter, and so on. The term "or" refers to a single element of indicated alternative elements or a combination of two or more elements, unless the context clearly indicates otherwise.

Unless explained otherwise, all technical and scientific terms used in this document have the same meaning as usually understood by a person skilled in the art to which the disclosure belongs. Although methods and materials similar or equivalent to those described herein may be used in the practice or assay of the present disclosure, methods and methods are described below.

45 suitable materials. The materials, methods and examples are illustrative only and are not intended to be limiting. Other features of the disclosure will be apparent from the following detailed description and claims.

Reference numbers: Reference numbers throughout this description are derived from the NCBI (National Center for Biotechnology Information) database maintained by the National Institute of Health, United States. Reference numbers are as provided in the database of February 1, 2008.

Enzyme Classification Numbers (EC): The EC numbers provided throughout this

55 description derives from the KEGG ligand database, maintained by the Kyoto Encyclopedia of Genes and Genomics, sponsored in part by the University of Tokyo. EC numbers are as provided in the database of February 1, 2008.

Amino acid: Nucleotide triplets, called codons, in DNA molecules encode an amino acid in a peptide. The term codon is also used for the corresponding (and complementary) sequences of three nucleotides in the mRNA in which the DNA sequence is transcribed.

Attenuate: The term as used herein generally refers to a functional deletion, including a mutation, partial or complete deletion, insertion or other variation made to a gene sequence or

A sequence that controls the transcription of a gene sequence, which reduces or inhibits the production of the gene product, or makes the gene product non-functional. In some cases a functional deletion is described as a cancellation mutation. Attenuation also includes amino acid sequence change by altering the nucleic acid sequence, placing the gene under the control of a less active promoter, by negative regulation, expressing interference RNA, ribozymes or antisense sequences that target the gene of interest, or by any other technique known in this field. In one example, the sensitivity of a particular enzyme to

5 feedback feedback inhibition or inhibition caused by a composition that is not a product or a reagent (non-route specific feedback) is reduced so that enzymatic activity is not affected by the presence of a compound. In other cases, an enzyme that has been altered to be less active may be termed attenuated.

"Carbon based products of interest" includes alcohols such as ethanol, propanol, isopropanol, butanol, fatty alcohols, fatty acid esters, wax esters; hydrocarbons and alkanes such as propane, octane, diesel, jet propellant 8, polymers such as terephthalate, 1,3-propanediol, 1,4-butanediol, polyols, polyhydroxyalkanoates (PHA), polyhydroxybutyrates (PHB), acrylate, adipic acid, ! -caprolactone, isoprene, caprolactam, gum; Consumer chemicals such as lactate, docosahexaenoic acid (DHA), 3

15 hydroxypropionate, ∀-valerolactone, lysine, serine, aspartate, aspartic acid, sorbitol, ascorbate, ascorbic acid, isopentenol, lanosterol, DHA omega-3, lycopene, itaconate, 1,3-butadiene, ethylene, propylene, succinate, citrate, citric acid, glutamate, malate, 3-hydroxyprionic acid (HPA), lactic acid, THF, gamma butyrolactone, pyrrolidones, hydroxybutyrate, glutamic acid, levulinic acid, acrylic acid, malonic acid; specialty chemicals such as carotenoids, isoprenoids, itaconic acid; Pharmaceuticals and pharmaceutical intermediates such as 7-aminodeacetoxycephalosporonic acid, cephalosporin, erythromycin, polyketides, statins, paclitaxel, docetaxel, terpenes, peptides, steroids, omega fatty acids and other suitable products of interest of this type. These products are useful in the context of biofuels, industrial and specialty chemicals, as intermediates used to make additional products, such as nutritional supplements, nutraceuticals, polymers, paraffin replacements, personal care products and pharmaceuticals.

Deletion: The removal of one or more nucleotides from a nucleic acid molecule or one or more amino acids from a protein, joining the regions on both sides.

DNA: Deoxyribonucleic acid. DNA is a long chain polymer that includes the genetic material of most living organisms (some viruses have genes that include ribonucleic acid, RNA). The units repeated in DNA polymers are four different nucleotides, each of which includes one of the four bases, adenine, guanine, cytosine and thymine linked with a deoxyribose sugar to which a phosphate group binds.

Endogenous: As used herein with reference to a nucleic acid molecule and a cell or

Particular microorganism refers to a nucleic acid or peptide sequence that is in the cell and has not been introduced into the cell (or its progenitors) using recombinant engineering techniques. For example, a gene that was present in the cell when the cell was originally isolated from nature. A gene is still considered endogenous if control sequences, such as a promoter or enhancer sequence that activates transcription or translation, have been altered by recombinant techniques.

"An enzymatic activity": As used herein, the term "an enzymatic activity" means that the indicated enzyme (for example, "an alcohol dehydrogenase activity") has measurable attributes with respect to, for example, specific activity of optimal substrate, pH and temperature, and other conventional measures of enzymatic activity such as activity encoded by a reference enzyme (e.g. alcohol

45 dehydrogenase). In addition, the enzyme is at least 90% identical to the nucleic or amino acid level to the reference enzyme sequence as measured by a BLAST search.

Exogenous: As used herein with respect to a nucleic acid molecule and a particular cell or microorganism refers to a nucleic acid or peptide sequence that was not present in the cell when the cell was originally isolated from nature. For example, a nucleic acid that originated from a different microorganism and was engineered into an alternative cell using recombinant DNA techniques or other methods to deliver said nucleic acid is exogenous.

Expression: The process by which the information encoded by a gene becomes the structures and functions of

A cell, such as a protein, transfer RNA or ribosomal RNA. The genes expressed include those that are transcribed into mRNA and then translated into protein and those that are transcribed into RNA but do not translate into protein (eg, transfer and ribosomal RNA).

Expression control sequence: As used herein refers to polynucleotide sequences that are necessary to affect the expression of coding sequences with which they are operatively linked. Expression control sequences are sequences that control transcription, post-transcriptional events and the translation of nucleic acid sequences. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; effective RNA processing signals such as splicing and polyadenylation signals; 65 sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (for example, ribosome binding sites); sequences that enhance protein stability; and when desired, sequences that

enhance protein secretion. The nature of such control sequences differs depending on the host organism; in prokaryotes, such control sequences generally include promoter, ribosome binding site and transcription termination sequence. The term "control sequences" is intended to include, at a minimum, all components whose presence is essential for expression, and may also

5 include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences.

Overexpression: When a gene is transcribed at a high rate compared to the endogenous transcription rate for that gene. In some examples, overexpression additionally includes a high rate of gene translation compared to the endogenous translation rate for that gene. Methods for assaying for overexpression are well known in the art, for example levels of transcribed RNA can be evaluated using reverse transcriptase polymerase chain reaction (RT-PCR) and protein levels can be evaluated using electrophoresis analysis in sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE). In addition, it is considered that a gene that is overexpressed when it shows high activity compared to its endogenous activity, which can appear, for example, by reducing the concentration

or activity of its inhibitor, or by expression of the mutant version with high activity. In preferred embodiments, when the host cell encodes an endogenous gene with a desired biochemical activity, it is useful to overexpress an exogenous gene, which allows more explicit regulatory control in fermentation and a means to potentially mitigate the effects of central metabolism regulation, which focuses explicitly around native genes.

Negative regulation: When a gene is caused to be transcribed at a reduced rate compared to the endogenous gene transcription rate for that gene. In some examples, the negative regulation additionally includes a reduced level of gene translation compared to the endogenous translation rate for that gene. Be

25 are well known to those skilled in the art for testing methods regarding negative regulation. For example, transcribed RNA levels can be evaluated using RT-PCR, and protein levels can be evaluated using SDS-PAGE analysis.

Cancellation: A gene whose level of expression or activity has been reduced to zero. In some examples, a gene is deleted by deletion of part or all of its coding sequence. In other examples, a gene is deleted by introducing one or more nucleotides in its open reading phase, which results in translation of a nonsense or otherwise non-functional protein product.

Autotroph: Autotrophs (or autotrophic organisms) are organisms that produce organic compounds

35 complexes from simple inorganic molecules and an external energy source, such as light (photoautotrophs) or chemical reactions of inorganic compounds.

Hydrocarbon: The term generally refers to a chemical compound consisting of the elements carbon (C), hydrogen (H) and optionally oxygen (O). There are essentially three types of hydrocarbons, for example, aromatic hydrocarbons, saturated hydrocarbons and unsaturated hydrocarbons such as alkenes, alkynes and dienes. The term also includes fuels, biofuels, plastics, waxes, solvents and oils. Hydrocarbons include biofuels, as well as plastics, waxes, solvents and oils.

"Immiscible" or "immiscibility" refers to the relative inability of a compound to dissolve in water and is

45 defined by the partition coefficient of the compounds. The partition coefficient, P, is defined as the equilibrium concentration of compound in an organic phase (in a biphasic system the organic phase is usually the phase formed by the fatty acid derivative during the production process, however, in some Examples may be provided an organic phase (such as an octane layer to facilitate the separation of products) divided by equilibrium concentration in an aqueous phase (i.e. fermentation broth) When a two phase system is described the P It is usually analyzed with respect to logP A compound with logP of 10 would divide 10: 1 with the organic phase, while a logP compound of 0.1 would divide 10: 1 with the aqueous phase.

Biosynthetic pathway: Also called "metabolic pathway", refers to a set of biochemical reactions

55 anabolic or catabolic to convert (transmute) one chemical species into another. For example, a biosynthetic hydrocarbon route refers to the set of biochemical reactions that convert inputs and / or metabolites into intermediates of the hydrocarbon product type and then into hydrocarbons or hydrocarbon products. Anabolic pathways involve building a larger molecule from smaller molecules, a process that requires energy. Catabolic pathways involve degrading larger molecules, often releasing energy.

Cellulose: Cellulose [(C6H10O5) n] is a long-chain, beta-glucose polymeric polysaccharide carbohydrate. It forms the primary structural component of plants and is not digestible by humans. Cellulose is a common material in plant cell walls and was first observed as such in 1838. It appears naturally almost purely only in cotton fiber; in combination with lignin and any hemicellulose, it

65 found in all plant material.

Biofuel: A biofuel is any fuel that derives from a biological source. The biofuel refers to one or more hydrocarbons, one or more alcohols, one or more fatty esters or a mixture thereof. Preferably, liquid hydrocarbons are used.

"Fuel component" is any compound or a mixture of compounds that are used to formulate a fuel composition. There are "major fuel components" and "minor fuel components." A major fuel component is present in a fuel composition at least 50% by volume; and a minor fuel component is present in a fuel composition in less than 50%. Fuel additives are minor fuel components. The isoprenoid compounds disclosed herein may be a major component or a minor component, alone or in a mixture with other fuel components.

As used herein, a composition that is a "substantially pure" compound is substantially without one or more additional compounds, that is, the composition contains more than 80% in

15 volume, more than 90% by volume, more than 95% by volume, more than 96% by volume, more than 97% by volume, more than 98% by volume, more than 99% by volume, more than 99.5 % by volume, more than 99.6% by volume, more than 99.7% by volume, more than 99.8% by volume or more than 99.9% by volume of the compound; or of 20% by volume, less than 10% by volume, less than 5% by volume, less than 3% by volume, less than 1% by volume, less than 0.5% by volume, less than 0.1 % by volume or less than 0.01% by volume of the additional compound (s), based on the total volume of the composition.

Nucleic acid molecule: The expression encompasses both RNA and DNA molecules including, without limitation, cDNA, genomic DNA and mRNA and also includes synthetic nucleic acid molecules, such as those that are chemically synthesized or recombinantly produced. The nucleic acid molecule can be

25 single-chain or double-stranded, circular or linear. If it is single stranded, the nucleic acid molecule may be the sense chain or the antisense chain.

Genetically engineered nucleic acid: A "genetically engineered nucleic acid" is a nucleic acid molecule that includes at least one difference from a naturally occurring nucleic acid molecule. A genetically engineered nucleic acid includes all exogenous unmodified and modified heterologous sequences (i.e., sequences derived from an organism or cell other than the one that houses the genetically engineered nucleic acid) as well as endogenous genes, operons, coding sequences or non-coding sequences, which have been modified, mutated or that include deletions or insertions compared to a sequence of natural origin. Genetically engineered nucleic acids also include all sequences, regardless of origin, that are linked to an inducible promoter.

or with another control sequence with which they are not naturally associated.

Suitable fermentation conditions. The term generally refers to fermentation media and conditions adjustable with pH, temperature, aeration levels, etc., preferably optimal conditions that allow microorganisms to produce carbon-based products of interest. To determine if the culture conditions allow the production of the product, the microorganism can be grown for approximately 24 hours up to one week after inoculation and a sample can be obtained and analyzed. The cells in the sample or the medium in which the cells are grown are tested for the presence of the desired product.

Isolated: An "isolated" nucleic acid or polynucleotide (for example, an RNA, DNA or a mixed polymer) is one that is substantially separated from other cellular components that naturally accompany the native polynucleotide in its natural host cell, for example , ribosomes, polymerases and genomic sequences with which it is naturally associated. The term encompasses a nucleic acid or polynucleotide that (1) has been removed from its naturally occurring environment, (2) is not associated with all or a part of a polynucleotide in which the "isolated polynucleotide" is found in nature, (3) is operatively linked with a polynucleotide with which it is not bound in nature, or (4) does not appear in nature. The term "isolated" or "substantially pure" can also be used in reference to recombinant or cloned DNA isolates, chemically synthesized polynucleotide analogs or polynucleotide analogs that are biologically synthesized by heterologous systems.

However, "isolated" does not necessarily require that the nucleic acid or polynucleotide described in this way be physically removed from its native environment. For example, an endogenous nucleic acid sequence in the genome of an organism is considered "isolated" herein if a heterologous sequence is placed (that is, a sequence that is not naturally adjacent to this endogenous nucleic acid sequence ) adjacent to the endogenous nucleic acid sequence, so that the expression of this endogenous nucleic acid sequence is altered. As an example, a non-native promoter sequence can replace (for example, homologous recombination) the native promoter of a gene in the genome of a human cell, so that this gene has an altered expression pattern. This gene would now be "isolated" because it is separated from at least some of the sequences that flank it naturally. A nucleic acid is also considered "isolated" if it contains any modification that does not appear naturally in the corresponding nucleic acid in a genome. By

For example, an endogenous coding sequence is considered "isolated" if it contains an insertion, deletion or a point mutation introduced artificially, for example, by human intervention. An "isolated nucleic acid" also includes a nucleic acid integrated into a host cell chromosome at a heterologous site, as well as a nucleic acid construct present as an episome. In addition, an "isolated nucleic acid" may be substantially without other cellular material, or substantially without culture medium when produced by recombinant techniques, or substantially without chemical precursors or other chemicals.

5 when chemically synthesized. The expression also encompasses nucleic acid molecules and proteins prepared by recombinant expression in a host cell as well as nucleic acid molecules and chemically synthesized proteins.

