US008986,963B2

(12) United States Patent (10) Patent No.: US 8,986,963 B2 Lee (45) Date of Patent: Mar. 24, 2015

(54) DESIGNER CALVIN-CYCLE-CHANNELED FOREIGN PATENT DOCUMENTS PRODUCTION OF BUTANOL AND RELATED WO 2005100.582 A2 10, 2005 HIGHER ALCOHOLS WO 2006119066 11, 2006 WO 2007032837 A2 3, 2007 (76) Inventor: James Weifu Lee, Cockeysville, MD WO 2007047148 4/2007 (US) WO 2007065035 6, 2007 WO 2007134340 A2 11/2007 WO 2008OO6038 A2 1, 2008 (*) Notice: Subject to any disclaimer, the term of this WO 201OO68821 A1 6, 2010 patent is extended or adjusted under 35 U.S.C. 154(b) by 714 days. OTHER PUBLICATIONS Appl. No.: 13/075,153 Chen et al., Photo. Res., 2005, 86:165-173.* (21) Raines et al., 2003, Photosynthesis Res., 75:1-10.* Filed: Mar. 29, 2011 Pickett-Heaps et al., 1999, Am. J. of Botany, 86:153-172.* (22) Shen et al., 2008, Metabolic Engineering, 10:312-320.* Prior Publication Data Sanderson, 2006, Nature, 444:673-676.* (65) Keneko Takakazu et al., Complete Genomic Sequence of the Fila US 2011/O177571 A1 Jul. 21, 2011 mentous Nitrogen-fixing Cyanobacterium Anabaena sp. Strain PCC 7120, DNA Research 8,205-213 (2001). Ramesh V. Nair et al., Regulation of the Sol Locus Genes for Butanol and Acetone Formation in Clostridium acetobutylicum ATCC824 by Related U.S. Application Data a Putative Transcriptional Repressor, Journal of Bacteriology, Jan. (63) Continuation-in-part of application No. 12/918,784, 1999, vol. 181, No. 1, pp. 319-330. filed as application No. PCT/US2009/034801 on Feb. (Gfeller and Gibbs (1984) “Fermentative metabolism of Chlamydomonas reinhardtii, Plant Physiol. 75:212-218). 21, 2009, now Pat. No. 8,735,651. (Lee, Blankinship and Greenbaum (1995), “Temperature effect on (60) Provisional application No. 61/066,845, filed on Feb. production of hydrogen and oxygen by Chlamydomonas cold strain 23, 2008, provisional application No. 61/066,835, CCMP1619 and wild type 137c. Applied Biochemistry and filed on Feb. 23, 2008, provisional application No. Biotechnology 51/52:379-386). Lee et al., "Discovery of an Alternative Oxygen Sensitivity in Algal 61/426,147, filed on Dec. 22, 2010. Photosynthetic H2 Production”. Proceedings of the 2000 U.S. DOE Hydrogen Program Review, NREL/CP-570-28890. (51) Int. C. (Lee, Mets, and Greenbaum (2002). “Improvement of photosynthetic CI2P 7/16 (2006.01) efficiency at high light intensity through reduction of chlorophyll CI2P 7/04 (2006.01) antenna size.” Applied Biochemistry and Biotechnology, 98-100: CI2N IS/II3 (2010.01) 37-48). CI2O I/00 (2006.01) (Nakajima, Tsuzuki, and Ueda (1999) “Reduced photoinhibition of a (52) U.S. C. phycocyanin-deficient mutant of Synechocystis PCC 6714'. Journal CPC. CI2P 7/16 (2013.01); C12P 7/04 (2013.01); of Applied Phycology 10: 447-452). CI2Y 204/01021 (2013.01); C12N 15/I 137 (Continued) (2013.01): CI2O I/00 (2013.01); Y02E 50/10 (2013.01); C12N 23 10/11 (2013.01); C12N Primary Examiner — Ashwin Mehta 23 10/14 (2013.01) Assistant Examiner — Jason DeVeau Rosen USPC ...... 435/160 (74) Attorney, Agent, or Firm — August Law, LLC; George (58) Field of Classification Search Willinghan None See application file for complete search history. (57) ABSTRACT (56) References Cited Designer Calvin-cycle-channeled and photosynthetic NADPH-enhanced pathways, the associated designer genes U.S. PATENT DOCUMENTS and designer transgenic photosynthetic organisms for photo biological production of butanol and related higher alcohols 6,699,696 B2 * 3/2004 Woods et al...... 435,161 7.682,821 B2 * 3/2010 Woods et al...... 435,292.1 from carbon dioxide and water are provided. The butanol and 2007/0037196 A1 2/2007 Gibson et al. related higher alcohols include 1-butanol, 2-methyl-1-bu 2007/0037197 A1 2/2007 Young et al. tanol, isobutanol, 3-methyl-1-butanol, 1-hexanol, 1-octanol, 2007/O122826 A1 5, 2007 Glass et al. 1-pentanol, 1-heptanol, 3-methyl-1-pentanol, 4-methyl-1- 2007.0128649 A1 6/2007 Young hexanol, 5-methyl-1-heptanol, 4-methyl-1-pentanol, 5-me 2007,0264688 A1 11/2007 Venter et al. 2007,0269.862 A1 11, 2007 Glass et al. thyl-1-hexanol, and 6-methyl-1-heptanol. The designer pho 2009,0081746 A1 3/2009 Liao et al. tosynthetic organisms such as designer transgenic 2009/011 1154 A1 4/2009 Liao et al. oxyphotobacteria and algae comprise designer Calvin-cycle 2009,0176280 A1 7/2009 Hutchinson, III et al. channeled and photosynthetic NADPH-enhanced pathway 2009/0203070 A1 8, 2009 Devroe et al. gene(s) and biosafety-guarding technology for enhanced 2010/0105103 A1 4/2010 Juan et al. 2010, O151545 A1 6, 2010 Roessler et al. photobiological production of butanol and related higher 2010/0209986 A1 8, 2010 Liao et al. alcohols from carbon dioxide and water. 2010, 0221800 A1 9, 2010 Liao et al. 2010/0330637 A1* 12/2010 Lee ...... 435,160 7 Claims, 12 Drawing Sheets US 8,986,963 B2 Page 2

(56) References Cited (Casey and Grossman (1994) “In vivo and in vitro characterization of the light-regulated cpcB2A2 promoter of Fremyella diplosiphont” OTHER PUBLICATIONS Journal of Bacteriology, 176(20):6362-6374). (Domain, Houot, Chauvat, and Cassier-Chauvat (2004) “Function (Quinn, Barraco, Ericksson and Merchant (2000). “Coordinate cop and regulation of the cyanobacterial genes leXA, recA and ruvB: per- and oxygen-responsive Cyc6 and CpX1 expression in LeXA is critical to the Survival of cells facing inorganic carbon Chlamydomonas is mediated by the same element.” JBiol Chem 275: starvation.” Molecular Microbiology, 53 (1):65-80). 6080-6089). (Keppetipola, Coffman, and etal (2003). Rapid detection of in vitro (Loppes and Radoux (2002) “Two short regions of the promoter are expressed proteins using LumioTM technology, Gene Expression, essential for activation and repression of the nitrate reductase gene in 25.3: 7-11). Chlamydomonas reinhardtii, Mol Genet Genomics 268: 42-48). (Griffin, Adams, and Tsien (1998), "Specific covalent labeling of (Sjoholm, Oliveira, and Lindblad (2007) “Transcription and regula recombinant protein molecules inside live cells'. Science, 281:269 tion of the bidirectional hydrogenase in the Cyanobacterium Nostoc 272). sp. strain PCC 7120.” Applied and Environmental Microbiology, (Pattanayak and Chatterjee (1998) “Nicotinamide adenine 73(17): 5435-5446). dinucleotide phosphate phosphatase facilitates dark reduction of (Qi, Hao, Ng, Slater, Baszis, Weiss, and Valentin (2005)"Application nitrate: regulation by nitrate and ammonia. Biologia Plantarium of the Synechococcus nirA promoter to establish an inducible expres 41(1):75-84). sion system for engineering the Synechocystis tocopherol pathway.” (Muto, Miyachi, Usuda, Edwards and Bassham (1981) “Light-in Applied and Environmental Microbiology, 71 (10): 5678-5684. duced conversion of nicotinamide adenine dinucleotide to Maeda, Kawaguchi, Ohe, and Omata (1998) “cis-Acting sequences nicotinamide adenine dinucleotide phosphate in higher plant leaves.” required for NtcB-dependent, nitrite-responsive positive regulation Plant Physiology 68(2):324-328. of the nitrate assimilation operon in the Cyanobacterium Matsumura-Kadota, Muto, Miyachi (1982) “Light-induced conver Synechococcus sp. strain PCC 7942,” Journal of Bacteriology, sion of NAD+ to NADP+ in Chlorella cells.” Biochimica Biophysica 180(16):4080-4088). Acta 679(2), pp. 300-307. Kojima and Nakamoto (2007) “A novel light- and heat-responsive (Liszewski (Jun. 1, 2003) Progress in RNA interference, Genetic regulation of the groE transcription in the absence of HrcA or CIRCE Engineering News, vol. 23, No. 11, pp. 8-17. in cyanobacteria.” FEBS Letters 581:1871-1880). (Fire, Xu, Montgomery, Kostas, Driver, Mello (1998) “Potent and 7942 (Erbe, Adams, Taylor and Hall (1996)“Cyanobacteria carrying specific genetic interference by double-stranded RNA in an Smit-lux transcriptional fusion as biosensors for the detection of Caenorhabditis elegans'. Nature 391 (6669):806-11. heavy metal cations,” Journal of Industrial Microbiology, 17:80-83). (Fuhrmann, Stahlberg, Govorunova, Rank and Hegemann (2001) (Michel, Pistorius, and Golden (2001)“Unusual regulatory elements Journal of Cell Science 114:3857-3863). for iron deficiency induction of the idiA gene of Synechococcus (Durre, P. 1998 Appl Microbiol Biotechnol 49: 639-648). elongatus PCC 7942” Journal of Bacteriology, 183(17):5015-5024). Qureshi, Hughes, Maddox, and Cotta 2005 Bioprocess Biosyst Eng (Patterson-Fortin, Colvin and Owttrim (2006) “A LexA-related pro 27:215-222). tein regulates redox-sensitive expression of the cyanobacterial RNA (Deng and Coleman (1999) “Ethanol synthesis by genetic engineer helicase, crh R', Nucleic Acids Research, 34(12):3446-3454). ing in cyanobacteria. * Applied and Environmental Microbiology, (Fang and Barnum (2004) “Expression of the heat shock gene 65(2):523-528). hsp16.6 and promoter analysis in the Cyanobacterium, (Hirano, Ueda, Hirayama, and Ogushi (1997) “CO2 fixation and Synechocystis sp. PCC 6803.” Current Microbiology 49:192-198). ethanol production with microalgal photosynthesis and intracellular (Nakamoto, Suzuki, and Roy (2000) “Constitutive expression of a anaerobic fermentation' Energy 22(2/3): 137-142). Small heat-shock protein confers cellular thermotolerance and ther The Eurasian Patent Office, Search Report, Apr. 15, 2011. mal protection to the photosynthetic apparatus in cyanobacteria.” FEBS Letters 483: 169-174). * cited by examiner U.S. Patent Mar. 24, 2015 Sheet 1 of 12 US 8,986,963 B2

