The present invention [2, 3] is directed to a photobiological butanol and related high alcohols production technology based on designer photosynthetic organisms such as designer transgenic plants (e. g., algae and oxyphotobacteria) or plant cells. In this context throughout this specification, a “higher alcohol” or “related higher alco­hol” refers to an alcohol that comprises at least four carbon atoms, which includes both straight and branched alcohols such as 1-butanol and 2-methyl-1-butanol. The Calvin-cycle-channeled and photosynthetic-NADPH-enhanced pathways are con­structed with designer enzymes expressed through use of designer genes in host photosynthetic organisms such as algae and oxyphotobacteria (including cyanobac­teria and oxychlorobacteria) organisms for photobiological production of butanol and related higher alcohols. The said butanol and related higher alcohols are selected from the group consisting of: 1-butanol, 2-methyl-1-butanol, isobutanol, 3-methyl-

1- butanol, 1-hexanol, 1-octanol, 1-pentanol, 1-heptanol, 3-methyl-1-pentanol,

4- methyl-1-hexanol, 5-methyl-1-heptanol, 4-methyl-1-pentanol, 5-methyl-1- hexanol, and 6-methyl-1-heptanol. The designer plants and plant cells are created using genetic engineering techniques such that the endogenous photosynthesis reg­ulation mechanism is tamed, and the reducing power (NADPH) and energy (ATP) acquired from the photosynthetic water splitting and proton gradient-coupled elec­tron transport process can be used for immediate synthesis of higher alcohols, such as 1-butanol (CH3CH2CH2CH2OH) and 2-methyl-1-butanol (CH3CH2CH(CH3)

CH2OH), from carbon dioxide (CO2) and water (H2O) according to the following generalized process reaction (where m, n, x, and y are its molar coefficients) in accordance with the present invention:

m(CO2) + n(H2O) ^ x(higher alcohols) + y(O2) (2)

The photobiological higher alcohols production methods of the present invention completely eliminate the problem of recalcitrant lignocellulosics by bypassing the bottleneck problem of the biomass technology. As shown in Fig. 1, for example, the photosynthetic process in a designer organism effectively uses the reducing power (NADPH) and energy (ATP) from the photosynthetic water splitting and proton gradient-coupled electron transport process for immediate synthesis of butanol (CH3CH2CH2CH2OH) directly from carbon dioxide (CO2) and water (H2O) without being drained into the other pathways for synthesis of the undesirable lignocellu — losic materials that are very hard and often inefficient for the biorefinery industry to use. This approach is also different from the existing “cornstarch butanol produc­tion” process. In accordance with this invention, butanol can be produced directly from carbon dioxide (CO2) and water (H2O) without having to go through many of the energy consuming steps that the cornstarch butanol-production process has to go through, including corn crop cultivation, corn-grain harvesting, corn-grain corn­starch processing, and starch-to-sugar-to-butanol fermentation. As a result, the pho­tosynthetic butanol-production technology of the present invention is expected to have a much (more than ten times) higher solar-to-butanol energy-conversion efficiency than the current technology. Assuming a 10% solar energy conversion efficiency for the envisioned photosynthetic butanol production process, the maxi­mal theoretical productivity (yield) could be about 72,700 kg of butanol per acre per year, which could support about 70 cars (per year per acre). Therefore, this inven­tion could bring a significant capability to the society in helping to ensure energy security. The present invention could also help protect the Earth’s environment from the dangerous accumulation of CO2 in the atmosphere, because the present methods convert CO2 directly into clean advanced biofuels (e. g., butanol) energy.

A fundamental feature of the present methodology is utilizing a plant (e. g., an alga or oxyphotobacterium) or plant cells, introducing into the plant or plant cells nucleic acid molecules encoding for a set of enzymes that can act on an intermediate product of the Calvin cycle and convert the intermediate product into butanol as illustrated in Fig. 1, instead of making starch and other complicated cellular (bio­mass) materials as the end products by the wild-type photosynthetic pathways. Accordingly, the present invention provides, inter alia, methods for producing butanol and/or related higher alcohols based on a designer plant (such as a designer alga and a designer oxyphotobacterium), designer plant tissue, or designer plant cells, DNA constructs encoding genes of a designer butanol — and/or related higher alcohols-production pathway(s), as well as the designer algae, designer oxyphoto — bacteria (including designer cyanobacteria), designer plants, designer plant tissues, and designer plant cells created. The various aspects of the present Host Photosynthetic Organisms