Operationally linked: A first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, the operably linked DNA sequences are contiguous and, when necessary to join two protein coding regions, in the same reading phase. The separate gene configurations that are transcribed in tandem

15 as a single messenger RNA are indicated as operons. Therefore, placing genes in close proximity, for example in a plasmid vector, under the transcriptional regulation of a single promoter, constitutes a synthetic operon.

Purified: The term purified does not require absolute purity: instead, it is understood as a relative term. Therefore, for example, a purified product preparation is one in which the product is more concentrated than the product is in its environment within a cell. For example, a purified wax is one that is substantially separated from cellular components (nucleic acids, lipids, carbohydrates and other peptides) that may accompany it. In another example, a purified wax preparation is one in which the wax is substantially free of contaminants, such as those that may be present after fermentation.

Detectable: Able to determine its existence or presence using various analytical methods as described throughout the description or otherwise known by a person skilled in the art.

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

35 encoders found in the organism from which the nucleic acid was isolated.

The term "recombinant host cell" ("expression host cell", "expression host system", "expression system", or simply "host cell"), as used herein, refers to a cell in which has introduced a recombinant vector, for example, a vector comprising acyl-CoA synthase. It should be understood that these terms are intended to refer not only to the particular object cell but also to the offspring of said cell. Because certain modifications may appear in successive generations due to mutation or environmental influences, such offspring may, in fact, not be identical to the parental cell, but still be included within the scope of the term "host cell" as used in This document. A recombinant host cell can be an isolated cell or line

A cell grown in culture or it can be a cell that resides in a living tissue or organism.

Release: The movement of a compound from inside a cell (intracellular) to the outside of a cell (extracellular). The movement can be active or passive. When the release is active it can be facilitated by one or more transporter peptides and in some examples it can consume energy. When the release is passive, it can be by diffusion through the membrane and can be facilitated by the continuous collection of the desired compound from the extracellular environment, thereby promoting further diffusion. The release of a compound can also be achieved by lysing a cell.

Surfactants: Substances capable of reducing the surface tension of a liquid in which they dissolve.

Typically they are composed of a water soluble head and a hydrocarbon chain or tail. The water soluble group is hydrophilic and can be ionic or non-ionic, and the hydrocarbon chain is hydrophobic. Surfactants are used in a variety of products, including detergents and cleaning products, and are also used as adjuvants for textiles, leather and paper, in chemical processes, in cosmetic and pharmaceutical products, in the food industry and in agriculture. In addition, they can be used to aid in the extraction and isolation of crude oils found in difficult-to-reach environments or as emulsions in water.

There are four types of surfactants characterized by various uses. Anionic surfactants have detergent type activity and are generally used for cleaning applications. Cationic surfactants contain long chain hydrocarbons and are often used to treat synthetic proteins and polymers or are 65 components of fabric softeners and hair conditioners. Amphoteric surfactants also contain long chain hydrocarbons and are typically used in shampoos. Nonionic surfactants are used

Generally in cleaning products.

Vector: The term "vector" as used herein refers to a nucleic acid molecule capable of transporting another nucleic acid with which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop in which additional segments of DNA can be ligated. Other vectors include cosmids, artificial bacterial chromosomes (BAC) and artificial yeast chromosomes (YAC). Another type of vector is a viral vector, in which additional DNA segments can be ligated into the viral genome (analyzed in more detail below). Certain vectors are capable of autonomous replication in a host cell into which they are introduced (for example, vectors that have an origin of replication that acts in the host cell). Other vectors can be integrated into the genome of a host cell after its introduction into the host cell, and thus replicated together with the host genome. In addition, certain preferred vectors are capable of directing the expression of genes with which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors"). A vector may also include one or more selectable marker genes and other elements.

15 genetics known in the art.

Wax: A variety of fatty acid esters that form solid or flexible substances in an identified set of physical conditions. Fatty acid esters called waxes generally have longer carbon chains than fatty acid esters that are not waxes. For example, a wax generally forms a flexible substance at room temperature.

Fatty ester: Includes any ester composed of a fatty acid. Carbon chains in fatty acids may contain any combination of the modifications described herein. For example, the carbon chain may contain one or more unsaturation points, one or more branching points, including

25 cyclic branching, and can be engineered to be short or long. Any alcohol can be used to form fatty acid esters, for example alcohols derived from the biosynthetic route of fatty acids, alcohols produced by the production host by means of biosynthetic routes of non-fatty acids, and alcohols provided in the fermentation broth.

Fatty acid: Includes products or derivatives thereof made in part from the biosynthetic route of fatty acids of the host organism. The biosynthetic route of fatty acids includes fatty acid synthase enzymes that 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 characteristics of desired carbon chain. Fatty acid derivatives

Examples include, for example, short and long chain alcohols, hydrocarbons and fatty acid esters including waxes.

General methods for genetically engineered microorganisms to produce carbon-based products

Methods for genetically engineered microorganisms to produce carbon-based products are based on metabolic engineering principles, and uses, for example, genetically engineered routes as described in, for example, WO 2007/136762 and WO 2007 / 139925 to produce products from energy captured by photoautotrophic organisms. Generally, carbon-based products of interest are produced by expressing a gene or set of genes as described in Figure 1 in a photoautotrophic microorganism, for example, cyanobacteria, as described herein. Plasmids are constructed to express various proteins that are useful in the production of carbon-based products, as described in the Examples herein, for example, Example 1. Constructions can be made synthetically or made using conventional molecular biology methods and all Cloned genes are placed under the control of constitutive promoters or inducible promoters. Plasmids containing the genes of interest are transformed into the host and corresponding transformants are selected on LB plate supplemented with antibiotics such as spectinomycin, carbenicillin, etc. Using conventional molecular biology techniques, cells are transformed into which a nucleic acid molecule has been introduced to express or overexpress desired genes while other nucleic acid molecules are attenuated or nullified.

55 functionally. Transformation techniques by which a nucleic acid molecule can be introduced into said cell include, but is not limited to, transfection with viral vectors, conjugation, transformation with plasmid vectors and introduction of naked DNA by electroporation, lipofection and particle gun acceleration. . The transformants are inoculated in a suitable medium. Samples containing the transformants are grown at suitable temperatures on a shaker until they reach a certain OD. The cells are then sedimented by centrifugation and the cell pellets are suspended. Separation techniques allow the sample to undergo GC / MS analysis. Total yield is determined.

Microorganisms selected or modified by genetic engineering for the production of carbon-based products of interest

65 Microorganism: Includes prokaryotic and eukaryotic microbial species from the Archaea, Bacteria and Eucarya domains, including the latter yeast and filamentous fungi, protozoa, algae or higher Protists. The terms "microbial cells" and "microbes" are used interchangeably with the term microorganism.

A variety of host organisms can be transformed to produce a product of interest. The 5 photoautotrophic organisms include algae, as well as prokaryotic cyanobacteria, green sulfur bacteria, green non-sulfur bacteria, purple sulfur bacteria and purple non-sulfur bacteria.

Suitable organisms include extremophiles that support various environmental parameters such as temperature, radiation, pressure, gravity, vacuum, desiccation, salinity, pH, oxygen tension and chemicals. They include hyperthermophiles, which grow at or above 80 ° C such as Pyrolobus fumarii; thermophiles, which grow between 60-80 ° C such as Synechococcus lividis; mesophiles, which grow between 15-60 ° C and psychrophiles, which grow at or below 15 ° C such as Psychrobacter and some insects. Radiation tolerant organisms include Deinococcus radiodurans. Pressure tolerant organisms include piezophiles or barophiles that tolerate pressure of 130 MPa. Hypergravity tolerant organisms (for example,> 1 g) and hypogravity (for example, <1 g) are also contemplated. Vacuum tolerant organisms include tardigrades, insects, microbes and seeds. Desiccation-tolerant and anhydrobotic organisms include xerophiles such as Artemia salina, nematodes, microbes, fungi and lichens. Salinity tolerant organisms include halophiles (for example, 2-5 M NaCl) Halobacteriacea and Dunaliella salina. PH tolerant organisms include alkalophiles such as Natronobacterium, Bacillus firmus OF4, Spirulina spp. (for example, pH> 9) and acidophilic such as Cyanidium caldarium, Ferroplasma sp. (for example, low pH). Anaerobes are also contemplated, which cannot tolerate O2 such as Methanococcus jannaschii; microaerophils, which tolerate some O2 such as Clostridium and aerobes, which require O2. Gas tolerant organisms, which tolerate pure CO2 include Cyanidium caldarium and metal tolerant organisms include metallotolents such as Ferroplasma acidarmanus (eg, Cu, As, Cd, Zn), Ralstonia sp. CH34 (for example, Zn, Co, Cd, Hg, Pb). Gross, Michael. Life on the Edge: Amazing 25 Creatures Thriving in Extreme Environments. New York: Plenum (1998) and Seckbach, J. “Search for Life in the Universe with Terrestrial Microbes Which Thrive Under Extreme Conditions” in Cristiano Batalli Cosmovici, Stuart Bowyer, and Dan Wertheimer, eds., Astronomical and Biochemical Origins and the Search for Life in the Universe, p.

511. Milan: Editrice Compositori (1997).

Algae and cyanobacteria include, but are not limited to the following genera:

Acanthoceras, Acanthococcus, Acaryochloris, Achnanthes, Achnanthidium, Actinastrum, Actinochloris, Actinocyclus, Actinotaenium, Amphichrysis, Amphidinium, Amphikrikos, Amphipleura, Amphiprora, Amphithrix, Amphora, Anabaena, Anabaencanous, Apnemomapus, Anumadesausonus, Apismouseum, Anumadiscustous, Apismouseaus, Apnechromesus, Anumadiscustous, Apismouseum, Anumousamus, Applesaus, Applesaus, Applesaus, Applesaus, Applesaus, Apology , aphanochaete, Aphanothece, Apiocystis, Apistonema, Arthrodesmus, Artherospira, Ascochloris, Asterionella, Asterococcus, Audouinella, Aulacoseira, Bacillaria, Balbiania, Bambusina, Bangia, Basichlamys, Batrachospermum, Binuclearia, Bitrichia, Blidingia, Botrdiopsis, Botrydium, Botryococcus, Botryosphaerella, Brachiomonas , Brachysira, Brachytrichia, Brebissonia, Bulbochaete, Bumilleria, Bumilleriopsis, Caloneis, Calothrix, Campylodiscus, Capsosiphon, Carteria, Catena, Cavinula, Centritractus, Centronella, Ceratium, Chaetoceros, Chaetochloris, Chaetomorpha, Chaetonella, Chaetonema, Chaetopeltis, Chaetophora, Chaetosphaeridium, Chamaesiphon , Chara, Characiochloris, Characiopsis, Characium, Charales, Chilomonas, Chlainomonas, Chlamydoblepharis, Chlamydocapsa, Chlamydomonas, Chlamydomonopsis, Chlamydomyxa, Chlamydonephris, Chlorangiella, Chlorangiopsis, Chlorella, Chlorobotrys, Chlorobrachis, Chlorochytrium, Chlorococcum, Chlorogloea, Chlorogloeopsis, Chlorogonium, Chlorolobion, Chloromonas, Chlorophysema, Chlorophyta, Chlorosaccus, 45 Chlorosarcina, Choricystis, Chromophyton, Chromulina, Chroococcidiopsis, Chroococcus, Chroodactylon, Chroomonas, Chroothece, Chrysamoeba, Chrysapsis, Chrysidiastrum, Chrysocapsa, Chrysocapsella, Chrysochaete, Chrysochromulina, Chrysococcus, Chrysocrinus, Chrysolepidomonas, Chrysolykos, Chrysonebula, Chrysophyta, Chrysopyxis, Chrysosaccus , Chrysophaerella, Chrysostephanosphaera, Clodophora, Clastidium, Closteriopsis, Closterium, Coccomyxa, Cocconeis, Coelastrella, Coelastrum, Coelosphaerium, Coenochloris, Coenococcus, Coenocystis, Colacium, Coleochaete, Collodictaugon Conjugation, Conjloattaugonung haete, Coronastrum, Cosmarium, Cosmioneis, Cosmocladium, Crateriportula, Craticula, Crinalium, Crucigenia, Crucigeniella, Cryptoaulax, Cryptomonas, Cryptophyta, Ctenophora, Cyanodictyon, Cyanonephron, Cyanophora Cyanophyta, Cyanothece, Cyanothomonas, Cyclonexis, Cyclostephanos, Cyclotella, Cylindrocapsa, Cylindrocystis, Cylindrospermum , Cylindrotheca, Cymatopleura, Cymbella, Cymbellonitzschia, Cystodinium, Dactylococcopsis, 55 Debarya, Denticula, Dermatochrysis, Dermocarpa, Dermocarpella, Desmatractum, Desmidium, Desmococcus, Desmonema, Desmosiphon, Diacatomos, Diacahotoma, Diacahotoma, Diacahotoma, Diacahotoma, Diacahotoma, Diacahotoma, Diacahotoma, Diacahotoma, Diacatomosia, Diachotama, Diacahotoma, Diachotama, Diachotama, Diachotama, Diacahotoma, Diacahotoma, Diacahotoma, Diacathomaceous, Diachotamus, Diacahotoma, Diacathomaceous, Diachotamus, Diachotamus, Diachotamus, Diacathomaceous, Diacathomaceous, Diacathomaceous, Diachromatum, Diachotamus, Diachotamus, Diachotamus, Diachotamus Dicranochaete, Dictyochloris, Dictyococcus, Dictyosphaerium, Didymocystis, Didymogenes, Didymosphenia, Dilabifilum, Dimorphococcus, Dinobryon, Dinococcus, Diplochloris, Diploneis, Diplostauron, Distrionella, Docidium, Draparnaldia, Dunaliella, Dysmorphococcus, Ecballocystis, Elakatothrix, Ellerbeckia, Encyonema, Enteromorpha, Entocladia, Entomone is, Entophysalis, Epichrysis, Epipyxis, Epithemia, Eremosphaera, Euastropsis, Euastrum, Eucapsis, Eucocconeis, Eudorina, Euglena, Euglenophyta, Eunotia, Eustigmatophyta, Eutreptia, Fallacia, Fischerella, Fragilariaminia, Fragilariaminia, Fragilariaminia, Genus Glaucocystis, Glaucophyta, Glenodiniopsis, Glenodinium, Gloeocapsa, Gloeochaete, Gloeochrysis, Gloeococcus, Gloeocystis, Gloeodendron, Gloeomonas, Gloeoplax, Gloeothece, Gloeotila, Gloeotrichia, Gloiodictyon, Golenkiniapomonia, Gomkinomopia, Gomkinomophalae, Gomnomophagonia, Gomnomophagonia , Goniochloris, Gonium, Gonyostomum, Granulochloris, Granulocystopsis, Groenbladia, Gymnodinium, Gymnozyga, Buwen, Haematococcus, Hafniomonas, Hallassia, Hammatoidea, Hannaea, Hantzschia, Hapalosiphon, Haplotaenium, Haptophyta, Haslea, Hemidinium, Hemitoma, Heribaudiella, Heteromastix, Heterothrix, Hibberdia , Hildenbrandia, Hillea, Holopedium, Homoeothrix, Hormanthonema, Hormoti la, Hyalobrachion, Hyalocardium, Hyalodiscus, Hyalogonium, Hyalotheca, Hydrianum, Hydrococcus, Hydrocoleum, Hydrocoryne, 5 Hydrodictyon, Hydrosera, Hydrurus, Hyella, Hymenomonas, Isthmochloron, Johannesbaptistia, Juranyiellaium, Keyephiolaphyllum, Keraphielodiola, Keraphielachodium, Keraphielachiolaphyllum, Keraphielachodium, Keraphielachodium, Keraphielachiolaphyllum, Keraphielachodium, Keraphielachioe, Keraphielachodiola, Keraphielachioelaphyllum, Kyrgyalachiolachiolachioe, Keraphielachiolachiola, Keraphielachiolachioelachioe, Kylenaphiolachodium , Kolbesia, Koliella, Komarekia, Korshikoviella, Kraskella, Lagerheimia, Lagynion, Lamprothamnium, Lemanea, Lepocinclis, Leptosira, Lobococcus, Lobocystis, Lobomonas, Luticola, Lyngbya, Malleochloris, Mallomonas, Mantoniellemia, Martylopeus, Mantonielle, Martyelloomyelia, Merntonyelia, Martyelos, Merntonyelia, Martyelloomyelia, Merntonyelia, Martyelos, Merntonyelia, Martyelos, Merntonyelia, Martyelos, Merntonyelia, Martyelos, Meltonylopemia, Mantonielloomyelia, Martyelos, Martyelloomyelia, Melonie, Chloegalae, Martyls , Mesostigma, Mesotaenium, Micractinium, 10 Micrasterias, Microchaete, Microcoleus, Microcystis, Microglena, Micromonas, Microspora, Microthamnion, Mischococcus, Monochrysis, Monodus, Monomastix, Monoraphidium, Monostroma, Mougeotia, Mougeotiopsis, Chrysotrops, Mygeloxium, Chrysotliogenesis, Mygeloxium, Chrysotliopsis, Mygethioxysopharyngealis, Mychlorethis, Chrysotrops, Myco-Chlorosis Nautococcus, Navicula, Neglectella, Neidium, Nephroclamys, Nephrocytium , Nephrodiella, Nephroselmis, Netrium, Nitella, Nitellopsis, Nitzschia, Nodularia, Nostoc, Ochromonas, Oedogonium, Oligochaetophora, Onychonema, Oocardium, Oocystis, Opephora, Ophiocytium, 15 Orthoseira, Oscillatoria, Oscillatoria, Palcillaina, Oscillatoria, Palcillaina, Oscillatoria, Palcillaina, Oscillatoria, Palcillaina, Oscillatoria, Palcillaina, Oscillatoria, Palcillaina, Oscillatoria, Palcillaina, Oscillatoria, Palcillaina, Oscillatoria, Palcillaina, Ocilla Paralia, Pascherina, Paulschulzia, Pediastrum, Pedinella, Pedinomonas, Pedinopera, Pelagodictyon, Penium, Peranema, Peridiniopsis, Peridinium, Peronia, Petroneis, Phacotus, Phacus, Phaeaster, Phaeodermatium, Phaeophyta, Phaeosphaera, Phaeothamnion, Phormidium, Phycopeltis, Phyllariochloris, Phyllocardium, Phyllomitas, Pinnularia, Pitophora, Placoneis, Planctonema, Planktosphaeria, Planothidium, Plectonema, Pleodorina, Pleurastrum, Pleurocapsa, 20 Pleurocladia, Pleurodiscus, Pleurosigma, Pleurosira, Pleurotaenium, Pocillomonas, Podohedra, Polyblepharides, Polychaetophora, Polyedriella, Polyedriopsis, Polygoniochloris, Polyepidomonas, polytaenia , Polytoma, Polytomella, Porphyridium, Posteriochromonas, Prasin ochloris, Prasinocladus, Prasinophyta Prasiola, Prochlorphyta, Prochlorothrix, protoderm, Protosiphon, Provasoliella, Prymnesium, Psammodictyon, Psammothidium, Pseudanabaena, Pseudenoclonium, Psuedocarteria, Pseudochate, Pseudocharacium, Pseudococcomyxa, 25 Pseudodictyosphaerium, Pseudokephyrion, Pseudoncobyrsa, Pseudoquadrigula, Pseudosphaerocystis, Pseudostaurastrum, Pseudostaurosira , Pseudotetrastrum, Pteromonas, Punctastruata, Pyramichlamys, Pyramimonas, Pyrrophyta, Quadrichloris, Quadricoccus, Quadrigula, Radiococcus, Radiofilum, Raphidiopsis, Raphidocelis, Raphidonema, Raphidophyta, Peimeria, Rhabdoderma, Rhabdomonas, Rhizoclonium, Rhodomonas, Rhodophyta, Rhoicosphenia, Rhopalodia Rivularia, Rosenvingiella , Rossithidium, Roya, Scenedesmus, Scherffelia, 30 Schizochlamydella, Schizochlamys, Schizomeris, Schizothrix, Schroederia, Scolioneis, Scotiella, Scotiellopsis, Scourfieldia, Scytonema, Selenastrum, Selenochloris, Sellaphora, Semiorbis, Sideroenis, Dimroonsis, Sideroenys, Dimroonsis ia, Siphononema, Sirocladium, Sirogonium, Skeletonema, Sorastrum, Spermatozopsis, Sphaerellocystis, Sphaerellopsis, Sphaerodinium, Sphaeroplea, Sphaerozosma, Spiniferomonas, Spirogyra, Spirotaenia, Spirulina, Spondylomorum, Spondylosium, Sporotetras, Spumella, Staurastrum, Stauerodesmus, Stauroneis, Staurosira, 35 Staurosirella , Stenopterobia, Stephanocostis, Stephanodiscus, Stephanoporos, Stephanosphaera, Stichococcus, Stichogloea, Stigeoclonium, Stigonema, Stipitococcus, Stokesiella, Strombomonas, Stylochrysalis, Stylodinium, Styloyxis, Stylosphaeridca, Syirenecum Syndrome, Symphonyx Syndrome, Symphonyx Syndrome, Symphonyx Syndrome, Syndrome, Syndrome, Symphony, Symphony, Symphony, Syndrome, Syndrome, Syndrome, Symphony, Syndrome, Syndrome, Syndrome, Syndrome, Syndrome, Syndrome, Syndrome, Syndrome, Syndrome, Syndrome, Syndrome, Symphony, Syndrome , Tabularia, Teilingia, Temnogametum, Tetmemorus, Tetrachlorella, Tetracyclus, Tetradesmus, Tetraedriella, Tetraedron, Tetraselmis, Tetraspora, Tetrastrum, Thalassiosira, Thamniochaete,

40 Thorakochloris, Thorea, Tolypella, Tolypothrix, Trachelomonas, Trachydiscus, Trebouxia, Trentepholia, Treubaria, Tribonema, Trichodesmium, Trichodiscus, Trochiscia, Tryblionella, Ulothrix, Uroglena, Uronema, Urosolenia, Urospolalaria, Urospolalaria, Volt, Volvalaia , Woloszynskia, Xanthidium, Xanthophyta, Xenococcus, Zygnema, Zygnemopsis and Zygonium.

45 Green non-sulfur bacteria include but are not limited to the following genera: Chloroflexus, Chloronema, Oscillochloris, Heliothrix, Herpetosiphon, Roseiflexus and Thermomicrobium.

Green sulfur bacteria include but not limited to the following genera: Chlorobium, Clathrochloris, and Prosthecochloris.

50 Purple sulfur bacteria include but are not limited to the following genera: Allochromatium, Chromatium, Halochromatium, Isochromatium, Marichromatium, Rhodovulum, Thermochromatium, Thiocapsa, Thiorhodococcus, and Thiocystis.

55 Purple non-sulfur bacteria include but are not limited to the following genera: Phaeospirillum, Rhodobaca, Rhodobacter, Rhodomicrobium, Rhodopila, Rhodopseudomonas, Rhodothalassium, Rhodospirillum, Rodovibrio and Roseospira.

Aerobic chemolithotrophic bacteria include but are not limited to nitrifying bacteria such as

60 Nitrobacteraceae sp, Nitrobacter sp., Nitrospina sp, Nitrococcus sp, Nitrospira sp, Nitrosomonas sp, Nitrosococcus sp, Nitrosospira sp, Nitrosolobus sp, Nitrosovibrio sp; colorless sulfur bacteria such as, Thiovulum sp., Thiobacillus sp., Thiomicrospira sp., Thiosphaera sp., Thermothrix sp; Mandatory chemolithotrophic hydrogen bacteria such as Hydrogenobacter. sp., oxidizing bacteria and / or depositories of iron and manganese such as Siderococcus sp., and magnetotactic bacteria such as Aquaspirillum sp.

Archaeobacteria include but are not limited to methanogenic archeobacteria such as Methanobacterium sp., Methanobrevibacter sp., Methanothermus sp., Methanococcus sp., Methanomicrobium sp., Methanospirillum sp., Methanogenium sp., Methanosarcina sp., Methanolorix sp. Methanococcoides sp., Methanoplanus sp .; Extremely thermophilic sulfur metabolizers such as Thermoproteus sp.,

5 Pyrodictium sp., Sulfolobus sp., Acidianus sp. and other microorganisms such as, Bacillus subtilis, Saccharomyces cerevisiae, Streptomyces sp., Ralstonia sp., Rhodococcus sp., Corynebacteria sp., Brevibacteria sp., Mycobacteria sp. and oil yeast.

Hyperphotynthetic conversion requires extensive genetic modification; therefore, in preferred embodiments, the parental photoautotrophic organism can be transformed with exogenous DNA.

Preferred organisms for hyperphotynthetic conversion include: Botryococcus braunii, Chlamydomonas reinhardtii and Dunaliela saline (algae), Synechococcus sp PCC 7002, Synechococcus sp. PCC 7942, Synechocystis sp. PCC 6803 and Thermosynechococcus elongatus BP-1 (cyanobacteria), Chlorobium tepidum (green bacteria from

15 sulfur), Chloroflexus auranticus (green non-sulfur bacteria), Chromatium tepidum and Chromatium vinoum (purple sulfur bacteria), Rhodospirillum rubrum, Rhodobacter capsulatus and Rhodopseudomonas palusris (non-sulfur purple bacteria).

Further suitable organisms include synthetic cells or cells produced by synthetic genomes as described in Venter et al. Published US Patent No. 2007/0264688, and cell-type systems or synthetic cells as described in Glass et al. United States Patent Published No. 2007/0269862.

Other suitable organisms further include microorganisms that can be engineered to fix carbon dioxide, bacteria such as Escherichia coli, Acetobacter aceti, Bacillus subtilis, yeast and fungi

25 such as Clostridium ljungdahlii, Clostridium thermocellum, Penicillium chrysogenum, Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pseudomonas fluorescens or Zymomonas mobilis.

A common theme in the genetic engineering selection or modification of a suitable organism is the autotrophic fixation of carbon, such as CO2 to products. This would encompass photosynthesis and methanogenesis. Acetogenesis is also covered, covering all three types of CO2 fixation; Calvin cycle, acetyl CoA route and TCA reducing route. The ability to use carbon dioxide as the sole source of cellular carbon (autotrophy) is found in almost all major prokaryotic groups. CO2 fixation routes differ between groups, and there is no clear pattern of distribution of the four autotrophic routes known today. Fuchs, G. 1989. Alternative pathways of autotrophic CO2 fixation, p. 365-382. In HG Schlegel, and B. Bowien (ed.), Autotrophic

35 Springer-Verlag bacteria, Berlin, Germany. The pentose phosphate reducing cycle (Calvin-Bassham-Benson cycle) represents the route of CO2 fixation in almost all aerobic autotrophic bacteria, for example, cyanobacteria.

Propagation of selected microorganisms

Methods for the cultivation of photosynthetic organisms in liquid medium and on plates containing agarose are well known to those skilled in the art (see, for example, websites associated with ATCC, and with the Pasteur Institute). For example, the cells of Synechococcus sp. PCC 7002 (available from the Cytobacterial Pasteur Culture Collection) are grown in BG-11 medium (17.65 mM NaNO3, 0.18 mM K2HPO4, 0.3 mM MgSO4, 0.25 CaCl2

45 mM, 0.03 mM citric acid, 0.03 mM ferric ammonium citrate, 0.003 mM EDTA, 0.19 mM Na2CO3, H3BO3 2.86 mg / l, MnCl2 1.8 mg / l, ZnSO4 0.222 mg / l , Na2MoO4 0.390 mg / l, CuSO4 0.079 mg / l and Co (NO3) 2 0.049 mg / l, pH 7.4) supplemented with 16 # g / l biotin, 20 mM MgSO4, 8 mM KCl and 300 mM NaCl (see, for example, a website associated with the Pasteur Institute and Price GD, Woodger FJ, Badger MR, Howitt SM, Tucker L. “Identification of a SulPtype bicarbonate transporter in marine cyanobacteria.” Proc Natl. Acad. Sci. USA (2004) 101 (52): 18228-33). Typically, the cultures are maintained at 28 ° C and are continuously bubbled with 5% CO2 under a light intensity of 120 #mol of photons / m2 / s. Alternatively, as described in Example 1, Synechococcus sp. PCC 7002 are grown in A + medium as previously described [Frigaard NU et al. (2004) “Gene inactivation in the cyanobacteriaum Synechococcus sp. PCC 7002 and the green sulfur bacterium Chlorobium tepidum using in vitro-made DNA constructs and natural transformation ”Methods Mol. Biol., 274: 325-340].

55 Thermosynechococcus elongatus BP-1 (available from Kazusa DNA Research Institute, Japan) is propagated in BG11 medium supplemented with 20 mM TES-KOH (pH 8.2) as previously described [Iwai M, Katoh H, Katayama M, Ikeuchi M. "Improved genetic transformation of the thermophilic cyanobacterium, Thermosynechococcus elongatus BP-1" Plant Cell Physiol (2004). 45 (2): 171-175)]. Typically, the cultures are maintained at 50 ° C and are continuously bubbled with 5% CO2 under a light intensity of 38 #mol of photons / m2 / s. T. elongatus BP-1 can be grown in A + medium also as described in Example 2.

Chlamydomonas reinhardtii (available from the culture collection of the Chlamydomonas center maintained by Duke University, Durham, North Carolina) are grown in minimal saline medium consisting of K2HPO4 143 65 mg / l, KH2PO4 73 mg / l, NH4NO3 400 mg / l, MgSO4 100 mg / l, CaCl2-2H2O 50 mg / l, trace element reserve 1 ml / l and MOPS 2.0 M 10 ml / l titrated with Tris base at pH 7.6 as described (Geraghty AM , Anderson JC,

Spalding MH. "A 36 kilodalton limiting-CO2 induced polypeptide of Chlamydomonas is distinct from the 37 kilodalton periplasmic anhydrase" Plant Physiol (1990) .93: 116-121). Typically, the cultures are maintained at 24 ° C and bubbled with 5% CO2 in air, under a light intensity of 60 #mol of photons / m2 / s.