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orf sef sef US 8,986,963 B2 1. 2 DESIGNER CALVIN-CYCLE-CHANNELED an additive to gasoline up to about 85% (E-85) and then only PRODUCTION OF BUTANOLAND RELATED after significant modification to the engine (while butanol can HIGHER ALCOHOLS work as a 100% replacement fuel without having to modify the current car engine). CROSS-REFERENCE TO RELATED A significant potential market for butanol and/or related APPLICATIONS higher alcohols as a liquid fuel already exists in the current transportation and energy systems. Butanol is also used as an This application is a continuation-in-part of co-pending industrial solvent. In the United States, currently, butanol is U.S. patent application Ser. No. 12/918,784 filed on Aug. 20. manufactured primarily from petroleum. Historically (1900s 2010, which is the National Stage of International Applica 10 1950s), biobutanol was manufactured from corn and molas tion No. PCT/US2009/034801 filed on Feb. 21, 2009, which ses in a fermentation process that also produced acetone and claims the benefit of U.S. Provisional Application No. ethanol and was known as an ABE (acetone, butanol, ethanol) 61/066,845 filed on Feb. 23, 2008, and U.S. Provisional fermentation typically with certain butanol-producing bacte Application No. 61/066,835 filed on Feb. 23, 2008. This ria Such as Clostridium acetobutyllicum and Clostridium application also claims the benefit of U.S. Provisional Appli 15 beijerinckii. When the USA lost its low-cost sugar supply cation No. 61/426,147 filed on Dec. 22, 2010. The entire from Cuba around 1954, however, butanol production by disclosures of all of these applications are incorporated herein fermentation declined mainly because the price of petroleum by reference. dropped below that of sugar. Recently, there is renewed R&D interest in producing butanol and/or ethanol from biomass FIELD OF THE INVENTION Such as corn Starch using Clostridia- and/or yeast-fermenta tion process. However, similarly to the situation of “corn The present invention generally relates to biosafety starch ethanol production, the “cornstarch butanol produc guarded biofuel energy production technology. More specifi tion' process also requires a number of energy-consuming cally, the present invention provides a photobiological steps including agricultural corn-crop cultivation, corn-grain advanced-biofuels production methodology based on 25 harvesting, corn-grain starch processing, and starch-to-Sugar designer transgenic plants, such as transgenic algae, blue to-butanol fermentation. The “cornstarch butanol produc green algae (cyanobacteria and oxychlorobacteria), or plant tion' process could also probably cost nearly as much energy cells that are created to use the reducing power (NADPH) and as the energy value of its product butanol. This is not surpris energy (ATP) acquired from the photosynthetic process for ing, understandably because the cornstarch that the current photoautotrophic synthesis of butanol and/or related higher 30 technology can use represents only a small fraction of the alcohols from carbon dioxide (CO) and water (HO). corn crop biomass that includes the corn stalks, leaves and roots. The cornstovers are commonly discarded in the agri REFERENCE TO SEQUENCE LISTING cultural fields where they slowly decompose back to CO., because they represent largely lignocellulosic biomass mate The present invention contains references to amino acid 35 rials that the current biorefinery industry cannot efficiently sequences and/or nucleic acid sequences which have been use for ethanol or butanol production. There are research Submitted concurrently herewith as the sequence listing text efforts in trying to make ethanol or butanol from lignocellu file “JWL 004 US 1 SeqListingFull ST25.txt, file size losic plant biomass materials—a concept called “cellulosic 429 KB, created on Mar. 29, 2011, in electronic format using ethanol or “cellulosic butanol. However, plant biomass has the Electronic Filing System of the U.S. Patent and Trade 40 evolved effective mechanisms for resisting assault on its cell mark Office. The aforementioned sequence listing was pre wall structural Sugars from the microbial and animal king pared with PatentIn 3.5, which complies with all format doms. This property underlies a natural recalcitrance, creat requirements specified in World Intellectual Property Orga ing roadblocks to the cost-effective transformation of nization Standard (WIPO) ST.25 and the related United lignocellulosic biomass to fermentable Sugars. Therefore, States (US) final rule, and is incorporated herein by reference 45 one of its problems known as the “lignocellulosic recalci in its entirety including pursuant to 37 C.F.R. S.1.52(e)(5) trance' represents a formidable technical barrier to the cost where applicable. effective conversion of plant biomass to fermentable Sugars. That is, because of the recalcitrance problem, lignocellulosic BACKGROUND OF THE INVENTION biomasses (such as cornstover, Switchgrass, and woody plant 50 materials) could not be readily converted to fermentable sug Butanol and/or related higher alcohols can be used as a ars to make ethanol or butanol without certain pretreatment, liquid fuel to run engines Such as cars. Butanol can replace which is often associated with high processing cost. Despite gasoline and the energy contents of the two fuels are nearly more than 50 years of R&D efforts in lignocellulosic biomass the same (110,000 Btu per gallon forbutanol; 115,000 Btu per pretreatment and fermentative butanol-production process gallon for gasoline). Butanol has many Superior properties as 55 ing, the problem of recalcitrant lignocellulosics still remains an alternative fuel when compared to ethanol as well. These as a formidable technical barrier that has not yet been elimi include: 1) Butanol has higher energy content (110,000 Btu nated so far. Furthermore, the steps of lignocellulosic biom per gallon butanol) than ethanol (84,000 Btu per gallon etha ass cultivation, harvesting, pretreatment processing, and cel nol); 2) Butanol is six times less "evaporative than ethanol lulose-to-sugar-to-butanol fermentation all cost energy. and 13.5 times less evaporative than gasoline, making it safer 60 Therefore, any new technology that could bypass these to use as an oxygenate and thereby eliminating the need for bottleneck problems of the biomass technology would be very special blends during the Summer and winter seasons; 3) useful. Butanol can be transported through the existing fuel infra Oxyphotobacteria (also known as blue-green algae includ structure including the gasoline pipelines whereas ethanol ing cyanobacteria and oxychlorobacteria) and algae (such as must be shipped via rail, barge or truck; and 4) Butanol can be 65 Chlamydomonas reinhardtii, Platymonas subcordiformis, used as replacement for gasoline gallon for gallon e.g. 100% Chlorella fisca, Dunaliella Salina, Ankistrodesmus