According to the present invention, a designer organism or cell for the photosyn­thetic butanol and/or related higher alcohols production of the invention can be created utilizing as host, any plant (including alga and oxyphotobacterium), plant tissue, or plant cells that have a photosynthetic capability, i. e., an active photosyn­thetic apparatus and enzymatic pathway that captures light energy through photo­synthesis, using this energy to convert inorganic substances into organic matter. Preferably, the host organism should have an adequate photosynthetic CO2 fixation rate, for example, to support photosynthetic butanol (and/or related higher alcohols) production from CO2 and H2 O at least about 1,450 kg butanol per acre per year, more preferably, 7,270 kg butanol per acre per year, or even more preferably,

72,700 kg butanol per acre per year.

In a preferred embodiment, an aquatic plant is utilized to create a designer plant. Aquatic plants, also called hydrophytic plants, are plants that live in or on aquatic environments, such as in water (including on or under the water surface) or perma­nently saturated soil. As used herein, aquatic plants include, for example, algae, blue-green algae (cyanobacteria and oxychlorobacteria), submersed aquatic herbs (Hydrilla verticillata, Elodea densa, Hippuris vulgaris, Aponogeton boivinianus, Aponogeton rigidifolius, Aponogeton longiplumulosus, Didiplis diandra, Vesicularia dubyana, Hygrophilia augustifolia, Micranthemum umbrosum, Eichhornia azurea, Saururus cernuus, Cryptocoryne lingua, Hydrotriche hottoniiflora, Eustralis stel — lata, Vallisneria rubra, Hygrophila salicifolia, Cyperus helferi, Cryptocoryne petchii, Vallisneria americana, Vallisneria torta, H. hottoniiflora, Crassula helmsii, Limnophila sessiliflora, Potamogetonperfoliatus, Rotala wallichii, Cryptocoryne becketii, Blyxa aubertii, Hygrophila difformmis), duckweeds (Spirodela polyrrhiza, Wolffia globosa, Lemna trisulca, Lemna gibba, Lemna minor, Landoltiapunctata), water cabbage (Pistia stratiotes), buttercups (Ranunculus), water caltrop (Trapa natans and Trapa bicornis), water lily (Nymphaea lotus, Nymphaeaceae and Nelumbonaceae), water hyacinth (Eichhornia crassipes), Bolbitis heudelotii, Cabomba sp., seagrasses (Heteranthera zosterifolia, Posidoniaceae, Zosteraceae, Hydrocharitaceae, and Cymodoceaceae). Butanol (and/or related higher alcohols) produced from an aquatic plant can diffuse into water, permitting normal growth of the plants and more robust production of butanol from the plants. Liquid cultures of aquatic plant tissues (including, but not limited to, multicellular algae) or cells (including, but not limited to, unicellular algae) are also highly preferred for use, since the butanol (and/or related higher alcohols) molecules produced from a designer butanol (and/or related higher alcohols) production pathway(s) can readily diffuse out of the cells or tissues into the liquid water medium, which can serve as a large pool to store the product butanol (and/or related higher alcohols) that can be subsequently harvested by filtration and/or distillation/evaporation techniques.

Although aquatic plants or cells are preferred host organisms for use in the meth­ods of the present invention, tissue and cells of nonaquatic plants, which are photo­synthetic and can be cultured in a liquid culture medium, can also be used to create designer tissue or cells for photosynthetic butanol (and/or related higher alcohols) production. For example, the following tissue or cells of nonaquatic plants can also be selected for use as a host organism in this invention: the photoautotrophic shoot tissue culture of wood apple tree Feronia limonia, the chlorophyllous callus-cultures of corn plant Zea mays, the green root cultures of Asteraceae and Solanaceae spe­cies, the tissue culture of sugarcane stalk parenchyma, the tissue culture of bryo — phyte Physcomitrella patens, the photosynthetic cell suspension cultures of soybean plant (Glycine max), the photoautotrophic and photomixotrophic culture of green Tobacco (Nicofiana tabacum L.) cells, the cell suspension culture of Gisekiapharna — ceoides (a C4 plant), the photosynthetic suspension cultured lines of Amaranthuspow — ellii Wats.,Datura innoxia Mill., Gossypium hirsutum L., and N. tabacumx Nicotiana glutinosa L. fusion hybrid.