5 The above defines typical propagation conditions. As appropriate, incubations are performed using alternative gas compositions or media, temperatures (5 -75 ° C) and / or light fluxes (0-5500 #mol of photons / m2 / s).

Light is supplied through a variety of mechanisms, including natural lighting (sunlight), light bulbs

10 conventional incandescent, fluorescent or halogen, or by propagation in specially designed illuminated culture chambers (for example the LI15 Illuminated Culture Chamber Model (Sheldon Manufacturing, Inc. Cornelius, OR)) For experiments that require wavelengths and / or intensities specific, the light is distributed by light emitting diodes (LED), in which the wavelength and intensity spectra can be carefully controlled (Philips).

Carbon dioxide is provided by inclusion of solid media supplements (ie, sodium bicarbonate) or as a gas by distribution to the culture medium incubator. Most experiments are performed using concentrated carbon dioxide gas, at concentrations between 1 and 30%, which are bubbled directly into the growth medium at speeds sufficient to provide mixing for the organisms.

20 When concentrated carbon dioxide gas is used, the gas originates in pure form from commercially available cylinders, or preferably from concentrated sources including gaseous discharge or exhaust gas from coal plants, refineries, gas installations cement production, natural gas facilities, breweries and the like.

25 Transformation of selected microorganisms

Synechococcus sp. Cells are transformed. PCC 7002 according to the optimized protocol previously described [Essich ES, Stevens Jr E, Porter RD "Chromosomal Transformation in the Cyanobacterium Agmenellum quadruplicatum". J Bacteriol (1990). 172 (4): 1916-1922]. The cells are cultured in medium A (18 g / l NaCl, 30 mg / l MgSO4 5 mg / l Na2EDTA, 600 mg / l KCl, Ca2.22 H2O 370 mg / l, 1 g / l NaNO3, KH2PO4 50 mg / l, Trizma base 1 g / l pH 8.2, vitamin B12 4 # g / l, FeCl3.6 H2O 3.89 mg / l, H3BO3 34.3 mg / l, MnCl2.4H2O 4.3 mg / l, ZnCl2. 315 # g / l, MoO3 30 # g / l, CuSO4.5H2O 3 # g / l, CoCl2.6H2O 13.2 # g / l) [Stevens SE, Patterson COP, and Myers J. "The production of hydrogen peroxide by green algae: a survey “J. Phycology (1973). 9: 427-430] plus 5 g / l NaNO3 at approximately 108 cells / ml. Nine volumes of cells are mixed with 1 volume of DNA 1-10 # g / ml in 0.15 M NaCl / 0.015 M Na3 citrate and incubated at 27-30 ° C for 3 hours before adding 1 volume of DNaseI at a final concentration of 10 # g / ml. The cells are seeded in 2.5 ml agar with overlapping of 0.6% medium A that was frightened at 45 ° C and incubated. The cells were exposed to antibiotics by placing 2.0 ml of agar of 0.6% medium A containing below appropriate concentration of antibiotic with a sterile Pasteur pipette. The transformants were selected 3-4 later. Selections are typically made using Kanamycin 200

40 # g / ml, chloramphenicol 8 # g / ml, spectinomycin 10 # g / ml in solid medium, while kanamycin 150 # g / ml, chloramphenicol 7 # g / ml and spectinomycin 5 # g / ml are used in liquid medium .

T. elongatus BP-1 cells are transformed according to the optimized protocol previously described (Iwai M, Katoh H, Katayama H, and Ikeuchi).

E. Coli are transformed using conventional techniques known to those skilled in the art, including thermal shock of chemically competent cells and electroporation [Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology volume 152 Academic Press, Inc., San Diego , California .; Sambrook et al. (1989) Molecular Cloning -A Laboratory Manual (2nd ed.) Vol 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor

50 Press, NY .; and Current Protocols in Molecular Biology, F.M. Ausubel et al., Eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (up to and including the 1997 Supplement)].

Biosynthetic pathways as described herein are first tested and optimized.

55 using episodic plasmids described above. Non-limiting optimizations include promoter exchange and adjustment, manipulation of the ribosome binding site, alteration of the gene order (eg, ABC gene versus BAC, CBA, CAB, BCA), co-expression of molecular chaperones, random mutagenesis or targeting of gene sequences to increase or reduce allosteric activity, folding or regulation, expression of gene sequences of alternative species, codon manipulation, addition or removal of address sequences

Intracellular 60 such as signal sequences, and the like.

Each gene or genetically modified nucleic acid is optimized individually or, alternatively, in parallel. Functional promoter and gene sequences are subsequently integrated into the E. coli chromosome to allow stable propagation in the absence of selective pressure (i.e., inclusion of antibiotics) using techniques

65 conventional known to those skilled in the art.

Figure 1 lists genes involved in the production of carbon-based products of interest, related to associated pathways, Enzyme Commission Numbers (EC), exemplary gene names, source organism, GenBank reference numbers, and source counterparts alternatives. When the parental organism encodes a gene with the indicated enzymatic activity, it is useful to overexpress these components or at least attenuate these

5 components as indicated. In some circumstances, the native enzyme sequence may be overexpressed or attenuated. In other circumstances, it is useful to overexpress or attenuate an exogenous gene, which allows for more explicit regulatory control in the biological process and a means to potentially mitigate the effects of central metabolic regulation, which centers around native genes in a way explicit

Ethanol Production

Figure 2 provides a route to produce ethanol, succinate and derivatives thereof.

To date, the current yields of ethanol produced in cyanobacteria are not suitable for production

15 commercial at 1.3 mM for OD730 per day, as disclosed in WO 2007/084477, or 1.7 #mol of ethanol per mg of chlorophyll per hour, as shown in US Patent No. 6,699. 696.

The present invention, therefore, provides a host cell capable of fixing CO2 that produces ethanol at a commercial level, for example, between about 50 and 150 g / l over a period of about 48 hours. In certain embodiments, the rate of ethanol productivity is in the range of about 2.5 g / l-h to about 5 g / l-h. In one embodiment, a host cell capable of fixing CO2 such as a Synechococcus sp. PCC 7002 to express genes such as pdc and / or adh as disclosed. Said recombinant microorganism encodes PDC activity that converts pyruvic acid into acetaldehyde and / or ADH activity that converts acetaldehyde into ethanol. The capacity of the microorganism

25 transformed to fix CO2 avoids the need to supplement with sugars or biomass. Consequently, the microorganisms of the present invention are attractive alternatives for producing biofuels. The present invention provides the w / v of ethanol to be at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 125 or at least 150 g / what is produced from another way on a commercial scale.

Selection of enzymes and optimal enzymes

At present, fermentation products such as ethanol, butanol, lactic acid, formate, acetate produced in biological organisms employ a NADH-dependent process. NAD is used to degrade glucose or other sources of sugars to form NADH. NADH is recycled during fermentation to NAD + to allow

Additional degradation of sugars, which results in secondary fermentation products. During photosynthesis, however, the cell forms NADPH, which is mainly used for biosynthetic operations in biological organisms, for example, cell for culture, division and to accumulate chemical stores such as glycogen, sucrose and other macromolecules. Light fermentation products are produced, but in small quantities.

Using natural or genetically engineered enzymes that use NADPH as a source to reduce energy instead of NADH would allow the direct use of photosynthetic reducing energy for the formation of normally fermentative by-products. Accordingly, the present invention provides a method for producing ethanol comprising culturing a host cell that is genetically modified to

45 comprise an exogenous genetically engineered nucleic acid encoding an NADPH-dependent alcohol dehydrogenase activity. This is an improvement over previous methods of using organisms such as algae to accumulate chemical stores, which are subsequently used to make fermentation products at night or the external use of separate organisms. In fact, fermentative products are formed at greater efficiencies directly to light during photosynthesis. In addition, mandatory production of high concentration macromolecules is relieved during the day, producing such products directly during the day.

NADPH-dependent enzymes that normally produce fermented products are rare in nature. Consequently, in certain aspects of the invention, ethanol is produced in organisms that express or

55 modified to express HUC22-1 of Moorella sp. or a homologue thereof that includes at least three alcohol dehydrogenases such as AdhA (NCBI reference YP_430754). It has been previously shown that this enzyme preferably uses NADP as a cofactor unlike NAD and produces ethanol at high rates from acetaldehyde ["Characterization of enzymes wrapped in the ethanol production of Moorella sp. HUC22-1 ”]. By coexpressing this gene in selected organisms, such as cyanobacteria, the NADPH2 formed during photosynthesis can be used directly to form ethanol in these organisms. Alternatively, enzymes that naturally use NADH can be engineered using genetic engineering techniques established to require NADPH2 instead of NADH.

In certain embodiments, Moorella's NADPH-dependent AdHA is co-expressed with pyruvate decarboxylase from

65 Zymomonas mobilis in cyanobacteria to derive an effective process for the production of NADPH-dependent ethanol as a cofactor instead of traditional NADH. Such transgenic organisms are capable of performing ethanol using NADPH-dependent processes.

Isolated polynucleotides

5 Isolated nucleic acid molecules for the adHA gene and variants thereof are described herein. The full length nucleic acid sequence for this gene, which encodes the NADP-dependent alcohol dehydrogenase enzyme, EC. 1.1.1.2, has been identified and sequenced. SEQ ID NO. 1 represents the coding sequence optimized for expression and codon for the adhAZ gene of Moorella sp. HUC22-1.

The cell of the present invention may comprise a nucleic acid molecule that comprises or consists of a sequence that is an optimized version for expression and codon of the wild-type adHA gene. In a further embodiment, the cell of the present invention may comprise a nucleic acid molecule and homologues, variants and derivatives of SEQ ID NO: 1 that comprise or consist of a sequence that is a variant of the adHA gene having at least 77.1 % identity with SEQ ID No. 1. The nucleic acid sequence

15 may preferably be 78%, 79%, 80%, 81% -85%, 90% -95%, 96% -98%, 99%, 99.9% or even greater identity with SEQ ID No. 1.

In another embodiment, the cell of the invention comprises a nucleic acid molecule encoding a polypeptide having the amino acid sequence of SEQ ID NO: 2.

Nucleic acid molecules can hybridize under stringent conditions with the nucleic acid molecules described above. As defined above, and as is well known in the art, rigorous hybridizations are performed at approximately 25 ° C below the thermal melting point (Tm) for the specific DNA hybrid under a set of particular conditions, in which Tm is the temperature at which 50% of the

25 hybrid target sequence with a perfectly matching probe. Rigorous washing is performed at temperatures approximately 5 ° C lower than the Tm for the specific DNA hybrid under a particular set of conditions.

Nucleic acid molecules comprising a fragment of any one of the nucleic acid sequences described above are also described. These fragments preferably contain at least 20 contiguous nucleotides. More preferably, the fragments of the nucleic acid sequences contain at least 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 or even more contiguous nucleotides.

Nucleic acid sequence fragments have utility in a variety of systems and methods. By

For example, the fragments can be used as probes in various hybridization techniques. Depending on the method, the target nucleic acid sequences may be DNA or RNA. The target nucleic acid sequences can be fractionated (for example, by gel electrophoresis) before hybridization, or hybridization can be performed on in situ samples. One skilled in the art will appreciate that nucleic acid probes of known sequence find utility in determining chromosomal structure (for example, by Southern blot) and in measuring gene expression (e.g., by Northern blotting) . In such experiments, the sequence fragments are preferably labeled detectably, so that their specific hybridization with target sequences can be detected and optionally quantified. One skilled in the art will appreciate that nucleic acid fragments can be used in a wide variety of transfer techniques not specifically described herein.

It should also be appreciated that the nucleic acid sequence fragments disclosed herein also find utility as probes when immobilized in microarrays. Methods for creating microarrays by deposition and fixation of nucleic acids on support substrates are well known in the art. Revised in DNA Microarrays: A Practical Approach (Practical Approach Series), Schena (ed.), Oxford University Press (1999) (ISBN: 0199637768); Nature Genet 21 (1) (supl): 1-60 (1999); Microarray Biochip: Tools and Technology, Schena (ed.), Eaton Publishing Company / BioTechniques Books Division (2000) (ISBN: 1881299376). The analysis of, for example, gene expression using microarrays comprising nucleic acid sequence fragments, such as nucleic acid sequence fragments disclosed herein, is a well established utility for sequence fragments in the field of Cellular and molecular biology. Others

55 uses for fragments of immobilized sequences in microarrays are described in Gerhold et al., Trends Biochem. Sci. 24: 168-173 (1999) and Zweiger, Trends Biotechnol. 17: 429-436 (1999); DNA Microarrays: A Practical Approach (Practical Approach Series), Schena (ed.), Oxford University Press (1999) (ISBN: 0199637768); Nature Genet 21 (1) (supl): 1-60 (1999); Microarray Biochip: Tools and Technology, Schena (ed.), Eaton Publishing Company / BioTechniques Books Division (2000) (ISBN: 1881299376).

Isolated nucleic acid molecules that encode the NADPH-dependent AdHA polypeptide comprising alcohol dehydrogenase activity are disclosed. As is well known in the art, enzymatic activities can be measured in various ways. For example, the pyrophosphorolysis of OMP can be followed spectroscopically. Grubmeyer et al., J. Biol. Chem. 268: 20299-20304 (1993). Alternatively, the activity of the enzyme can be followed using chromatographic techniques, such as high performance liquid chromatography. Chung and Sloan, J. Chromatogr. 371: 71-81 (1986). As another alternative, the activity can be measured indirectly by determining the levels of product produced from the enzymatic activity. These levels can be measured with techniques that include aqueous methanol / chloroform extraction as are known and described in the art (see M. Kates (1986) Techniques of Lipidology; Isolation, analysis and identification of Lipids. Elsevier Science Publishers, New York ( ISBN: 0444807322)). More modern techniques include the use of gas chromatography 5 linked to mass spectrometry (Niessen, W. M. A. (2001). Current practice of gas chomatography— mass spectometry. New York, N.Y: Marcel Dekker. (ISBN: 0824704738)). Additional modern techniques can be used for identification of recombinant protein activity and products include mass spectrometry liquid chromatography (LCMS), high performance liquid chromatography (HPLC), capillary electrophoresis, mass spectometry-desorption and laser ionization assisted by time-matrix flight (MALDI-TOF MS), nuclear magnetic resonance imaging (NMR), near infrared spectroscopy (NIR), viscosimetry (Knothe, G., RO Dunn, and MO Bagby. 1997. Biodiesel: The use of vegetable oils and their derivatives as alternative diesel fuels, Am. Chem. Soc. Symp. Series 666: 172-208), titration to determine free fatty acids (Komers, K., F. Skopal and R. Stloukal. 1997. Determination of the neutralization number for biodiesel fuel production Fett / Lipid 99 (2): 52-54), enzymatic methods (Bailer, J., and K. de Hueber. 1991. Determination of saponifiable glycerol in "bio-diesel".