braunii, or any other percentage, whereas ethanol can only be used as and Scenedesmus obliquus), which can perform photosyn US 8,986,963 B2 3 4 thetic assimilation of CO with O. evolution from water in a for enhanced photobiological production of butanol and liquid culture medium with a maximal theoretical Solar-to related higher alcohols from carbon dioxide and water. biomass energy conversion of about 10%, have tremendous According to another embodiment, the transgenic photo potential to be a clean and renewable energy resource. How synthetic organism comprises a transgenic designer plant or ever, the wild-type oxygenic photosynthetic green plants, plant cells selected from the group consisting of aquatic Such as blue-green algae and eukaryotic algae, do not possess plants, plant cells, green algae, red algae, brown algae, blue the ability to produce butanol directly from CO, and H.O. green algae (oxyphotobacteria including cyanobacteria and The wild-type photosynthesis uses the reducing power oxychlorobacteria), diatoms, marine algae, freshwater algae, (NADPH) and energy (ATP) from the photosynthetic water salt-tolerant algal strains, cold-tolerant algal strains, heat splitting and proton gradient-coupled electron transport pro 10 tolerant algal strains, antenna-pigment-deficient mutants, cess through the algal thylakoid membrane system to reduce butanol-tolerant algal strains, higher-alcohols-tolerant algal CO into carbohydrates (CHO), such as starch with a series strains, butanol-tolerant oxyphotobacteria, higher-alcohols of enzymes collectively called the “Calvin cycle' at the tolerant oxyphotobacteria, and combinations thereof. stroma region in an algal or green-plant chloroplast. The net According to one of the various embodiments, a designer result of the wild-type photosynthetic process is the conver 15 Calvin-cycle-channeled photosynthetic NADPH-enhanced sion of CO, and HO into carbohydrates (CHO), and O. pathway that takes the Calvin-cycle intermediate product, using Sunlight energy according to the following process 3-phosphoglycerate, and converts it into 1-butanol comprises reaction: a set of enzymes selected from the group consisting of NADPH-dependent glyceraldehyde-3-phosphate dehydro genase, NAD-dependent glyceraldehyde-3-phosphate dehy The carbohydrates (CHO), are then further converted to all drogenase, phosphoglycerate mutase, enolase, pyruvate kinds of complicated cellular (biomass) materials including kinase, citramalate synthase, 2-methylmalate dehydratase, proteins, lipids, and cellulose and other cell-wall materials 3-isopropylmalate dehydratase, 3-isopropylmalate dehydro during cell metabolism and growth. genase, 2-isopropylmalate synthase, isopropylmalate In certain alga Such as Chlamydomonas reinhardtii, some 25 isomerase, 2-keto acid decarboxylase, alcohol dehydroge of the organic reserves such as starch could be slowly metabo nase, NADPH-dependent alcohol dehydrogenase, and lized to ethanol (but not to butanol) through a secondary butanol dehydrogenase. fermentative metabolic pathway. The algal fermentative According to one of the various embodiments, another metabolic pathway is similar to the yeast-fermentation pro designer Calvin-cycle-channeled photosynthetic NADPH cess, by which starch is breakdown to Smaller Sugars such as 30 enhanced 1-butanol-production pathway comprises a set of glucose that is, in turn, transformed into pyruvate by a glyco enzymes selected from the group consisting of NADPH lysis process. Pyruvate may then be converted to formate, dependent glyceraldehyde-3-phosphate dehydrogenase, acetate, and ethanol by a number of additional metabolic NAD-dependent glyceraldehyde-3-phosphate dehydroge steps (Gfeller and Gibbs (1984) “Fermentative metabolism of nase, phosphoglycerate mutase, enolase, phosphoenolpyru Chlamydomonas reinhardtii,' Plant Physiol. 75:212-218). 35 vate carboxylase, aspartate aminotransferase, aspartokinase, The efficiency of this secondary metabolic process is quite aspartate-semialdehyde dehydrogenase, homoserine dehy limited, probably because it could use only a small fraction of drogenase, homoserine kinase, threonine synthase, threonine the limited organic reserve such as starch in an algal cell. ammonia-lyase, 2-isopropylmalate synthase, isopropyl Furthermore, the native algal secondary metabolic process malate isomerase, 3-isopropylmalate dehydrogenase, 2-keto could not produce any butanol. As mentioned above, butanol 40 acid decarboxylase, and NAD-dependentalcoholdehydroge (and/or related higher alcohols) has many Superior physical nase, NADPH-dependent alcohol dehydrogenase, and properties to serve as a replacement for gasoline as a fuel. butanol dehydrogenase. Therefore, a new photobiological butanol (and/or related According to another embodiment, a designer Calvin higher alcohols)-producing mechanism with a high Solar-to cycle-channeled photosynthetic NADPH-enhanced pathway biofuel energy efficiency is needed. 45 that takes the Calvin-cycle intermediate product, 3-phospho International Application No. PCT/US2009/034801 dis glycerate, and converts it into 2-methyl-1-butanol, comprises closes a set of methods on designer photosynthetic organisms a set of enzymes selected from the group consisting of (such as designer transgenic plant, plant cells, algae and oxy NADPH-dependent glyceraldehyde-3-phosphate dehydro photobacteria) for photobiological production of butanol genase, NAD-dependent glyceraldehyde-3-phosphate dehy from carbon dioxide (CO) and water (H2O). 50 drogenase, phosphoglycerate mutase, enolase, pyruvate kinase, citramalate synthase, 2-methylmalate dehydratase, SUMMARY OF THE INVENTION 3-isopropylmalate dehydratase, 3-isopropylmalate dehydro genase, acetolactate synthase, ketol-acid reductoisomerase, The present invention discloses designer Calvin-cycle dihydroxy-acid dehydratase, 2-keto acid decarboxylase, channeled and photosynthetic NADPH-enhanced pathways, 55 NAD-dependent alcohol dehydrogenase, NADPH-depen the associated designer genes and designer transgenic photo dent alcohol dehydrogenase, and 2-methylbutyraldehyde synthetic organisms for photobiological production of reductase. butanol and/or related higher alcohols that are selected from According to another embodiment, a designer Calvin the group that consists of 1-butanol, 2-methyl-1-butanol, cycle-channeled photosynthetic NADPH-enhanced pathway isobutanol, 3-methyl-1-butanol, 1-hexanol, 1-octanol. 1-pen 60 for photobiological production of 2-methyl-1-butanol pro tanol, 1-heptanol, 3-methyl-1-pentanol, 4-methyl-1-hexanol, duction comprises a set of enzymes selected from the group 5-methyl-1-heptanol, 4-methyl-1-pentanol, 5-methyl-1-hex consisting of NADPH-dependent glyceraldehyde-3-phos anol, 6-methyl-1-heptanol, and combinations thereof. phate dehydrogenase, NAD-dependent glyceraldehyde-3- The designer photosynthetic organisms such as designer phosphate dehydrogenase, phosphoglycerate mutase, eno transgenic oxyphotobacteria and algae comprise designer 65 lase, phosphoenolpyruvate carboxylase, aspartate Calvin-cycle-channeled and photosynthetic NADPH-en aminotransferase, aspartokinase, aspartate-semialdehyde hanced pathway gene(s) and biosafety-guarding technology dehydrogenase, homoserine dehydrogenase, homoserine US 8,986,963 B2 5 6 kinase, threonine synthase, threonine ammonia-lyase, aceto FIG. 3A illustrates a cell-division-controllable designer lactate synthase, ketol-acid reductoisomerase, dihydroxy organism that contains two key functions: designer biosafety acid dehydratase, 2-keto acid decarboxylase, and NAD mechanism(s) and designer biofuel-production pathway(s). dependent alcohol dehydrogenase, NADPH dependent alco FIG. 3B illustrates a cell-division-controllable designer holdehydrogenase, and 2-methylbutyraldehyde reductase. organism for photobiological production of butanol According to another embodiment, a designer Calvin (CHCHCHCH-OH) from carbon dioxide (CO) and water cycle-channeled photosynthetic NADPH-enhanced pathway (H2O) with designer biosafety mechanism(s). FIG. 3C illustrates a cell-division-controllable designer for photobiological production of isobutanol comprises a set