By “liquid medium” is meant liquid water plus relatively small amounts of inor­ganic nutrients (e. g., N, P, K, etc., commonly in their salt forms) for photoautotrophic cultures; and sometimes also including certain organic substrates (e. g., sucrose, glu­cose, or acetate) for photomixotrophic and/or photoheterotrophic cultures.

In an especially preferred embodiment, the plant utilized in the butanol (and/or related higher alcohols) production method of the present invention is an alga or a blue-green alga. The use of algae and/or blue-green algae has several advantages. They can be grown in an open pond at large amounts and low costs. Harvest and purification of butanol (and/or related higher alcohols) from the water phase is also easily accomplished by distillation/evaporation or membrane separation.

Algae suitable for use in the present invention include both unicellular algae and multiunicellular algae. Multicellular algae that can be selected for use in this inven­tion include, but are not limited to, seaweeds such as Ulva latissima (sea lettuce), Ascophyllum nodosum, Codium fragile, Fucus vesiculosus, Eucheuma denticula — tum, Gracilaria gracilis, Hydrodictyon reticulatum, Laminaria japonica, Undaria pinntifida, Saccharinajaponica, Porphyra yezoensis, and Porphyra tenera. Suitable algae can also be chosen from the following divisions of algae: green algae (Chlorophyta), red algae (Rhodophyta), brown algae (Phaeophyta), diatoms (Bacillariophyta), and blue-green algae (Oxyphotobacteria including Cyanophyta and Prochlorophytes). Suitable orders of green algae include Ulvales, Ulotrichales, Volvocales, Chlorellales, Schizogoniales, Oedogoniales, Zygnematales, Cladophorales, Siphonales, and Dasycladales. Suitable genera of Rhodophyta are Porphyra, Chondrus, Cyanidioschyzon, Porphyridium, Gracilaria, Kappaphycus, Gelidium, and Agardhiella. Suitable genera of Phaeophyta are Laminaria, Undaria, Macrocystis, Sargassum, and Dictyosiphon. Suitable genera of Cyanophyta (also known as Cyanobacteria) include (but not limited to) Phoridium, Synechocystis, Syncechococcus, Oscillatoria, and Anabaena. Suitable genera of Prochlorophytes (also known as oxychlorobacteria) include (but not limited to) Prochloron, Prochlorothrix, and Prochlorococcus. Suitable genera of Bacillariophyta are Cyclotella, Cylindrotheca, Navicula, Thalassiosira, and Phaeodactylum. Preferred species of algae for use in the present invention include: C. reinhardtii, P. subcordi — formis, C. fusca, Chlorella sorokiniana, Chlorella vulgaris, “Chlorella” ellipsoidea, Chlorella spp., D. salina, Dunaliella viridis, Dunaliella bardowil, Haematococcus pluvialis; Parachlorella kessleri, Betaphycus gelatinum, Chondrus crispus, Cyanidioschyzon merolae, Cyanidium caldarium, Galdieria sulphuraria, Gelidiella acerosa, Gracilaria changii, Kappaphycus alvarezii, Porphyra miniata, Ostreococcus tauri, P. yezoensis, Porphyridium sp., Palmariapalmata, Gracilaria spp., Isochrysis galbana, Kappaphycus spp., L. japonica, Laminaria spp., Monostroma spp., Nannochloropsis oculata, Porphyra spp., Porphyridium spp., Undaria pinnatifida, Ulva lactuca, Ulva spp., Undaria spp., Phaeodactylum tricornutum, Navicula sap — rophila, Crypthecodinium cohnii, Cylindrotheca fusiformis, Cyclotella cryptica, Euglena gracilis, Amphidinium sp., Symbiodinium microadriaticum, Macrocystis pyrifera, A. braunii, and S. obliquus.