15 Fresenius J. Anal. Chem. 340 (3): 186), methods based on physical properties, wet chemical methods, etc. to analyze the levels and identity of the product produced by the organisms of the present invention. Other methods and techniques for measuring enzymatic activity may also be suitable, as will be known by one skilled in the art.

Vectors, including expression vectors, comprising the above nucleic acid molecules are also described. The vectors may include the aforementioned nucleic acid molecules operatively linked with one or more expression control sequences. Vectors can therefore be used to express an NADPH-dependent AdHA polypeptide comprising alcohol dehydrogenase activity.

25 Isolated Polypeptides

Isolated polypeptides (including muteins, allelic variants, fragments, derivatives and the like) encoded by the nucleic acid molecules described above are also described. The isolated polypeptide may comprise the polypeptide sequence corresponding to SEQ ID No. 2. The isolated polypeptide may comprise a polypeptide sequence at least 71.1% identical to SEQ ID No. 2. Preferably the isolated polypeptide has 72%, 73% -75% , 76% -80%, 81% -90%, 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99, 6%, 99.7%, 99.8%, 99.9% or even greater identity with SEQ ID No. 2.

Isolated polypeptides comprising a fragment of the polypeptide sequences described above are described. These fragments preferably include at least 20 contiguous amino acids, more preferably at least 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 or even more contiguous amino acids.

The polypeptides also include fusions between the polypeptide sequences described above and heterologous polypeptides. Heterologous sequences may, for example, include sequences designed to facilitate purification, for example, histidine markers and / or recombinantly expressed protein visualization. Other non-limiting examples of protein fusions include those that allow the presentation of the encoded protein on the surface of a phage or a cell, fusions with intrinsically fluorescent proteins, such as green fluorescent protein (GFP) and fusions with the Fc region of IgG.

Optimal Enzyme Results

Increased level of ethanol is observed by genetically modifying host cells so that they have NADPH-dependent alcohol dehydrogenase activity. Methods for producing increased ethanol level comprise the expression of said NADPH-dependent adHA genes as described herein.

In certain aspects of the invention, increased levels of ethanol of at least about 249 mg / l of ethanol are produced for 72 hours. More preferably, at least about 297 mg / l of ethanol is produced

55 for 72 hours (Figure 6).

In other aspects of the invention, reduced levels of acetaldehyde are produced. In preferred embodiments, less than about 14 mg / l acetaldehyde is produced (Figure 7).

In addition, in other aspects of the invention, methods are produced to produce more ethanol in relation to increased OD. In preferred embodiments, at least about 36 mg / l ethanol is produced per OD. More preferably, at least about 47 mg / l of ethanol are produced per OD (Figure 8).

Accordingly, it is shown herein that the expression of said enzymes dependent on

65 NADPH to generate fermentative products such as ethanol increases ethanol levels, reduces acetaldehyde levels and in fact allows increased ethanol production as a function of DO.

Nutrient Independence

In another aspect, in addition to CO2 and light, photoautotrophic organisms typically require sources of inorganic nutrients and vitamins. The required nutrients are generally used to complement the growth medium during the experimental scale propagation of said organisms. However, such nutrients are prohibitively expensive in the context of industrial-scale bioprocessing.

Vitamin B12 is a vitamin cofactor that facilitates radical-based reaction catalysis. Many organisms, including Synechococcus sp. PCC 7002, require external sources of vitamin B12 for growth, which is prohibitively expensive in large-scale industrial bioprocessing. In one embodiment, the need for vitamin B12 is avoided by genetically engineered photoautotrophic cells to express the route of vitamin B12 biosynthesis as disclosed in document PCT / US2008 / 083056, filed on November 10, 2008. A route of Exemplary biosynthesis found in Salmonella typhimurium is overexpressed, including but not limited to the following genes encoding the amino acid sequences exposed in 15 (Uroporfirin-III C-methyltransferase (CysG), EC 2.1.1.107, locus NP_462380), (Sirohydrochlorin cobaltokelatase (CbiK ), EC 4.99.1.3, locus NP_460970), (Precorrin-2 C20 methyltransferase (CbiL), EC 2.1.1.130, locus NP_460969), (Precorrin3B methylase (CbiH), EC 2.1.1.131, locus NP_460972), (CbiG / precorrin bifunctional methyltransferase (CbiG), locus NP_460973), (Precorrin-4 C11-methyltransferase (CbiF), EC 2.1.1.133, locus NP_460974), (cobalamin biosynthesis protein (CbiD), locus NP_460977), (precorrinie-6 NADPH (CbiJ), EC 20 1.3.1.54, locus NP_460971), (precorrin-6B C5,15-methyltransferase (CbiE), EC 2.1.1.132, locus NP_460976), (precorrin-6B C12 decarboxylase (CbiT), EC 2.1.1.132, locus NP_460975), (precorrin-8X-methylmutase (CbiC), EC 5.4.1.2, locus NP_460978), (cobyric acid A, C-diamide-synthase (CbiA), EC 6.3.1 -, locus NP_460980) , (Cob acid

(I) irinic a, c-diamide adenosyltransferase (BtuR), EC 2.5.1.17, locus NP_460677), (cobyrinic acid synthase (CbiP), EC 6.3.5.10, locus NP_460964), (cobyric acid decarboxylase (CobD), EC 4.1 .1.81, locus NP_459636),

25 (adenosylcobinamide phosphate synthase (CbiB), EC 6.3.1.10, locus NP_460979), (alpha ribazol-5'-P phosphatase (CobC), EC 3.1.3.73, locus NP_459635), (cobalamin (5'-phosphate) - synthase (CobS), EC 2.7.8.26, locus NP_460962), (Cobinamide phosphate guanylyl transferase (CobU), EC 2.7.7.62, locus NP_460963) and (nicotinate-nucleotide dimethylbenzimidazole-P phosphoribosyl transferase (CobT), EC 2.4.21, EC 2.4.21, EC 2.4.21 locus NP_460961)].

30 In addition, to allow cobalt uptake and incorporation into vitamin B12, the genes encoding the cobalt transporter are overexpressed. The exemplary cobalt transporter protein found in Salmonella typhimurium is overexpressed and is encoded by amino acid sequences exposed in (ABC type Co2 + transport system, permease component (CbiM), locus NP_460968), (cobalt type transport system ABC, periplasmic component (CbiN), locus NP_460967) and (type cobalt transport system

35 ABC, permease component (CbiQ), locus NP_461989).

In a preferred embodiment, photoautotrophic organisms are genetically engineered to overexpress independent enzymes of vitamin B12 to avoid the need for this cofactor completely. In most photoautotrophic organisms, only methionine synthase (EC 2.1.1.13) and ribonucleotide reductases of class II require vitamin B12. An exemplary vitamin B12 methionine synthase (EC 2.1.1.14) from Thermotoga maritime is therefore overexpressed, as set forth in PCT / US2008 / 083.506 (5-methyltetrahydrofteroyltriglutamate-homocysteine methyltransferase (MetE), locus NP_229090). In addition, an exemplary class I ribonucleotide reductase (nrdAB) from Synechocystis sp. PCC 6803 encoding the amino acid sequences exposed in (ribonucleoside diphosphate reductase, alpha subunit (NrdA), locus

45 NP_441654), (ribonucleoside diphosphate reductase, beta subunit (NrdB), locus NP_443040).

By genetically engineering an organism with the enzymes listed above and in Figure 1, a photoethanolgene is produced as described more specifically in Example 3. Accordingly, the present invention provides a host cell capable of fixing CO2 such as a Synechococcus cyanobacterium. sp.

50 PCC 7002 that is genetically engineered to express genes such as pdc and / or adh to produce ethanol, and genetically engineered to be nutrient independent.

Ethanol production in continuous lighting

55 Normally, glycogen is formed in microorganisms in the light, to consume to produce energy in the dark. With continuous lighting, however, it would be disadvantageous to accumulate significant amounts of glycogen, which would move carbon away from the desired route or routes, especially since there could not be a dark period long enough to use glycogen. In certain embodiments to avoid glycogen synthesis during illumination, the genes encoding enzymatic activities related to the synthesis of

60 glycogen are attenuated or completely eliminated, to the extent that the organism continues to remain easily in a viable state and is robust under fermentation conditions. Accordingly, the cell of the present invention may be at least attenuated in enzymatic activities including, without limitation: glucose-1-phosphate adenyltransferase (EC 2.7.7.27), glycogen synthase (EC 2.4.1.21 and EC 2.4.1.11), glucose-1-phosphate uridylyltransferase (EC 2.7.7.9) and branching enzyme 1,4-alpha-glucan (EC 2.4.1.18).

65 In certain aspects for the production of ethanol, the carbon that is available from the CO2 fixation is directed to pyruvate as efficiently as possible. Cyanobacteria can synthesize some pyruvate from carbon fixation during illumination, using glyceraldehyde-3-phosphate derived from the Calvin cycle, because they must still perform biosynthetic precursors from it. However, they do so only to the extent that

5 require for growth. To increase the flow to pyruvate from the intermediates of the Calvin cycle, it is desirable to constitutively express the genes encoding glycolytic enzymes, from the native host or from a non-native host. The choice of genes is based on whether allosteric regulatory effects are projected to prevent them from exercising their full activities in the expected metabolic context of the host. Constitutive capacity could be achieved by eliminating transcriptional regulation where it exists, or by expressing enzymes from constitutive promoters with which they are not normally associated. Accordingly, the cell of the present invention may comprise additional enzyme activities including, but not limited to: glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.12 or EC 1.2.1.13), phosphoglycerate kinase (EC 2.7.2.3), phosphoglycerate mutase (EC 5.4.2.1), enolasa (EC 4.2.1.11) and pyruvate kinase (EC 2.7.1.40).

The cell of the present invention may also comprise additional enzymatic activities for the conversion of pyruvate to ethanol. In certain embodiments, said conversion can be carried out by at least four different routes: 1) the pyruvate decarboxylase route, 2) the pyruvate dehydrogenase route, 3) the pyruvate oxidase route and 4) the route of the pyruvate-lyase format. The enzymatic activities required for the pyruvate decarboxylase pathway are: pyruvate decarboxylase (EC 4.1.1.1) and alcohol dehydrogenase (EC 1.1.1.1 or EC 1.1.1.2). The enzymatic activities required for the pyruvate dehydrogenase route are: acetaldehyde dehydrogenase (EC 1.2.1.10) and alcohol dehydrogenase (EC 1.1.1.1 or EC 1.1.1.2). The enzymatic activities required for the pyruvate oxidase route are: pyruvate oxidase (EC 1.2.2.2), acetyl-CoA synthetase (EC 6.2.1.1), acetaldehyde dehydrogenase (EC 1.2.1.10) and alcohol dehydrogenase (EC 1.1.1.1 or EC 1.1.1.2). The enzymatic activities required for the pyruvate-lyase format route are: pyruvate-lyase format (EC 2.3.1.54), format

25 hydrogen-lyase (no EC number), acetaldehyde dehydrogenase (EC 1.2.1.10), and alcohol dehydrogenase (EC

1.1.1.1 or EC 1.1.1.2). Preferably, one or more of these routes is expressed constitutively or under some other controlled regulation.

In addition to providing exogenous genes or endogenous genes with new regulation, the optimization of ethanol production in microorganisms preferably requires the elimination or attenuation of certain host enzymatic activities. These include, but are not limited to, pyruvate oxidase (EC 1.2.2.2), D-lactate dehydrogenase (EC 1.1.1.28), acetate kinase (EC 2.7.2.1), phosphate acetyltransferase (EC 2.3.1.8), citrate synthase (EC 2.3 .3.1), phosphoenolpyruvate carboxylase (EC 4.1.1.31). The degree to which these manipulations are necessary is determined by the observed by-products found in the bioreactor or shake flask. For example,

35 the observation of acetate would suggest the suppression of enzymatic activities pyruvate oxidase, acetate kinase and / or phosphotransacetylase. In another example, the observation of D-lactate would suggest the suppression of enzymatic activities D-lactate dehydrogenase, while the observation of succinate, malate, fumarate, oxaloacetate or citrate would suggest the suppression of the enzymatic activities citrate synthase and / or PEP carboxylase.

Ethanol production in light-dark cycle

In alternative embodiments, the present invention is adapted so that microorganisms are used in systems that are suitable for processing with a light-dark cycle, in which several hours of constant illumination are followed by several hours of relative darkness. Using said cycle, the complete elimination of the

The ability to synthesize glycogen may not be a viable strategy, because the cells will require some glycogen to survive the dark period. In this case, one of two alternative strategies can be implemented: 1) attenuation, but not elimination, of glycogen synthesis enzymes, so that some glycogen is still made during the light phase; or 2) maximization of glycogen production during the light phase.

In one embodiment, the microorganisms are attenuated but not eliminated in glycogen synthesis enzyme activities. Such methods are implemented by suppressing the activities of the native glycogen synthesis gene or genes and replacing them with analogs that have non-native regulation and expression at a lower level than in the native host.

55 In another embodiment, microorganisms maximize glycogen production during the light phase. Such methods are implemented by scanning for strains with the highest glycogen content among a collection of wild type strains or a library of mutants of a particular strain, after which strains have been cultured to an adequate cell concentration and have been subjected subsequently to the limitation of a key nutrient such as nitrogen, phosphorus or potassium. It would be more advantageous to use a light-dark cycle so that the glycogen is synthesized at the maximum possible amount with respect to dry weight of the cells during the light cycle, then ethanol is almost completely metabolized as possible during the cycle of darkness.

During the dark cycle, glycogen is converted into pyruvate by endogenous enzymes, but, as with

65 continuous illumination, these enzymes can be regulated in the host so that the overall conversion happens at a suboptimal rate. To increase the rate of this conversion, said regulation is counteracted, either by mutating and exploring strains with respect to rapid use of glycogen in the dark or by providing the host with the necessary enzymes at higher levels of expression and / or by providing exogenous genes encoding enzymes that are less subject to allosteric regulation than those of the host. Consequently, preferred enzyme activities to achieve this effect include those listed above for

5 conversion of glyceraldehyde-3-phosphate into ethanol, in addition to enzymatic activities that convert glycogen to glyceraldehyde-3-phosphate: glycogen phosphorylase (EC 2.4.1.1), phosphoglucomutase (EC 5.4.2.2), glucose-6-phosphate isomerase (EC 5.3.1.9), phosphofructokinase (EC 2.7.1.11), fructose-6-phosphate aldolase (EC 4.1.2.13) and triosaphosphate isomerase (EC 5.3.1.1).