organism for biosafety-guarded photobiological production of enzymes selected from the group consisting of NADPH of other biofuels such as ethanol (CHCH-OH) from carbon dependent glyceraldehyde-3-phosphate dehydrogenase, 10 dioxide (CO) and water (H2O). NAD-dependent glyceraldehyde-3-phosphate dehydroge FIG. 4 presents designer Calvin-cycle-channeled and pho nase, phosphoglycerate mutase, enolase, pyruvate kinase, tosynthetic NADPH-enhanced pathways using the reducing acetolactate synthase, ketol-acid reductoisomerase, dihy power (NADPH) and energy (ATP) from the photosynthetic droxy-acid dehydratase, 2-keto acid decarboxylase, and water splitting and proton gradient-coupled electron transport NAD-dependent alcohol dehydrogenase, and NADPH-de 15 process to reduce carbon dioxide (CO) into 1-butanol pendent alcohol dehydrogenase. (CHCHCHCH-OH) with a series of enzymatic reactions. Likewise, a number of other designer Calvin-cycle-chan FIG. 5 presents designer Calvin-cycle-channeled and pho neled photosynthetic NADPH-enhanced pathways are also tosynthetic NADPH-enhanced pathways using NADPH and disclosed according to one of the various embodiments for ATP from the photosynthetic water splitting and proton gra photobiological production of butanol and/or related higher dient-coupled electron transport process to reduce carbon alcohols such as 3-methyl-1-butanol, 1-hexanol, 1-octanol, dioxide (CO) into 2-methyl-1-butanol (CHCH-CH(CH) 1-pentanol, 1-heptanol, 3-methyl-1-pentanol, 4-methyl-1- CHOH) with a series of enzymatic reactions. hexanol, 5-methyl-1-heptanol, 4-methyl-1-pentanol, 5-me FIG. 6 presents designer Calvin-cycle-channeled and pho thyl-1-hexanol, and/or 6-methyl-1-heptanol. tosynthetic NADPH-enhanced pathways using NADPH and According to one of various embodiments, a method for 25 ATP from the photosynthetic water splitting and proton gra photobiological production and harvesting of butanol and dient-coupled electron transport process to reduce carbon dioxide (CO) into isobutanol ((CH) CHCH-OH) and 3-me related higher alcohols comprises: a) introducing a transgenic thyl-1-butanol (CHCH(CH)CHCH-OH) with a series of photosynthetic organism into a photobiological reactor sys enzymatic reactions. tem, the transgenic photosynthetic organism comprising FIG.7 presents designer Calvin-cycle-channeled and pho transgenes coding for a set of enzymes configured to act on an 30 tosynthetic NADPH-enhanced pathways using NADPH and intermediate product of a Calvin cycle and to convert the ATP from the photosynthetic water splitting and proton gra intermediate product into butanol and/or related higher alco dient-coupled electron transport process to reduce carbon hols; b) using reducing power NADPH and energy ATPasso dioxide (CO) into 1-hexanol ciated with the transgenic photosynthetic organism acquired (CHCHCHCHCHCH-OH) and 1-octanol from photosynthetic water splitting and proton gradient 35 (CHCHCHCHCHCHCHCH-OH) with a series of coupled electron transport process in the photobioreactor to enzymatic reactions. synthesize butanol and/or related higher alcohols from car FIG. 8 presents designer Calvin-cycle-channeled and pho bon dioxide and water, and c) using a product separation tosynthetic NADPH-enhanced pathways using NADPH and process to harvest the synthesized butanol and/or related ATP from the photosynthetic water splitting and proton gra higher alcohols from the photobioreactor. 40 dient-coupled electron transport process to reduce carbon dioxide (CO) into 1-pentanol (CHCHCHCHCH-OH), BRIEF DESCRIPTION OF THE DRAWINGS 1-hexanol (CHCH2CH2CH2CHCH-OH), and 1-heptanol (CHCH2CH2CH2CH2CHCH-OH) with a series of enzy FIG. 1 presents designer butanol-production pathways matic reactions. branched from the Calvin cycle using the reducing power 45 FIG. 9 presents designer Calvin-cycle-channeled and pho (NADPH) and energy (ATP) from the photosynthetic water tosynthetic NADPH-enhanced pathways using NADPH and splitting and proton gradient-coupled electron transport pro ATP from the photosynthetic water splitting and proton gra cess to reduce carbon dioxide (CO) into butanol dient-coupled electron transport process to reduce carbon CHCHCHCH-OH with a series of enzymatic reactions. dioxide (CO) into 3-methyl-1-pentanol (CHCH-CH(CH) FIG. 2A presents a DNA construct for designer butanol 50 CHCH-OH), 4-methyl-1-hexanol (CHCH-CH(CH) production-pathway gene(s). CHCHCH-OH), and 5-methyl-1-heptanol (CHCHCH FIG. 2B presents a DNA construct for NADPH/NADH (CH)CHCHCHCH-OH) with a series of enzymatic conversion designer gene for NADPH/NADH inter-conver reactions. S1O. FIG. 10 presents designer Calvin-cycle-channeled and FIG. 2C presents a DNA construct for a designer iRNA 55 photosynthetic NADPH-enhanced pathways using NADPH starch/glycogen-synthesis inhibitor(s) gene. and ATP from the photosynthetic water splitting and proton FIG. 2D presents a DNA construct for a designer starch gradient-coupled electron transport process to reduce carbon degradation-glycolysis gene(s). dioxide (CO) into 4-methyl-1-pentanol (CHCH(CH) FIG. 2E presents a DNA construct of a designer butanol CHCHCH-OH), 5-methyl-1-hexanol (CHCH(CH) production-pathway gene(s) for cytosolic expression. 60 CHCHCHCH-OH), and 6-methyl-1-heptanol (CHCH FIG. 2F presents a DNA construct of a designer butanol (CH)CHCHCHCHCH-OH) with a series of enzymatic production-pathway gene(s) with two recombination sites for reactions. integrative genetic transformation in oxyphotobacteria. FIG. 2G presents a DNA construct of a designer biosafety DETAILED DESCRIPTION OF THE INVENTION control gene(s). 65 FIG. 2H presents a DNA construct of a designer proton The present invention is directed to a photobiological channel gene(s). butanol and related high alcohols production technology US 8,986,963 B2 7 8 based on designer photosynthetic organisms such as designer cells, introducing into the plant or plant cells nucleic acid transgenic plants (e.g., algae and oxyphotobacteria) or plant molecules encoding for a set of enzymes that can act on an cells. In this context throughout this specification, a “higher intermediate product of the Calvin cycle and convert the alcohol or “related higher alcohol refers to an alcohol that intermediate product into butanol as illustrated in FIG. 1, comprises at least four carbon atoms, which includes both 5 instead of making starch and other complicated cellular (bio straight and branched alcohols such as 1-butanol and 2-me mass) materials as the end products by the wild-type photo thyl-1-butanol. The Calvin-cycle-channeled and photosyn synthetic pathway. Accordingly, the present invention pro thetic-NADPH-enhanced pathways are constructed with vides, interalia, methods for producing butanol and/or related designer enzymes expressed through use of designer genes in higher alcohols based on a designer plant (Such as a designer host photosynthetic organisms such as algae and oxyphoto 10 alga and a designer oxyphotobacterium), designer plant tis bacteria (including cyanobacteria and oxychlorobacteria) Sue, or designer plant cells, DNA constructs encoding genes organisms for photobiological production of butanol and of a designer butanol- and/or related higher alcohols-produc related higher alcohols. The said butanol and related higher tion pathway(s), as well as the designer algae, designer oxy alcohols are selected from the group consisting of 1-butanol, photobacteria (including designer cyanobacteria), designer 2-methyl-1-butanol, isobutanol, 3-methyl-1-butanol, 1-hex 15 plants, designerplant tissues, and designer plant cells created. anol, 1-octanol. 1-pentanol, 1-heptanol, 3-methyl-1-pen The various aspects of the present invention are described in tanol, 4-methyl-1-hexanol, 5-methyl-1-heptanol, 4-methyl further detail hereinbelow. 