Preferred species of blue-green algae (oxyphotobacteria including cyanobacteria and oxychlorobacteria) for use in the present invention include: Thermosynechococcus elongatus BP-1, Nostoc sp. PCC 7120, Synechococcus elongatus PCC 6301, Syncechococcus sp. strain PCC 7942, Syncechococcus sp. strain PCC 7002, Syncechocystis sp. strain PCC 6803, Prochlorococcus marinus MED4, P. marinus MIT 9313, P. marinus NATL1A, Prochlorococcus SS120, Spirulina platensis (Arthrospira platensis), Spirulina pacifica, Lyngbya majuscule, Anabaena sp., Synechocystis sp., Synechococcus elongates, Synechococcus (MC-A),Trichodesmium sp., Richelia intracellularis, Synechococcus WH7803, Synechococcus WH8102, Nostocpunctiforme, Syncechococcus sp. strain PCC 7943, Synechocyitis PCC 6714 phycocyanin-deficient mutant PD-1, Cyanothece strain 51142, Cyanothece sp. CCY0110, Oscillatoria limosa, Lyngbya majuscula, Symploca muscorum, Gloeobacter violaceus, Prochloron didemni, Prochlorothrix hollandica, Synechococcus (MC-A), Trichodesmium sp., R. intracellularis, P. marinus, Prochlorococcus SS120, Synechococcus WH8102, L. majuscula, S. muscorum, Synechococcus bigranulatus, cryophilic Oscillatoria sp., Phormidium sp., Nostoc sp.-1, Calothrixparietina, ther­mophilic S. bigranulatus, Synechococcus lividus, thermophilic Mastigocladus laminosus, Chlorogloeopsis fritschii PCC 6912, Synechococcus vulcanus, Synechococcus sp. strain MA4, Synechococcus sp. strain MA19, and T. elongatus.

Proper selection of host photosynthetic organisms for their genetic backgrounds and certain special features is also beneficial. For example, a photosynthetic — butanol-producing designer alga created from cryophilic algae (psychrophiles) that can grow in snow and ice, and/or from cold-tolerant host strains such as Chlamydomonas cold strain CCMG1619, which has been characterized as capable of performing photosynthetic water splitting as cold as 4°C [4], permits photobio­logical butanol production even in cold seasons or regions such as Canada. Meanwhile, a designer alga created from a thermophilic/thermotolerant photosyn­thetic organism such as thermophilic algae C. caldarium and G. sulphuraria and/or thermophilic cyanobacteria (blue-green algae) such as T. elongatus BP-1 and

S. bigranulatus may permit the practice of this invention to be well extended into the hot seasons or areas such as Mexico and the Southwestern region of the United States including Nevada, California, Arizona, New Mexico, and Texas, where the weather can often be hot. Furthermore, a photosynthetic-butanol-producing designer alga created from a marine alga, such as P. subcordiformis, permits the practice of this invention using seawater, while the designer alga created from a freshwater alga such as C. reinhardtii can use freshwater. Additional optional features of a photosynthetic butanol (and/or related higher alcohols) producing designer alga include the benefits of reduced chlorophyll antenna size, which has been demon­strated to provide higher photosynthetic productivity [5] and butanol tolerance (and/ or related higher alcohol tolerance) that allows for more robust and efficient photo­synthetic production of butanol (and/or related higher alcohols) from CO2 and H2O. By use of a phycocyanin-deficient mutant of Synechocystis PCC 6714, it has been experimentally demonstrated that photoinhibition can be reduced also by reducing the content of light-harvesting pigments [6]. These optional features can be incor­porated into a designer alga, for example, by use of a butanol-tolerant and/or chlo­rophyll antenna-deficient mutant (e. g., C. reinhardtii strain DS521) as a host organism, for genetic transformation with the designer butanol-production-pathway genes. Therefore, in one of the various embodiments, a host alga is selected from the group consisting of green algae, red algae, brown algae, blue-green algae (oxy- photobacteria including cyanobacteria and prochlorophytes), diatoms, marine algae, freshwater algae, unicellular algae, multicellular algae, seaweeds, cold-tolerant algal strains, heat-tolerant algal strains, light-harvesting-antenna-pigment-deficient mutants, butanol-tolerant algal strains, higher alcohols-tolerant algal strains, and combinations thereof.