In yet another embodiment, at least one cytochrome oxidase activity is attenuated or functionally suppressed. Cytochrome oxidases act to transfer electrons to oxygen in the dark. Howitt et al., Performed cytochrome oxidases deletions (CtaI, CtaII and Cyd) and were able to obtain strains that did not breathe in darkness but grow normally in the light. (Quinol and Cytochrome Oxidases in the Cyanobacterium Synechocystis sp. PCC 6803, [Howitt et al., Biochemistry (1998) 37 (51): 17944-51]). Strains that lacked an oxidase breathed

15 at almost wild rates, while those lacking both CtaI and Cyd did not breathe. The inability to breathe in the dark means more fermentative products, including ethanol and perhaps also succinate as well as 1,4-butanediol. Accordingly, the present invention provides a carbon-fixing organism, for example, a cyanobacterium obtained by genetic engineering in which at least one cytochrome oxidase activity is attenuated, which increases the production of ethanol, succinate and / or 1,4-butanediol. .

Increase in fatty acid production

The introduction of heterologous nucleic acid sequences involved in a biosynthetic pathway for hydrocarbon production can be carried out stably or transiently in various host cells using

25 techniques well known in this field, for example, electroporation, precipitation with calcium phosphate, transfection mediated by DEAE-dextran, transfection mediated by liposomes, conjugation and transduction. For stable transformation, a DNA sequence may also include a selectable marker, such as antibiotic resistance, for example resistance to neomycin, tetracycline, chloramphenicol, kanamycin and genes that complement auxotrophic deficiencies.

Expression control sequences suitable for use in prokaryotic host cells include, but are not limited to, promoters capable of recognizing T4, T3, SP6 and T7 polymerases, PR and PL promoters of bacteriophage lambda, trp, recA, promoters. E. coli thermal shock and lacZ, alpha-amylase promoters and sigma-specific B. subtilis, Bacillus bacteriophage promoters, promoters of

Streptomyces, the int promoter of the bacteriophage lambda, the bla promoter of the beta-lactamase gene of pBR322, and the CAT promoter of the chloramphenicol acetyl transferase gene. Prokaryotic promoters are reviewed in Glick, J. Ind. Microbiol. 1: 277, 1987; Watson et al, MOLECULAR BIOLOGY OF THE GENES 4th Ed., Benjamin Cummins (1987); and Sambrook et al., mentioned above.

Non-limiting examples of eukaryotic promoters suitable for use within a eukaryotic host are those of viral origin and include the promoter of the mouse metallothionein I gene (Hamer et al., J. MoI. Appl. Gen. 1: 273, 1982 ); the Herpes virus TK promoter (McKnight, Cell 31: 355, 1982), the SV40 early promoter (Benoist et al, Nature (London) 290: 304, 1981), the Rous sarcoma virus promoter; the cytomegalovirus promoter (Foecking et al, Gene 45: 101, 1980); the yeast gal4 gene promoter (Johnston, et al, PNAS (USA) 79: 6971,

45 1982; Silver et al, PNAS (USA) 81: 5951, 1984); and the IgG promoter (Orlandi et al, PNAS (USA) 86: 3833, 1989).

In some examples a genetically modified host cell is genetically modified with a heterologous DNA sequence that encodes a biosynthetic pathway gene product that is operably linked to a constitutive promoter. Suitable constitutive promoters are known in the art and include the major late constitutive adenovirus promoter, a constitutive MPSV promoter and a constitutive CMV promoter. Suitable constitutive promoters applicable to Synechococcus sp. PCC 7002 include, for example, PtacI, P-EM7, Paph2 and PaadA.

The microbial host cell can be genetically modified with a nucleic acid sequence

A heterologous encoding a biosynthetic pathway gene product that is operably linked to an inducible promoter. Inducible promoters are well known in the art. Suitable inducible promoters include, but are not limited to, promoters that are affected by proteins, metabolites or chemicals. These include: a bovine leukemia virus promoter, a metallothionein promoter, a dexamethasone inducible MMTV promoter, an SV40 promoter, a polyIII MRP promoter, a tetracycline inducible CMV promoter (such as the immediate CMV promoter - human early), as well as those of the trp and lac operons.

When a host cell is genetically modified with heterologous nucleic acid sequences encoding two or more proteins involved in a biosynthetic pathway to produce carbon-based products of interest, the nucleic acid sequences can be directed by a single promoter in a single vector or at least one

65 promoter in separate expression vectors.

The intracellular concentration (for example, the concentration of the intermediate in the genetically modified host cell) of the biosynthetic pathway intermediate can be increased to further enhance the yield of the final product. For example, increasing the intracellular amount of a substrate (for example, a primary substrate) for an enzyme that is active in the biosynthetic pathway and the like.

Carbon Chain Modifications

Figure 1 provides a description of the various genes that can be modulated to alter the structure of the product derived from fatty acids and the encoded enzymes that can be used alone or in combination to make various fatty acids and hydrocarbons. The products can be produced in such a way that they contain branching points, saturation levels and carbon chain length, thus making these products desirable starting materials for use in many applications. Various carbon-based products of interest produced by microorganisms are described.

Figure 1 also lists enzymes that are directly involved in the synthesis of carbon-based products, including waxes, fatty acid esters and / or fatty alcohols. To increase the production of fatty acid esters / waxes, and fatty alcohols, one or more of the enzymes can be overexpressed or mutated to reduce feedback inhibition. Additionally, enzymes that metabolize intermediates to produce non-fatty acid based products (side reactions) can be functionally suppressed or attenuated to increase carbon flow through the biosynthetic pathway of fatty acids. The examples provided herein describe how to genetically engineer enzymes in the respective routes of host organisms to produce genetically engineered organisms that produce ethanol.

Photo-fermentation consolidated

25 In an alternative approach to directly produce carbon-based product of final interest as a result of photosynthesis, carbon-based products of interest can be produced by promoting other organisms that are more susceptible to making any particular product while cultivating the photosynthetic organism at the same time. Your carbon source. Consequently, the fermentation and production of carbon-based products of interest can be carried out separately from the production of carbon source in a photobioreactor.

The methods for producing said carbon-based products of interest may include two stages. The first stage includes photosynthetic organisms to convert carbon dioxide into photosynthetic products such as glucose. The second stage is to use photosynthetic products as a carbon source for cells that

35 produce carbon-based products of interest. The two stage approach may comprise a photobioreactor comprising photosynthetic cells; a second reactor comprising cells capable of fermentation; in which photosynthetic cells provide a carbon source such as glucose for fermentation capable cells to produce a carbon based product of interest. The second reactor may comprise more than one type of microorganism. The resulting carbon-based products of interest are separated and / or collected later.

The two stages can be combined in a single stage procedure whereby genetically engineered photosynthetic organisms convert light and CO2 directly into glucose and these organisms are capable of producing a variety of carbon-based products of interest.

Certain changes in the culture conditions of photosynthetic host cells, for example, cyanobacteria for sugar production can be optimized with respect to growth. For example, conditions are optimized with respect to light intensity, exposure to light, exposure time, daytime cycle, addition of supplements, nutrients, recirculation speed and flow rates that maintain a relationship of light and darkness. As will be apparent to those skilled in the art, sufficient conditions to achieve optimum growth will vary depending on the location, climate and other environmental factors, such as the day cycle, light intensity and time of exposure to light. Other adjustments may be required, for example, the ability of an organism to capture carbon. Increased carbon in the form of CO2 can be introduced into a bioreactor by gas sprinklers or aeration devices.

55 The advantages of consolidated photo-fermentation include a process in which there is separation of chemical end products, for example, glucose, spatial separation between end products (membranes) and time. Additionally, unlike the traditional or cellulosic production of biomass to biofuels, pretreatment, saccharification and cultivation plow are avoided.

The consolidated photo-fermentation process produces continuous products. The process may involve direct capture of light to product from initial organisms engineered to produce various products without the need to lyse the organisms. For example, organisms can use 3PGAL in light to make a desired fermentation product, for example, ethanol. As an alternative, the organisms

65 can accumulate glycogen in the light and metabolize ethanol in the dark to make more fermentation products. Such end products can be easily secreted unlike intracellular products such as oil and cellulose. Organisms can produce light sugars, which are secreted into the medium and said sugars are used in the dark during fermentation with the same or different organisms or a combination of both.

Fermentation conditions

5 The production and isolation of carbon-based products of interest can be enhanced using specific fermentation techniques. One method to maximize production by reducing costs is to increase the percentage of carbon that is converted into hydrocarbon products. During normal cell life cycles carbon is used in cellular functions including production of lipids, saccharides, proteins, organic acids and nucleic acids. Reducing the amount of carbon needed for growth-related activities can increase the efficiency of the conversion from carbon source to product. This can be achieved by first cultivating microorganisms to a desired density, such as a density achieved at the peak of the logarithmic growth phase. At that point, the replication control point genes can be exploited to stop the growth of the cells. Specifically, quorum detection mechanisms may be used [reviewed in

15 Camilli and Bassler, Science 311: 1113, (2006); Venturi FEMS Microbio Rev 30: 274-291 (2006); and Reading and Sperandio, FEMS Microbiol Lett, 254: 1-11, (2006)] to activate genes such as p53, p21 or other control point genes. Genes that can be activated to stop replication and cell growth in E. coli include the umuDC genes, whose overexpression stops stationary phase progression to exponential growth [Murli et al, J.of Bact., 182: 1127, (2000) ]. UmuC is a DNA polymerase that can carry out translesion synthesis on non-coding lesions, the mechanical basis of most chemical and UV mutagenesis. The umuDC gene products are used for the translesion synthesis procedure and also act as a DNA damage control point. UmuDC gene products include UmuC, UmuD, UmuD ', UmuD'2C, UmuD'2 and UmUD2. Simultaneously, the product producing genes are activated, thus minimizing the need for replication and maintenance routes to use while the fatty acid derivative is being performed.

In one aspect, the percentage of input carbons converted to hydrocarbon products is an efficient and economical process. Using carbon dioxide as the carbon source, oxygen is released in the form of O2, which leads to a maximum theoretical metabolic efficiency of ~ 34% (w / w) (for products derived from fatty acids).

This figure, however, changes for other hydrocarbon products and carbon sources. Typical efficiencies in the literature are ~ <5%. Genetically engineered microorganisms that produce hydrocarbon products may have more than 1, 3, 5, 10, 15, 20, 25 and 30% efficiency. In one example the microorganisms will show an efficiency of about 10% to about 25%. In other examples, said microorganisms will show an efficiency of about 25% to about 30%, and in others

35 examples such microorganisms will show> 30% efficiency.

In some examples when the final product is released from the cell, a continuous procedure can be employed. In this approach, a reactor with organisms that produce fatty acid derivatives can be assembled in multiple ways. In one example, a part of the medium is removed and allowed to separate. The fatty acid derivatives are separated from the aqueous layer, which in turn will be returned to the fermentation chamber.

In another example, the fermentation chamber will include a fermentation that undergoes continuous reduction. In this case, a stable reducing environment would be created. The electronic balance would be maintained by the release of oxygen. Attempts to increase the balance of NAD / H and NADP / H can also facilitate stabilization of the

45 electronic balance.

The availability of intracellular NADPH can also be enhanced by genetically engineered the production host to express an NADH: NADPH transhydrogenase. The expression of one or more NADH: NADPH transhydrogenases converts the NADH produced in glycolysis to NADPH that enhances the production of fatty acid derivatives.

For large-scale product production, genetically engineered microorganisms are grown in batches of 10 l, 100 l or larger, fermented and induced to express desired products based on specific genes encoded in plasmids as appropriate. Cells that harbor nucleic acids are incubated

55 genetically engineered to overexpress or attenuate gene products from a 500 ml planting culture for 10 l fermentations (5 l for 100 l fermentations) in LB medium (without glycerol) at 37 ° C stirred at> 200 rpm until the cultures reach a desired OD (typically 16 hours) incubated with kanamycin, ampicillin or the like. The medium is continuously supplemented to maintain a 25 mM sodium propionate at a suitable pH of approximately 8.0 to activate genetically engineered gene systems for production as well as to stop cell proliferation. The medium is continuously supplemented with carbon dioxide. Aliquots of no more than 10% of the total cell volume are removed every hour and allowed to stand without agitation to allow the hydrocarbon product to rise to the surface and experience a spontaneous phase separation. The hydrocarbon component is then collected and the aqueous phase is returned to the reaction chamber. The reaction chamber works continuously. For the production of

After wax isolation, after isolation, the wax esters are briefly washed in 1M HCl to break the ester bond and returned to pH 7 with thorough washing with distilled water.

Processing and separation

Carbon-based products produced by carbon dioxide fixing organisms during fermentation can be separated from fermentation media. Known techniques can be used to separate

5 fatty acid derivatives of aqueous media. An exemplary separation procedure provided herein is a two-phase (two-phase) separation procedure. This process involves fermenting the genetically engineered production hosts under conditions sufficient to produce, for example, a fatty acid, allowing the fatty acid to be collected in an organic phase and separating the organic phase from the aqueous fermentation medium. This method can be practiced in a situation of both discontinuous and continuous fermentation.

Biphasic separation uses the relative immiscibility of fatty acid to facilitate separation. One skilled in the art will appreciate that by choosing a fermentation medium and the organic phase so that the fatty acid derivative that is produced has a high logP value, even at very low concentrations the fatty acid will separate into

15 the organic phase in the fermentation bottle.

When fatty acids are produced by the methods described herein, said products will be relatively immiscible in the fermentation medium, as well as in the cytoplasm. Therefore, the fatty acid will be collected in an organic phase either intracellularly or extracellularly. The collection of products in an organic phase will reduce the impact of the fatty acid derivative on cellular function and allow the production host to produce more product.

Fatty alcohols, fatty acid esters, waxes and hydrocarbons produced as described herein allow the production of homogeneous compounds with respect to other compounds in which at

25 minus 50%, 60%, 70%, 80%, 90% or 95% of the fatty alcohols, fatty acid esters, waxes and hydrocarbons produced have carbon chain lengths that vary by less than 4 carbons, or less of 2 carbons. These compounds can also be produced so that they have a relatively uniform degree of saturation with respect to other compounds, for example at least 50%, 60%, 70%, 80%, 90% or 95% of the fatty alcohols, esters of Fatty acids, hydrocarbons and waxes are mono, di or tri-unsaturated.

Detection and analysis

Generally, the products of interest produced from the "solar bio-factories" described herein can be analyzed by any of the conventional analytical methods, for example, chromatography.

35 gas (GC), mass spectrometry (MS), gas chromatography-mass spectrometry (GCMS), and liquid chromatography-mass spectrometry (LCMS), high performance liquid chromatography (HPLC), capillary electrophoresis, spectrometry mass desorption and laser ionization assisted by matrix-time-of-flight (MALDI-TOF MS), nuclear magnetic resonance imaging (NMR), near infrared spectroscopy (NIR), viscometry [Knothe et al., Am. Chem. Soc. Sump. . Series, 666: 172-208 (1997)], titration to determine free fatty acids [Komers et al., Fett / Lipid 99 (2): 52-54 (1997)], enzymatic methods [Bailer et al, J Anal. Chem. 340 (3): 186 (1991)], methods based on physical properties, wet chemical methods, etc.