1-pentanol, 5-methyl-1-hexanol, and 6-methyl-1-heptanol. Host Photosynthetic Organisms The designer plants and plant cells are created using genetic According to the present invention, a designer organism or engineering techniques such that the endogenous photosyn cell for the photosynthetic butanol and/or related higher alco thesis regulation mechanism is tamed, and the reducing hols production of the invention can be created utilizing as power (NADPH) and energy (ATP) acquired from the photo host, any plant (including alga and oxyphotobacterium), plant synthetic water splitting and proton gradient-coupled elec tissue, or plant cells that have a photosynthetic capability, i.e., tron transport process can be used for immediate synthesis of an active photosynthetic apparatus and enzymatic pathway higher alcohols, such as 1-butanol (CHCH2CH2CH2OH) 25 that captures light energy through photosynthesis, using this and 2-methyl-1-butanol (CHCH-CH(CH)CH-OH), from energy to convert inorganic Substances into organic matter. carbon dioxide (CO) and water (HO) according to the fol Preferably, the host organism should have an adequate pho lowing generalized process reaction (where m, n, X and y are tosynthetic CO fixation rate, for example, to support photo its molar coefficients) in accordance of the present invention: synthetic butanol (and/or related higher alcohols) production 30 from CO, and HO at least about 1,450kg butanol per acreper year, more preferably, 7.270 kg butanol per acre per year, or The photobiological higher-alcohols-production methods of even more preferably, 72,700 kg butanol per acre per year. the present invention completely eliminate the problem of In a preferred embodiment, an aquatic plant is utilized to recalcitrant lignocellulosics by bypassing the bottleneck create a designer plant. Aquatic plants, also called hydro problem of the biomass technology. As shown in FIG. 1, for 35 phytic plants, are plants that live in or on aquatic environ example, the photosynthetic process in a designer organism ments, such as in water (including on or under the water effectively uses the reducing power (NADPH) and energy Surface) or permanently Saturated soil. As used herein, (ATP) from the photosynthetic water splitting and proton aquatic plants include, for example, algae, blue-green algae gradient-coupled electron transport process for immediate (cyanobacteria and oxychlorobacteria), Submersed aquatic synthesis of butanol (CHCHCHCH-OH) directly from 40 herbs (Hydrilla verticillate, Elodea densa, Hippuris vulgaris, carbon dioxide (CO) and water (H2O) without being drained Aponogeton Boivinianus Aponogeton Rigidifolius, Aponoge into the other pathway for synthesis of the undesirable ligno ton Longiplunulosus, Didiplis Diandra, Vesicularia cellulosic materials that are very hard and often inefficient for Dubyana, Hygrophilia Augustifolia, Micranthemum Umbro the biorefinery industry to use. This approach is also different sum, Eichhornia Azurea, Saururus Cernutus, Cryptocoryne from the existing "cornstarch butanol production' process. In 45 Lingua, Hydrotriche Hottoniiflora Eustralis Stellata, Vallis accordance with this invention, butanol can be produced neria Rubra, Hygrophila Salicifolia, Cyperus Helferi, Cryp directly from carbon dioxide (CO) and water (HO) without tocoryne Petchii, Vallisneria americana, Vallisneria Torta, having to go through many of the energy consuming steps that Hydrotriche Hottoniiflora, Crassula Helmsii, Limnophila the cornstarch butanol-production process has to go through, Sessiliflora, Potamogeton Perfoliatus, Rotala Wallichii, including corn crop cultivation, corn-grain harvesting, corn 50 Cryptocoryne Becketii, Blyxia Aubertii, Hygrophila Diform grain cornstarch processing, and starch-to-Sugar-to-butanol mis), duckweeds (Spirodella polyrrhiza, Wolfia globosa, fermentation. As a result, the photosynthetic butanol-produc Lenna trisulca, Lenna gibba, Lemma minor; Landoltia punc tion technology of the present invention is expected to have a tata), water cabbage (Pistia stratiotes), buttercups (Ranuncu much (more than 10-times) higher Solar-to-butanol energy lus), water caltrop (Trapa natans and Trapa bicornis), water conversion efficiency than the current technology. Assuming 55 lily (Nymphaea lotus, Nymphaeaceae and Nelumbonaceae), a 10% solar energy conversion efficiency for the proposed water hyacinth (Eichhornia crassipes), Bolbitis heudelotii, photosynthetic butanol production process, the maximal Cabomba sp., Seagrasses (Heteranthera Zosterifolia, Posi theoretical productivity (yield) could be about 72,700 kg of doniaceae, Zosteraceae, Hydrocharitaceae, and Cymod butanol per acre per year, which could support about 70 cars oceaceae). Butanol (and/or related higher alcohols) produced (per year per acre). Therefore, this invention could bring a 60 from an aquatic plant can diffuse into water, permitting nor significant capability to the Society in helping to ensure mal growth of the plants and more robust production of energy security. The present invention could also help protect butanol from the plants. Liquid cultures of aquatic plant tis the Earth's environment from the dangerous accumulation of Sues (including, but not limited to, multicellular algae) or CO in the atmosphere, because the present methods convert cells (including, but not limited to, unicellular algae) are also CO directly into clean butanol energy. 65 highly preferred for use, since the butanol (and/or related A fundamental feature of the present methodology is uti higher alcohols) molecules produced from a designer butanol lizing a plant (e.g., an alga or oxyphotobacterium) or plant (and/or related higher alcohols) production pathway(s) can US 8,986,963 B2 9 10 readily diffuse out of the cells or tissues into the liquid water Cylindrotheca, Navicula, Thalassiosira, and Phaeodactylum. medium, which can serve as a large pool to store the product Preferred species of algae for use in the present invention butanol (and/or related higher alcohols) that can be subse include Chlamydomonas reinhardtii, Platymonas subcordi quently harvested by filtration and/or distillation/evaporation formis, Chlorella fisca, Chlorella sorokiniana, Chlorella vul techniques. 5 garis, Chlorella ellipsoidea, Chlorella spp., Dunaliella Although aquatic plants or cells are preferred host organ salina, Dunaliella viridis, Dunaliella bardowil, Haemato isms for use in the methods of the present invention, tissue coccus pluvialis, Parachlorella kessleri, Betaphycus gelati and cells of non-aquatic plants, which are photosynthetic and num, Chondrus crispus, Cyanidioschyzon merolae, Cya can be cultured in a liquid culture medium, can also be used to nidium caldarium, Galdieria sulphuraria, Gelidiella create designer tissue or cells for photosynthetic butanol 10 acerosa, Graciliaria changii, Kappaphycus alvarezii, Por (and/or related higher alcohols) production. For example, the phyra miniata, Ostreococcus tauri, Porphyra yezoensis, Por following tissue or cells of non-aquatic plants can also be phyridium sp., Palmaria palmata, Graciliaria spp., Isochrysis selected for use as a host organism in this invention: the galbana, Kappaphycus spp., Laminaria japonica, Laminaria photoautotrophic shoot tissue culture of wood apple tree spp., Monostroma spp., Nannochloropsis oculata, Porphyra Feronia limonia, the chlorophyllous callus-cultures of corn 15 spp., Porphyridium spp., Undaria pinnatifida, Ulva lactuca, plant Zea mays, the green root cultures of Asteraceae and Ulva spp., Undaria spp., Phaeodactylum Tricornutum, Nav Solanaceae species, the tissue culture of Sugarcane stalk icula saprophila, Crypthecodinium cohnii, Cylindrotheca parenchyma, the tissue culture of bryophyte Physcomitrella fiusiformis, Cyclotella cryptica, Euglena gracilis, Amphi patens, the photosynthetic cell Suspension cultures of Soy dinium sp., Symbiodinium microadriaticum, Macrocystis bean plant (Glycine max), the photoautotrophic and photo pyrifera, Ankistrodesmus braunii, and Scenedesmus obliq mixotrophic culture of green Tobacco (Nico?iana tabacum L.) S. cells, the cell Suspension culture of Gisekiapharmaceoides (a Preferred species of blue-green algae (oxyphotobacteria C plant), the photosynthetic suspension cultured lines of including cyanobacteria and oxychlorobacteria) for use in the Amaranthus powellii Wats. Datura innoxia Mill. Gos present invention include Thermosynechococcus elongatus sypium hirsutum L., and Nicotiana tabacum X Nicotiana glu 25 BP-1, Nostoc sp. PCC 7120, Synechococcus elongatus PCC tinosa L. fusion hybrid. 6301, Syncechococcus sp. strain PCC 7942, Syncechococcus By “liquid medium' is meant liquid water plus relatively sp. strain PCC 7002, Syncechocystis sp. strain PCC 6803, Small amounts of inorganic nutrients (e.g., N. P. Ketc, com Prochlorococcus marinus MED4, Prochlorococcus marinus monly in their salt forms) for photoautotrophic cultures; and MIT 9313, Prochlorococcus marinus NATL1A, Prochloro Sometimes also including certain organic Substrates (e.g., 30 coccus SS120, Spirulina platensis (Arthrospira platensis), Sucrose, glucose, or acetate) for photomixotrophic and/or Spirulina pacifica, Lyngbya majuscule, Anabaena sp., Syn photoheterotrophic cultures. echocystis sp., Synechococcus elongates, Synechococcus In an especially preferred embodiment, the plant utilized in (MC-A), Trichodesmium sp., Richelia intracellularis, Syn the butanol (and/or related higher alcohols) production echococcus WH7803, Synechococcus WH8102, Nostoc method of the present invention is an alga or a blue-green 35 punctiforme, Syncechococcus sp. strain PCC 7943, Syn alga. The use of algae and/or blue-green algae has several echocyitis PCC 6714 phycocyanin-deficient mutant PD-1, advantages. They can be grown in an open pond at large Cyanothece strain 51142, Cyanothece sp. CCYO110, Oscil amounts and low costs. Harvest and purification of butanol latoria limosa, Lyngbya majuscula, Symploca muscorum, (and/or related higher alcohols) from the water phase is also Gloeobacter violaceus, Prochloron didemni, Prochlorothrix easily accomplished by distillation/evaporation or membrane 40 hollandica, Synechococcus (MC-A), Trichodesmium sp., separation. Richelia intracellularis, Prochlorococcus marinus, Prochlo Algae suitable for use in the present invention include both rococcus SS120, Synechococcus WH8102, Lyngbya maius unicellular algae and multi-unicellular algae. Multicellular cula, Symploca muscorum, Synechococcus bigranulatus, algae that can be selected for use in this invention include, but cryophilic Oscillatoria sp., Phormidium sp., Nostoc sp.-1, are not limited to, Seaweeds Such as Ulva latissima (sea let 45 Calothrix parietina, thermophilic Synechococcus bigranula tuce), Ascophyllum nodosum, Codium fragile, Fucus vesicu tus, Synechococcus lividus, thermophilic Mastigocladus losus, Eucheuma denticulatum, Graciliaria gracilis, Hydrod laminosus, Chlorogloeopsis fritschii PCC 6912, Synechococ ictyon reticulatum, Laminaria japonica, Undaria pinntifida, cus vulcanus, Synechococcus sp. strain MA4, Synechococcus Saccharina japonica, Porphyra yezoensis, and Porphyra ten sp. Strain MA19, and Thermosynechococcus elongatus. era. Suitable algae can also be chosen from the following 50 Proper selection of host photosynthetic organisms for their divisions of algae: green algae (Chlorophyta), red algae genetic backgrounds and certain special features is also ben (Rhodophyta), brown algae (Phaeophyta), diatoms (Bacilla eficial. For example, a photosynthetic-butanol-producing riophyta), and blue-green algae (Oxyphotobacteria including designer alga created from cryophilic algae (psychrophiles) Cyanophyta and Prochlorophytes). Suitable orders of green that can grow in Snow and ice, and/or from cold-tolerant host algae include Ulvales, Ulotrichales, Volvocales, Chlorellales, 55 strains such as Chlamydomonas cold strain CCMG1619, Schizogoniales, Oedogoniales, Zygnematales, Cladopho which has been characterized as capable of performing pho rales, Siphonales, and Dasycladales. Suitable genera of tosynthetic water splitting as cold as 4°C. (Lee, Blankinship Rhodophyta are Porphyra, Chondrus, Cyanidioschyzon, Por and Greenbaum (1995), “Temperature effect on production of phyridium, Graciliaria, Kappaphycus, Gielidium and Agar hydrogen and oxygen by Chlamydomonas cold strain dhiella. Suitable genera of Phaeophyta are Laminaria, 60 CCMP1619 and wild type 137c. Applied Biochemistry and Undaria, Macrocystis, Sargassum and Dictyosiphon. Suit Biotechnology 51/52:379-386), permits photobiological able genera of Cyanophyta (also known as Cyanobacteria) butanol production even in cold seasons or regions such as include (but not limited to) Phoridium, Synechocystis, Syn Canada. Meanwhile, a designer alga created from a thermo cechococcus, Oscillatoria, and Anabaena. Suitable genera of philic/thermotolerant photosynthetic organism such as ther Prochlorophytes (also known as oxychlorobacteria) include 65 mophilic algae Cyanidium caldarium and Galdieria sul (but not limited to) Prochloron, Prochlorothrix, and Prochlo phuraria and/or thermophilic cyanobacteria (blue-green rococcus. Suitable genera of Bacillariophyta are Cyclotella, algae) Such as Thermosynechococcus elongatus BP-1 and US 8,986,963 B2 11 12 Synechococcus bigranulatus may permit the practice of this ate product of the Calvin cycle the designer butanol-produc invention to be well extended into the hot seasons or areas tion pathway branches off from the Calvin cycle. such as Mexico and the Southwestern region of the United In one example, a designer pathway is created that takes States including Nevada, California, Arizona, New Mexico glyceraldehydes-3-phosphate and converts it into butanol by and Texas, where the weather can often be hot. Furthermore, using, for example, a set of enzymes consisting of as shown a photosynthetic-butanol-producing designer alga created with the numerical labels 01-12 in FIG. 1, glyceraldehyde-3- from a marine alga, such as Platymonas subcordiformis, per phosphate dehydrogenase 01, phosphoglycerate kinase 02, mits the practice of this invention using seawater, while the phosphoglycerate mutase 03, enolase 04, pyruvate kinase 05, designer alga created from a freshwater alga Such as Chlamy pyruvate-ferredoxin oxidoreductase 06, thiolase 07, 3-hy 10 droxybutyryl-CoA dehydrogenase 08, crotonase 09, butyryl domonas reinhardtii can use freshwater. Additional optional CoA dehydrogenase 10, butyraldehyde dehydrogenase 11, features of a photosynthetic butanol (and/or related higher and butanol dehydrogenase 12. In this glyceraldehydes-3- alcohols) producing designer alga include the benefits of phosphate-branched designer pathway, for conversion of two reduced chlorophyll-antenna size, which has been demon molecules of glyceraldehyde-3-phosphate to butanol, two strated to provide higher photosynthetic productivity (Lee, 15 NADH molecules are generated from NAD" at the step from Mets, and Greenbaum (2002). “Improvement of photosyn glyceraldehyde-3-phosphate to 1,3-diphosphoglycerate cata thetic efficiency at high light intensity through reduction of lyzed by glyceraldehyde-3-phosphate dehydrogenase 01: chlorophyll antenna size. Applied Biochemistry and Bio meanwhile two molecules of NADH are converted to NAD": technology, 98-100: 37-48) and butanol-tolerance (and/or one at the step catalyzed by 3-hydroxybutyryl-CoA dehydro related higher alcohols-tolerance) that allows for more robust genase 08 in reducing acetoacetyl-CoA to 3-hydroxybutyryl and efficient photosynthetic production of butanol (and/or CoA and another at the step catalyzed by butyryl-CoA dehy related higher alcohols) from CO and H2O. By use of a drogenase 10 in reducing crotonyl-CoA to butyryl-CoA. phycocyanin-deficient mutant of Synechocystis PCC 6714, it Consequently, in this glyceraldehydes-3-phosphate has been experimentally demonstrated that photoinhibition branched designer pathway (01-12), the number of NADH can be reduced also by reducing the content of light-harvest 25 molecules consumed is balanced with the number of NADH ing pigments (Nakajima, Tsuzuki, and Ueda (1999) molecules generated. Furthermore, both the pathway step “Reduced photoinhibition of a phycocyanin-deficient mutant catalyzed by butyraldehyde dehydrogenase 11 (in reducing of Synechocystis PCC 6714, Journal of Applied Phycology butyryl-CoA to butyraldehyde) and the terminal step cata 10: 447-452). These optional features can be incorporated lyzed by butanol dehydrogenase 12 (in reducing butyralde into a designer alga, for example, by use of abutanol-tolerant 30 hyde to butanol) can use NADPH, which can be regenerated and/or chlorophyll antenna-deficient mutant (e.g., Chlamy