Carbon identification

45 Biologically produced carbon-based products, for example, ethanol, fatty acids, alkanes, isoprenoids, represent a new raw material for fuels, such as alcohols, diesel and gasoline. These biofuels have not been produced using biomass but use CO2 as their carbon source. These new fuels can be distinguished from petrochemical carbon derived fuels based on the isotopic identification of double carbon. Said products, derivatives and mixtures thereof can be completely distinguished from their counterparts derived from petrochemical products based on the isotopic identification of double carbon and 14C (fM), indicating new compositions of matter.

There are three isotopes of natural carbon origin: 12C, 13C and 14C. These isotopes appear in total carbon on the ground in fractions of 0.999, 0.011, and 10-12, respectively. The 12C and 13C isotopes are stable, while 55 14C naturally degrades to 14N, a beta particle, and an anti-neutrino in a process with a half-life of

5,730 years. The 14C isotope originates in the atmosphere, mainly due to the 14N neutron bombardment ultimately caused by cosmic radiation. Due to its relatively short half-life (in geological terms), 14C appears at extremely low levels in fossil carbon. During the course of 1 million years without exposure to the atmosphere, only 1 part in 1050 will remain 14C.

The 13C: 12C ratio varies slightly but measurably between natural carbon sources. Generally these differences are expressed as deviations from the ratio of 13C: 12C in a conventional material. The international standard for carbon is the Belemnite Pee Dee, a form of limestone found in South Carolina, with a 13C fraction of 0.0112372. For a carbon source a, the deviation of the 13C: 12C ratio from that of the Belemnite

Pee Dee 65 is expressed as: ∃a = (Ra / Rs) -1, where Ra = 13C: 12C ratio in the natural source, and Rs = 13C: 12C ratio in Belemnite Pee Dee, the pattern.

For convenience, ∃a is expressed in parts per thousand, or ‰. A negative value of ∃a shows a deviation towards 12C over 13C compared to the Belemnite Pee Dee. Table 1 shows ∃a and the fraction of 14C for various natural carbon sources.

Table 1:

Variations of 13C: 12C in natural carbon sources

Source

- to (‰) References

Underground carbon

32.5 Farquhar et al.

Fossil fuels

26 Farquhar et al.

DEC * Oceanic

0-1.5 Goericke et al.

Ivlev

Atmospheric CO2

6-8 Ivlev, Farquhar et al.

DEC * of fresh water

6-14 Dettman et al.

Belemnite Pee Dee

0 Ivlev

* DEC = dissolved inorganic carbon

10 Biological processes often differentiate between carbon isotopes. The natural abundance of 14C is very small, and therefore the differentiation for or against 14C is difficult to measure. The biological differentiation between 13C and 12C, however, is well documented. For a biological product p, quantities similar to the previous ones can be defined:

15 ∃p = (Rp / Rs) -1, in which Rp = 13C: 12C ratio in the biological product, and Rs = 13C: 12C ratio in the Belemnite Pee Dee, the pattern.

Table 2 shows the deviations measured in the 13C: 12C ratio for some biological products.

20 Table 2: 13C: 12C variations in selected biological products

Product

- p (‰) -D (‰) * References

Atmospheric CO2 / vegetable starch

18-28 10-20 Ivlev

Marine DIC cyanobacterial biomass

18-31 16.5-31 Goericke et al., Sakata et al.

Marine DIC cyanobacterial lipid

39-40 37.5-40 Sakata et al.

Marine DIC Seaweed Lipid

17-28 15.5-28 Goericke et al., Abelseon et al.

Freshwater DIC seaweed biomass

17-36 3-30 Marty et al.

E.coli vegetable sugar lipid

15-27 almost 0 Monson et al.

Fossil carbon cyanobacterial lipid

63.5-66 37.5-40 -

Fossil carbon cyanobacterial biomass

42.5-57 16.5-31 -

* D = differentiation by a biological process in its use of 12C versus 13C (see text)

Table 2 introduces a new quantity, D. This is the differentiation by a biological process in its use of 12C versus 13C. The inventors define D as follows: D = (Rp / Ra) -1.

25 This amount is very similar to ∃a and ∃p, except that the biological product is now compared directly with the carbon source rather than with a standard. Using D, the deviation effects of a carbon source and a biological process can be combined to obtain the biological product deviation compared to the pattern. Solving for ∃p, you get: ∃p = (D) (∃a) + D + ∃a, and, because (D) (∃a) is generally very small compared to the other terms, ∃p≈ ∃a + D.

For a biological product that has a production process with a known D, it can therefore be estimated ∃p by adding ∃a and D. It is assumed that D acts independently of the carbon source.

This has been done in Table 2 for cyanobacterial lipid and biomass produced from fossil carbon. How

35 shown in the above tables, cyanobacterial products made from fossil carbon (in the form of, for example, exhaust gas or other emissions) will have a ∃p greater than those of comparable biological products made from other sources, distinguishing them based on in the composition of the matter of these other biological products. In addition, any product derived only from fossil carbon will have a negligible fraction of 14C, while products made from carbon on the ground will have a fraction of 14Cde

40 approximately 10-12.

Consequently, various carbon-based products of interest can be characterized as -∃p (‰) from about 63.5 to about 66 and -D (‰) from about 37.5 to about 40.

References

one.

Goericke, R., Montoya, J.P., and Fry, B. Physiology of isotopic fractionation in algae and cyanobacteria. Chapter 9 in "Stable Isotopes in Ecology and Environmental Science", by K. Lajtha and R. H. Michener, Blackwell Publishing, 1994.

2.

Monson, K.D. and Hayes, J.M. Biosynthetic control of the natural abundance of carbon 13 at specific positions within fatty acids in Escherichia coli. J. Biol. Chem. 255: 11435-41 (1980).

3.

Abelseon, P.H. and Hoering, T.C. Carbon isotope fractionation in formation of amino acids by photosynthetic organisms. Proc. Natl. Acad. Sci. 47: 623-32 (1961).

Four.

Sakata, S., Hayes, J.M., McTaggart, A.R., Evans, R.A., Leckrone, K.J., and Togasaki, R.K. Isotopic carbon

fractionation associated with lipid biosynthesis by a cyanobacterium: relevance for interpretation of biomarker 15 records. Geochim Cosmochim Minutes 61: 5379-89 (1997).

5.

Ivlev, A.A. Carbon isotope effects (13C / 12C) in biological systems. Separation Sci. Technol. 36: 1819-1914 (20010).

6.

Farquhar, G. D., Ehleringer, J. R., and Hubick, K. T. Carbon isotope discrimination and photosynthesis. Annu Rev. Plant Physiol. Plant Mol. Biol. 40: 503-37 (1989).

7.

Marty, J. and Planas, D. Comparison of methods to determine algal ∃13C in freshwater. Limnol Oceanogr .: Methods 6: 51-63 (2008).

8.

Dettman, D. L., Reische, A. K., and K. C. Lohmann. Controls on the stable isotope composition of seasonal growth bands in aragonitic fresh-water bivalves (unionidae). Geochim Cosmochim Minutes 63: 1049-1057 (1999).

25 All publications and patent documents cited herein are hereby incorporated by reference in their entirety for all purposes to the same extent as if each were so indicated individually.

Examples

Subsequent examples are provided herein for illustrative purposes and are not intended to be restrictive.

EXAMPLE 1 Plasmid construction for Synechococcus sp. PCC 7002

Construction of pJB5: The base plasmid pJB5 was designed as an empty expression vector for recombination in Synechococcus sp. PCC 7002. Two homology regions, the upstream homology region (UHR) and the downstream homology region were designed to flank the construction. These 500 bp homology regions correspond to positions 3301-3800 and 3801-4300 (Genbank reference NC_005025) for UHR and DHR respectively. The aadA promoter, gene sequence and terminator were designed to confer resistance to streptomycin and spectinomycin to the integrated construct. For expression, pJB5 was designed with the promoter of the kanamycin resistance cassette aph2 and the ribosome binding site (RBS). String down this

45 promoter and RBS, the inventors designed and inserted the restriction endonuclease recognition site for NdeI and EcoRI, as well as the sites for XhoI, BamHI, SpeI and PacI. After the EcoRI site, the natural terminator of the Zymomonas mobilis alcohol dehydrogenase (adhII) gene was included. Restriction sites of xbaI flank the UHR and DHR allowing the cleavage of the DNA that is intended to be used for recombination of the rest of the vector. PJB5 was constructed by contractual synthesis of DNA2.0 (Menlo Park, CA).

Construction of pJB5-PdcAdhII. The pyruvate decarboxylase (pdc) and alcohol dehydrogenase (adhII) genes were cloned into plasmid pJB5 with the following procedure. The pdc-adhII genes of Zymomonas mobilis (GenBank: DD161475, M15394) were designed with an NdeI site replacing the start of the pdc coding region. After the PDC gene, the inventors designed two restriction endonuclease sites (XhoI and BamHI). TO

Then, the complete adhII sequence subsequent to the restriction sites was designed and finally the natural adhII terminator, downstream of an inserted EcoRI site, was also included. This construct was constructed by contractual synthesis of DNA2.0 (Menlo Park, CA) and was inserted by restriction digestion with NdeI and EcoRI (New England Biolabs; Ipswitch, MA) in both pJB5 and the insert followed by ligation with a Quick kit Ligation (New England Biolabs, Ipswitch, MA). The linked construction was transformed into the competent E. coli F’Iq NEB 5-alpha (high efficiency) (New England Biolabs: Ipswitch, MA).

pJB5-PdcAdhII (TS): The pyruvate decarboxylase (pdc) genes of Zymobacter palmae (GenBank: AF474145) and alcohol dehydrogenase TS42 (adhII) as described in Rellos et al. (1998) "Thermostable variants of Zymomonas mobilis alcohol dehydrogenase obtained using PCR-mediated random mutagenesis" Protein Expr Purif 65 12: 61-61) were cloned into plasmid pJB5 with the following procedure. These genes were designed with an NdeI site replacing the start of the pdc coding region. After the pdc gene and before the adhII gene, it is present

a hole that includes XhoI and BamHI sites to allow promoters to be inserted later (total length of the hole: 39 bp) and the original RBS for adhII of Z. mobilis. The adhII gene (Z. mobilis) has the original terminator present afterwards, in which an EcoRI site has been located between the adhII gene and the terminator. After the terminator, SpeI and PacI sites were present for cloning. This construction was constructed by contractual synthesis of

5 DNA2.0 (Menlo Park, CA) and was inserted by restriction digestion with NdeI and EcoRI (New England Biolabs; Ipswitch, MA) both in pJB5 and in the insert followed by ligation with a Quick Ligation kit (New England Biolabs, Ipswitch, MA). The linked construction was transformed into the competent E. coli F'Iq NEB 5-alpha (high efficiency) (New England Biolabs, Ipswitch, MA).

pJB5-Pdc: The pyruvate decarboxylase (pdc) gene was cloned into plasmid pJB5 with the following procedure. The construction of pJB5-PdcAdhII of Example 2 was digested with BamHI and EcoRI (New England Biolabs; Ipswitch, MA). Incompatible 5 'and 3' DNA projections were removed using the Quick Blunting kit (New England Biolabs, MA), and then ligated using the Quick Ligation kit (New England Biolabs; Ipswitch, MA).

15 pJB5-AdhII: The alcohol dehydrogenase (adhII) gene was cloned into plasmid pJB5 with the following procedure. The construction of pJB5-PdcAdhII of Example 2 was digested with NdeI and BamHI (New England Biolabs; Ipswitch, MA). Incompatible 5 'and 3' DNA projections were removed using the Quick Blunting kit (New England Biolabs, MA) and then ligated using the Quick Ligation kit (New England Biolabs; Ipswitch, MA).

pJB5-metE (E. coli): The methionine synthase (metE) gene independent of vitamin B12 from E. coli (GenBank: NP_418273.1), was cloned into plasmid pJB5 by the following procedure. A construct was synthesized by contractual synthesis by DNA2.0 (Menlo Park, CA) to include an NdeI site to replace the start of the metE gene, and an EcoRI site at the end of the gene. This construct was inserted by restriction digestion with NdeI and EcoRI (New England Biolabs; Ipswitch, MA) in both pJB5 and the insert followed by ligation with a Quick Ligation kit (New

25 England Biolabs; Ipswitch, MA). The linked construct was transformed into the competent E. coli F'Iq NEB 5-alpha (high efficiency) (New England Biolabs: Ipswitch, MA).

pJB5-metE (T. elongates BP-1): The vitamin B12 methionine synthase (metE) gene independent of Thermosynechococcus elongates BP-1 (GenBank: NP_681881), was cloned into plasmid pJB5 by the following procedure. A construct was synthesized by contractual synthesis by DNA2.0 (Menlo Park, CA) to include an NdeI site to replace the start of the metE gene and an EcoRI site at the end of the gene. This construct was inserted by restriction digestion with NdeI and EcoRI (New England Biolabs; Ipswitch, MA) both in pJB5 and in the insert followed by ligation with a Quick Ligation kit (New England Biolabs; Ipswitch, MA). The linked construct was transformed into the competent E. coli F'Iq NEB 5-alpha (high efficiency) (New England Biolabs: Ipswitch, MA).

EXAMPLE 2

Plasmid construction for Thermosynechococcus elongatus BP-1

Thermosynechococcus elongatus BP-1 was selected as another exemplary CO2 fixative production host and modified by genetically engineered nucleic acids to functionally suppress certain genes and / or to express, overexpress certain genes.

Four plasmids (pJB518, pJB59, pJB20 and pJB21), all derived from pJB5, were constructed to allow

Homologous recombination in four different loci in the genome of Thermosynechococcus elongatus BP-1. Specifically, the 0.5 kb regions of upstream (UH) and downstream (DH) homology used for homologous recombination of Synechococcus sp. PCC 7002 in pJB5 were replaced by the following approximately 2.5 kb regions of T. elongatus BP-1 (reference NC_004113): coordinates 831908-834231 (UH) and 834232-836607 (DH) of the genome for pJB18, 454847-457252 (UH) and 457252-459740 (DH) for pJB19, 481310-483712 (UH) and 483709-486109 (DH) for pJB20 and 787356-789654 (UH) and 791080-793494 (DH) for pJB21. The first three homology regions are based on the TS1, TS3 and TS4 integration sites described in Onai K. et al. (2004). "Natural transformation of the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1: a simple and efficient method for gene transfer." Mol. Gen. Genomics 271: 50-59. The latter is designed to completely suppress the open reading phase of glgA, which encodes glycogen synthase: The purpose of this deletion is

55 Minimize the flow of competitively bound carbon to glycogen once the ethanol producing genes have been integrated into the chromosome.