by the photosynthetic water splitting and proton gradient domonas reinhardtii strain DS521) as a host organism, for coupled electron transport process. Therefore, this glyceral gene transformation with the designer butanol-production dehydes-3-phosphate-branched designer butanol-production pathway genes. Therefore, in one of the various embodi pathway can operate continuously. ments, a host alga is selected from the group consisting of 35 In another example, a designer pathway is created that green algae, red algae, brown algae, blue-green algae (oxy takes the intermediate product, 3-phosphoglycerate, and con photobacteria including cyanobacteria and prochlorophytes), verts it into butanol by using, for example, a set of enzymes diatoms, marine algae, freshwater algae, unicellular algae, consisting of (as shown with the numerical labels 03-12 in multicellular algae, seaweeds, cold-tolerant algal strains, FIG. 1) phosphoglycerate mutase 03, enolase 04, pyruvate heat-tolerant algal strains, light-harvesting-antenna-pig 40 kinase 05, pyruvate-ferredoxin oxidoreductase 06, thiolase ment-deficient mutants, butanol-tolerant algal strains, higher 07, 3-hydroxybutyryl-CoA dehydrogenase 08, crotonase 09. alcohols-tolerant algal strains, and combinations thereof. butyryl-CoA dehydrogenase 10, butyraldehyde dehydroge Creating a Designer Butanol-Production Pathway in a Host nase 11, and butanol dehydrogenase 12. It is worthwhile to Selecting Appropriate Designer Enzymes note that the last ten enzymes (03-12) of the glyceraldehydes One of the key features in the present invention is the 45 3-phosphate-branched designer butanol-producing pathway creation of a designer butanol-production pathway to tame (01-12) are identical with those utilized in the 3-phospho and work with the natural photosynthetic mechanisms to glycerate-branched designer pathway (03-12). In other achieve the desirable synthesis of butanol directly from CO words, the designer enzymes (01-12) of the glyceraldehydes and H.O.The natural photosynthetic mechanisms include (1) 3-phosphate-branched pathway permit butanol production the process of photosynthetic water splitting and proton gra 50 from both the point of 3-phosphoglycerate and the point dient-coupled electron transport through the thylakoid mem glyceraldehydes 3-phosphate in the Calvin cycle. These two brane, which produces the reducing power (NADPH) and pathways, however, have different characteristics. Unlike the energy (ATP), and (2) the Calvin cycle, which reduces CO by glyceraldehyde-3-phosphate-branched butanol-production consumption of the reducing power (NADPH) and energy pathway, the 3-phosphoglycerate-branched pathway which (ATP). 55 consists of the activities of only ten enzymes (03-12) could In accordance with the present invention, a series of not itself generate any NADH that is required for use at two enzymes are used to create a designer butanol-production places: one at the step catalyzed by 3-hydroxybutyryl-CoA pathway that takes an intermediate product of the Calvin dehydrogenase 08 in reducing acetoacetyl-CoA to 3-hy cycle and converts the intermediate product into butanol as droxybutyryl-CoA, and another at the step catalyzed by illustrated in FIG. 1. A “designer butanol-production-path 60 butyryl-CoA dehydrogenase 10 in reducing crotonyl-CoA to way enzyme’ is hereby defined as an enzyme that serves as a butyryl-CoA. That is, if (or when) a 3-hydroxybutyryl-CoA catalyst for at least one of the steps in a designer butanol dehydrogenase and/or abutyryl-CoA dehydrogenase that can production pathway. According to the present invention, a use strictly only NADH but not NADPH is employed, it number of intermediate products of the Calvin cycle can be would require a supply of NADH for the 3-phosphoglycerate utilized to create designer butanol-production pathway(s): 65 branched pathway (03-12) to operate. Consequently, in order and the enzymes required for a designer butanol-production for the 3-phosphoglycerate-branched butanol-production pathway are selected depending upon from which intermedi pathway to operate, it is important to use a 3-hydroxybutyryl US 8,986,963 B2 13 14 CoA dehydrogenase 08 and abutyryl-CoA dehydrogenase 10 catalytic function but may or may not have exactly the same that can use NADPH which can be supplied by the photo protein structures. The most essential feature of an enzyme is driven electron transport process. Therefore, it is a preferred its active site that catalyzes the enzymatic reaction. There practice to use a 3-hydroxybutyryl-CoA dehydrogenase and a fore, certain enzyme-protein fragment(s) or Subunit(s) that butyryl-CoA dehydrogenase that can use NADPH or both contains such an active catalytic site may also be selected for NADPH and NADH (i.e., NAD(P)H) for this 3-phosphoglyc use in this invention. For various reasons, some of the natural erate-branched designer butanol-production pathway (03-12 enzymes contain not only the essential catalytic structure but in FIG. 1). Alternatively, when a 3-hydroxybutyryl-CoA also other structure components that may or may not be dehydrogenase and a butyryl-CoA dehydrogenase that can desirable for a given application. With techniques of bioin use only NADH are employed, it is preferably here to use an 10 formatics-assisted molecular designing, it is possible to select additional embodiment that can confer an NADPH/NADH the essential catalytic structure(s) for use in construction of a conversion mechanism (to supply NADH by converting designer DNA construct encoding a desirable designer NADPH to NADH, see more detail later in the text) in the enzyme. Therefore, in one of the various embodiments, a designer organism to facilitate photosynthetic production of designer enzyme gene is created by artificial synthesis of a butanol through the 3-phosphoglycerate-branched designer 15 DNA construct according to bioinformatics-assisted molecu pathway. lar sequence design. With the computer-assisted synthetic In still another example, a designer pathway is created that biology approach, any DNA sequence (thus its protein struc takes fructose-1,6-diphosphate and converts it into butanol by ture) of a designer enzyme may be selectively modified to using, as shown with the numerical labels 20-33 in FIG. 1, a achieve more desirable results by design. Therefore, the terms set of enzymes consisting of aldolase 20, triose phosphate "designer modified sequences” and “designer modified isomerase 21, glyceraldehyde-3-phosphate dehydrogenase enzymes' are hereby defined as the DNA sequences and the 22, phosphoglycerate kinase 23, phosphoglycerate mutase enzyme proteins that are modified with bioinformatics-as 24, enolase 25, pyruvate kinase 26, pyruvate-NADP oxi sisted molecular design. For example, when a DNA construct doreductase (or pyruvate-ferredoxin oxidoreductase) 27, for a designer chloroplast-targeted enzyme is designed from thiolase 28, 3-hydroxybutyryl-CoA dehydrogenase 29, cro 25 the sequence of a mitochondrial enzyme, it is a preferred tonase 30, butyryl-CoA dehydrogenase 31, butyraldehyde practice to modify some of the protein structures, for dehydrogenase 32, and butanol dehydrogenase 33, with aldo example, by selectively cutting out certain structure compo lase 20 and triose phosphate isomerase 21 being the only two nent(s) Such as its mitochondrial transit-peptide sequence that additional enzymes relative to the glyceraldehydes-3-phos is not suitable for the given application, and/or by adding phate-branched designer pathway. The use of a pyruvate 30 certain peptide structures such as an exogenous chloroplast NADP" oxidoreductase 27 (instead of pyruvate-ferredoxin transit-peptide sequence (e.g., a 135-bp Rubisco small-Sub oxidoreductase) in catalyzing the conversion of a pyruvate unit transit peptide (RbcS2)) that is needed to confer the molecule to acetyl-CoA enables production of an NADPH, ability in the chloroplast-targeted insertion of the designer which can be used in some other steps of the butanol-produc protein. Therefore, one of the various embodiments flexibly tion pathway. The addition of yet one more enzyme in the 35 employs the enzymes, their