All homology regions of T. elongatus BP-1 were generated by PCR using Phusion ™ hot-boot high-fidelity DNA polymerase (developed and manufactured by Finnzymes Oy. Distributed by New England Biolabs, Ipswitch, MA) according to manufacturer's instructions The UH direct PCR primer has a 5'-terminal SbfI restriction site, the UH reverse PCR primer, a 5 'terminal NotI restriction site, the DH direct PCR primer, a 5'-terminal AscI restriction site and the DH reverse PCR primer, a 5'-terminal FseI restriction site. For pJB18, pJB59, pJB20 and pJB21, the UH region is first inserted into pJB5 by restriction digestion with SbfI and NotI (New England Biolabs, Ipswitch, MA) of both vector 65 and PCR generated insert, followed by a kit Quick Ligation (New England Biolabs; Ipswitch, MA). The linked construct is transformed into the competent E. coli NEB 5-alpha (high efficiency) (New England Biolabs: Ipswitch, MA). The

UH region sequence in pHB5 is validated by contractual sequencing with GENEWIZ (South Plainfield, NJ). For pJB18, pJB19, pJB20 and pJB21, the DH region is then inserted into the construction of pJB5-UH region exactly as it has been done for the UH region, except that the AscI and FseI restriction enzymes (New England Biolabs, Ipswitch, are used, MA). The sequence of the DH regions is confirmed by contractual sequencing with

5 GENEWIZ (South Plainfield, NJ).

In each of pJB18, pJB19, pJB20 and pJB21, two different versions of the pyruvate decarboxylase (pdc) / alcohol dehydrogenase (adhII) operons are cloned, creating a set of eight plasmids ready for integration into the T. elongatus BP genome. -one. In each case, the selectable marker is the aadA gene of pJB5 encoding resistance to spectinomycin and streptomycin. The first version of the operon comprises the pdc and adhII genes of Zymomonas mobilis (GenBank: DD161475, M15394) and is designed with an NdeI site that encompasses the starting codon of the pdc coding sequence. Following the pdc gene in order are: an XhoI restriction site, a BamHI restriction site, the adhII coding sequence, the natural Zymomonas mobilis adhII terminator, and finally an EcoRI restriction site. The second version of the operon, designed to encode relatively more versions

15 thermostable pyruvate decarboxylase and alcohol dehydrogenase comprised the Zymobacter palmae (GenBank: AF474145) pdc gene and the TS42 adhII mutant described in Rellos et al., Protein Expr. Purif., 12: 61-61 (1998), and is otherwise identical to the first construction in everything else. Both constructs are performed by contractual synthesis of DNA2.0 (Menlo Park, CA) and inserted by restriction digestion with NdeI and EcoRI (New England Biolabs; Ipswitch, MA) in pJB18, pJB59, pJB20, and pJB21, followed by ligation with a Quick Ligation kit (New England Biolabs; Ipswitch, MA). In this way eight plasmids of operons pdc-adhII are constructed: pJB22, pJB23, pJB24 and pJB25 containing version 1 of the operon, based on pJB18, PJB19, pJB20 and pJB21, respectively, and pJB26, pJB27, pJB28 and pJB29 containing operon version 2, based on pJB18, pJB19, pJB20 and pJB21, respectively.

In plasmids pJB22, pJB23, pJB24, pJB25, pJB26, pJB27, pJB28 and pJB29, the pdc-adhII operon is expressed by the constitutive PaphI promoter, which is flanked by unique NotIy NdeI restriction sites. These sites allow cloning other constitutive promoters, instead of the PaphII promoter, in case that promoter does not provide sufficient expression of the operon when it is integrated into the genome of T. elongatus BP-1. Separate plasmids (pJB9, pJB10, pJB11, pJB12, pJB13, pJB14, pJB15, pJB16 and pJB17) are constructed, all performed by contractual synthesis, for example, DNA2.0 (Menlo Park, CA), each carrying nine alternative constitutive promoters Candidates flanked by NotIy NdeI sites so that they can replace the PaphII promoter with conventional cloning methods. Seven of these promoters are native T. elongatus BP-1 promoters, corresponding to the upstream sequence of the following genes: cpcC, apcA, tsr2142, psaA, rbcL, hsp33 and trnE_UUC and two are E. coli type promoters: Ptac (as described in De Boer et al., Proc Natl Acad USA 80: 21-25 (1983)) and the

35 synthetic PEM7 promoter.

EXAMPLE 3

Genetically engineered microorganisms that produce ethanol

Synechococcus sp. PCC 7002 genetically modified: Each of the constructs as described in Example 1 is integrated into the genome of Synechococcus sp. PCC 7002 using the following protocol. Synechococcus 7002 was cultured for 48 hours from colonies in a shake flask incubated at 30 ° C at 1% CO2 to an OD730 of 1 A + medium described in Frigaard NU et al. (2004) "Gene inactivation in the cyanobacterium

45 Synechococcus sp. PCC 7002 and the green sulfur bacterium Chlorobium tepidum using in vitro-made DNA constructs and natural transformation "Methods Mol Biol 274: 325-340. 500 #l of culture was added to a test tube with 30 #lde 1-5 #g of DNA prepared from a Qiagen Qiaprep agitation Miniprep kit (Valencia, CA) for each construct.The cells were incubated by bubbling 1% CO2 at approximately 1 bubble every 2 seconds for 4 hours 200 #l of cells were seeded in A + medium plates with 1.5% agarose and grown at 30 ° C for two days in low light, 10 # g / ml of spectinomycin was applied to the plates as a base Resistant colonies were visible in 7-10 days.

Strain construction and AdhA expression of Moorella sp. HUC22-1: It has been shown that the sequence for Moorella sp. HUC22-1 is an alcohol dehydrogenase that uses NADP that is also heat stable and

55 preferred for the reduction of acetaldehyde [Inokuma et al., Arch. Microbiol., 188: 37-45 (2007)]. Although the sequence has not been published, the similarity of amino acids with AdhIV of Moorella thermoacetica (Reference number: ABC20211) was 100%. The Moorella thermoacetica AdhIV nucleic acid sequence (Reference number: CP000232) had codons optimized for expression and was constructed by DNA 2.0 and designated SEQ ID No. 1 (the amino acid encoded is SEQ ID NO: 2). The sequence is flanked with CTCGAGTTGGATCC at the 5 'end, which encodes the restriction sites Xho and BamHI, and at the 3' end with TTTCAAAACAGGAATTC at the 3 'end (similar to pJB5-3) that contains an EcoRI site for the purposes of cloning into expression vectors.

Moorella adhA was then cloned downstream of two pyruvate decarboxylase genes, one from Zymomonas mobilis (reference number: AAV89984) and one from Zymobacter palmae (reference number: AAM49566) to form the expression plasmids pJB136 and pJB133, respectively. As controls, expression plasmids were constructed for the Z. mobilis pyruvate decarboxylase gene with the Z. mobilis adhII gene (number of

reference: YP_163331) and the Z. palmae pyruvate decarboxylase gene with an improved thermotolerant TS42 adhII [Rellos et al., Protein Expression and Purification, 12: 61-66 (1998)] To form pJB5-3 and pJB5-4, respectively.

Plasmids pJB5-3, pJB5-4, pJB133, pJB136 were cloned into Synechococcus sp. PCC 7002 (JCC1) using conventional procedures and were designated JCC136, JCC137, JCC445, JCC446 respectively (Table 3).

Table 3

Host Construction of Pyruvate decarboxylase Alcohol dehydrogenase integration

JCC136 pJB5-3 Z. mobilis pdc Z. mobilis adhII

JCC137 pJB5-4 Z. palmae pdc Z. mobilis adhII TS42

JCC445 pJB133 Z. palmae pdc Morella adhA

JCC446 pJB136 Z. mobilis pdc Morella adhA

JCC1, JCC136, JCC137, JCC445, JCC446 were plated with A + medium (1.5% agar) with 100 # g / ml spectinomycin for transgenic strains. A single colony was grown in 10 ml of A + with 100 # g / ml spectinomycin in a test tube immersed in a 37 ° C bath with 1% CO2 introduced by bubbling. Cultures were allowed to grow to OD730 nm 5.0 or greater (Molecular Devices Spectramax M2e; it was previously determined that an OD730 nm

15 of 1 is equal to ~ 0.3 g of CDW), and then sedimented by centrifugation (21000 RCF, 20 ° C, 5 minutes), suspended in fresh A + medium to the original concentration, and then appropriately re-diluted up to DO730 nm 0.2 in 25 ml of A + in a 125 ml shake flask with baffles. Approximately 1 ml of culture was taken for each time point (0, 6, 24, 48, 72 hours after dilution; the 6 hour time point is not represented for reasons of time spacing), OD730 nm (diluted from appropriate way to

20 provide a reading between 0.04 and 0.4, which was previously determined to be the most accurate range in the Spectramax M2e). The samples were immediately pelleted by centrifugation at 4 ° C for 10 minutes at 21,000 RCF. The supernatant was placed in a new tube, and frozen at -80 ° C until it was ready for analysis.

25 The supernatant of each time point was analyzed for ethanol and acetaldehyde using an Agilent 7890 gas chromatograph equipped with a head space analyzer and a flame ionization detector (Agilent) using a J&W Scientific DB -ALC1 (catalog number: 123-9134; length: 30 m, internal diameter, 0.320 mm, film thickness: 1.80 #m). 100 #l of each sample was subjected to head space analysis. Controls were measured only with respect to A + as well as serial dilution of standards for

30 ethanol and acetaldehyde obtained from Sigma to obtain a calibration curve.

To measure the optical densities, concentrations of ethanol and acetaldehyde, the cultures were back-diluted from OD730 nm 5 or more to an OD 730 nm starting and time points were taken at 0, 24, 48 and 72 hours after dilution.

35 The optical densities of cultures of Synechococcus sp. wild type and various transgenics (Figure 5). The graph shows representations of OD 730nm measurements at each time point. The resulting OD measurements are shown in Table 4.

Table 4

OD (730 nm)

Weather

0 24 48 72

JCC1

0.257 2.19 5.7 10.1

JCC136

0.259 2.26 5.06 7.6

JCC137

0.263 2,265 5.02 8

JCC445

0.246 1.52 4.16 6.35

JCC446

0.227 1.71 4.52 6.95

40 Ethanol concentrations of cultures in the supernatant are shown showing increased ethanol concentrations over time in various cultures of transgenic Synechococcus species (Figure 6). Notably, a higher concentration of ethanol was measured in JCC445, the strain transformed with Moorella adHA at 72 hours (Table 5).

Table 5

EtOH (mg / l)

0

24 48 72

JCC1

0 0 1,704728 5,880188

JCC136

0 63,00976 140,7334 252,8226

JCC137

0 72,02925 137.0422 256.4378

JCC445

0 14,03474 153,5205 296,761

JCC446

0 16.06255 125.6418 249,6592

Additionally, reduced acetaldehyde concentrations at various time points were observed in strains transformed with Moorella adhA (Figure 7 and Table 6).

Table 6

Acetaldehyde (mg / l)

0 24 48

72

JCC1

0 0 0.411352 0.362828

JCC136

0 14,20144 34.95365 36,49536

JCC137

0 19.80197 36.05125 35,83849

JCC445

0 9,455919 10.82248 13,57957

JCC446

0 8.368128 9.070718 12,32025

At subsequent time points, the strains transformed with Moorella adhA (JCC445 and JCC446) show notable increases in the ratio of ethanol and acetaldehyde compared to alcohol dehydrogenases based on Z. mobilis as shown in the cultures over time. (Figure 8 and Table 7).

Table 7

EtOH / Acetaldehyde

0 24 48

72

JCC1

N / A N / A 4,144204 16,20655

JCC136

N / A 4.436856 4.026287 6.927526

JCC137

N / A 3,637479 3,801316 7,155373

JCC445

N / A 1,484228 14,18532 21,85348

JCC446

N / A 1,919491 13.85137 20,26414

Figure 9 represents ratios of ethanol and DO730nm of the crops over time. The

15 concentration ratios of ethanol and DO730nm in the supernatant at each time point. The ratio of strains transformed with adh from Z. mobilis (JCC 136 and JCC 137) quickly reaches a steady state while the ratio of strains transformed with Moorella adhA constructs (JCC445 and JCC446) increases over time (Table 8).

20 Table 8

EtOH / OD (mg / l / OD)

0 24 48

72

JCC1

0 0 0.299075 0.582197

JCC136

0 27,88043 27,81293 33,26613

JCC137

0 31,80099 27,29924 32.05472

JCC445

0 9.233379 36.90395 46.73402

JCC446

0 9.393303 27.79687 35,92218

Thermosynechococcus elongatus BP-1 genetically modified: from Example 2, pJB22, pJB23, pJB24, pJB25, pJB26, pJB27, pJB28 and pJB29 are integrated into the chromosome of T. elongatus BP-1 by homologous recombination using the detailed transformation method in Onai K. et al (2004). "Natural transformation of the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1: a simple and efficient method for gene transfer." Mol. Gen.

5 Genomics 271: 50-59. The selection antibiotics used are spectinomycin plus streptomycin.

Informal Sequence List

SEQ ID NO: 1 10 Moorella adhA gene with optimized codons

SEQ ID NO: 2 15 Amino acid sequence of Moorella sp. HUC22-1 and Moorella thermoacetica

Claims (7)

1. A genetically modified photosynthetic microbial host cell that produces ethanol, where the cell The host is genetically modified to comprise an exogenous genetic engineering modified nucleic acid encoding an NADPH-dependent alcohol dehydrogenase activity. 2. The cell of claim 1, wherein the NADPH-dependent alcohol dehydrogenase activity is adhA of Moorella sp. HUC22-1.

The cell of claim 1 or 2, which also encodes a pyruvate decarboxylase activity.

4. The cell of claim 3, wherein the pyruvate decarboxylase activity is selected from the pdc activity of Z. palmae and Z. Mobilis

The cell of claim 1, wherein the NADPH-dependent alcohol dehydrogenase coding sequence is at least 77.1% identical to SEQ ID NO: 1.

6. The cell of claim 1, wherein the NADPH-dependent alcohol dehydrogenase is at least 72% identical to SEQ ID No. 2. 20 7. The cell of any one of the preceding claims, wherein the NADPH-dependent alcohol dehydrogenase coding sequence and / or pyruvate decarboxylase coding sequence is under the control of an inducible promoter.

The cell of any one of the preceding claims, wherein the cell in culture is capable of producing ethanol in a yield of at least about 249 mg / l of culture medium in 72 hours, preferably where

(i)

the yield is at least about 296 mg / l of ethanol for 72 hours; 30 (ii) the yield is 2.5 to 5 g / l of culture medium per hour;

(iii) the level of acetaldehyde in said culture after 72 hours is less than about 14 mg / l;

(iv)

the cell in the culture produces at least about 36 mg / l of ethanol per OD; or

(v)

the cell in culture produces at least about 47 mg / l of ethanol per OD.

The cell of any one of the preceding claims, which is a cyanobacterium.

10. The cell of claim 9, which is Synechococcus sp. PCC 7002, Synechococcus sp. PCC 7942, Synechocystis sp. PCC 6803 or Thermosynechococcus elongatus BP-1. Use of the cell of any one of the preceding claims to produce ethanol. 12. A method of producing ethanol, comprising culturing the cell of any one of claims 1 to 10 and then isolating the ethanol from said cell or culture medium. 13. The method of claim 12, wherein the cell is cultured in a photobioreactor.