isozymes, functional analogs, designer organism, phosphofructose kinase 19, permits the designer modified enzymes, and/or the combinations thereof creation of another designer pathway which branches off in construction of the designer butanol-production path from the point of fructose-6-phosphate of the Calvin cycle for way(s). the production of butanol. Like the glyceraldehyde-3-phos As shown in Table 1, many genes of the enzymes identified phate-branched butanol-production pathway, both the fruc 40 above have been cloned and/or sequenced from various tose-1,6-diphosphate-branched pathway (20-33) and the organisms. Both genomic DNA and/or mRNA sequence data fructose-6-phosphate-branched pathway (19-33) can them can be used in designing and synthesizing the designer DNA selves generate NADH for use in the pathway at the step constructs for transformation of a host alga, oxyphotobacte catalyzed by 3-hydroxybutyryl-CoA dehydrogenase 29 to rium, plant, plant tissue or cells to create a designer organism reduce acetoacetyl-CoA to 3-hydroxybutyryl-CoA and at the 45 for photobiological butanol production (FIG. 1). However, step catalyzed by butyryl-CoA dehydrogenase 31 to reduce because of possible variations often associated with various crotonyl-CoA to butyryl-CoA. In each of these designer Source organisms and cellular compartments with respect to a butanol-production pathways, the numbers of NADH mol specific host organism and its chloroplast/thylakoid environ ecules consumed are balanced with the numbers of NADH ment where the butanol-production pathway(s) is designed to molecules generated; and both the butyraldehyde dehydroge 50 work with the Calvin cycle, certain molecular engineering art nase 32 (catalyzing the step in reducing butyryl-CoA to work in DNA construct design including codon-usage opti butyraldehyde) and the butanol dehydrogenase 33 (cata mization and sequence modification is often necessary for a lyzing the terminal step in reducing butyraldehyde to butanol) designer DNA construct (FIG. 2) to work well. For example, can all use NADPH, which can be regenerated by the photo in creating a butanol-producing designer eukaryotic alga, if synthetic water splitting and proton gradient-coupled elec 55 the source sequences are from cytosolic enzymes (se tron transport process. Therefore, these designer butanol quences), a functional chloroplast-targeting sequence may be production pathways can operate continuously. added to provide the capability for a designer unclear gene Table 1 lists examples of the enzymes including those encoded enzyme to insert into a host chloroplast to confer its identified above for construction of the designer butanol function for a designer butanol-production pathway. Further production pathways. Throughout this specification, when 60 more, to provide the switchability for a designer butanol reference is made to an enzyme, such as, for example, any of production pathway, it is also important to include a func the enzymes listed in Table 1, it includes their isozymes, tional inducible promoter sequence Such as the promoter of a functional analogs, and designer modified enzymes and com hydrogenase (Hyd1) or nitrate reductase (Nial) gene, or binations thereof. These enzymes can be selected for use in nitrite reductase (nirA) gene in certain designer DNA con construction of the designer butanol-production pathways 65 struct(s) as illustrated in FIG. 2A to control the expression of (such as those illustrated in FIG. 1). The “isozymes or func designer gene(s). In addition, as mentioned before, certain tional analogs’ refer to certain enzymes that have the same functional derivatives or fragments of these enzymes (se US 8,986,963 B2 15 16 quences), chloroplast-targeting transit peptide sequences, invention. The arts in creating and using the designer organ and inducible promoter sequences can also be selected foruse isms are further described hereinbelow. in full, in part or in combinations thereof, to create the Table 1 lists examples of enzymes for construction of designer organisms according to various embodiments of this designer butanol-production pathways.

GenBank Accession Number, JGI Protein ID or Enzyme Source (Organism) Citation Butanol dehydrogenase Clostridium GenBank: AB257439; Saccharoperbativiacetonictim; AJ508920; AF112135; Propionibacterium feudenreichii; AF388671; AF157307; M96.946, Trichomonas vaginalis; Aeromonas M96945 hydrophila: Clostridium beijerinckii; Clostridium acetobutyllicum Butyraldehyde Clostridium GenBank: AY251646 dehydrogenase Saccharoperbativiacetonictim Butyryl-CoA Clostridium beijerinckii; Butyrivibrio GenBank: AF4940.18: dehydrogenase fibrisolvens; Butyrate-producing AB190764; DQ987697; Z92974 bacterium L2-50; Thermoanaerobacterium thermosaccharolyticum; Crotonase Clostridium beijerinckii; Butyrivibrio GenBank: AF4940.18: fibrisolvens; Butyrate-producing AB190764; DQ987697; Z92974 bacterium L2-50; Thermoanaerobacterium thermosaccharolyticum; 3-Hydroxybutyryl-CoA Clostridium beijerinckii; Butyrivibrio GenBank: AF4940.18: dehydrogenase fibrisolvens. Aiellomyces capsulatus; AB190764; XM OO1537366; Aspergiiitisfiinigatiis; Aspergilius XM 741533; XM OO1274776: clavatus; Neosartorya fischeri: XM 001262361; DQ987697; Butyrate-producing bacterium L2-50; BTOO1208; Z92974 Arabidopsis thaliana; Thermoanaerobacterium thermosaccharolyticum; Thiolase Butyrivibriofibrisolvens; butyrate GenBank: AB190764: producing bacterium L2-50; DQ987697; Z92974 Thermoanaerobacterium thermosaccharolyticum; Glyceraldehyde-3- Mesostigma viride cytosol; Triticum GenBank: DQ873404; phosphate aestivum cytosol; Chlamydomonas EF5921.80; L27668; dehydrogenase reinhardtii chloroplast: Botryotinia XM OO1549497: JO1324; fickeiana; Saccharomyces cerevisiae; M18802; EU078558; Zymomonas mobilis; Karenia brevis; XM OO1539393; Aieliomyces capsulatus; Pichia Stipitis; XM OO138.6423, Pichia guilliermondii; Kluyveromyces XM OO1386568; marxiantis, Tritici in aestivitin: XM 001485596; DQ681.075; Arabidopsis thaliana; Zea mayS EF5921.80; NM 101214; cytosolic U45857, ZMU45856, U45855 Phosphoglycerate kinase Chlamydomonas reinhardtii GenBank: U14912, AF244144: chloroplast; Plasmodium vivax; XM OO1614707; Babesia bovis; Botryotinia fuckeiana; XM OO1610679; Monocercomonoides sp.: XM 001548271; DQ665858; Lodderomyces elongisportis; Pichia XM 001523843; guilliermondii; Arabidopsis thaliana; XM 001484377; NM 179576; Heianthus annuals; Oryza sativa; DQ835564; EF122488; Dictyostelium discoideum; Eugiena AF316577; AY647236; gracilis; Chondrits crispiis; AYO29776: AF 108452; Phaeodactylum tricornutum: Solanum AFO73473 itiberosum Phosphoglycerate Chlamydomonas reinhardtii JGI Chlre2 protein ID 161689, mutase cytoplasm; Aspergilius finigatus; GenBank: AF268078: (phosphoglyceromutase) Coccidioides immitis: Leishmania XM 747847; XM 749597; braziliensis; Aiellomyces capsulatus; XM OO1248115; Monocercomonoides sp.: Aspergilius XM 001569263; clavatiis; Arabidopsis thaliana; Zea XM 00153.9892; DQ665859; mayS XM OO1270940; NM 117020; M80912 Enolase Chlamydomonas reinhardtii GenBank: X66412, P31683; cytoplasm; Arabidopsis thaliana; AK222035; DQ221745; Leishmania Mexicana; Lodderomyces XM 001528071; elongisportis; Babesia bovis; XM OO1611873; Scierotinia Scierotiorum: Pichia XM OO1594215; guilliermondii; Spirotrichonympha XM 001483612; AB221057; leidvi; Oryza sativa: Trimastix EF122486, UO9450; DQ845796; pyriformis; Leticonostoc ABO88633; U82438; D64113; mesenteroides; Davidiella tassiana; U13799; AY307449; U17973 Aspergilius oryzae; Schizosaccharomyces pombe; Brassica naptis; Zea mays US 8,986,963 B2 17 18 -continued

GenBank Accession Number, JGI Protein ID or Enzyme Source (Organism) Citation Pyruvate kinase Chlamydomonas reinhardtii JGI Chlre3 protein ID 138105; cytoplasm; Arabidopsis thaliana; GenBank: AK229638; Saccharomyces cerevisiae; Babesia AY949876, AY949890, bovis; Scierotinia Scierotiorum: AY949888; XM OO1612087; Trichomonas vaginalis; Pichia XM OO1594.710; guilliermondii; Pichia stipitis; XM OO1329865; Lodderomyces elongisports; XM 001487289; Coccidioides immitis; Timastix XM OO1384591; pyriformis; Glycine max (soybean) XM OO1528210; XM 001240868; DQ845797; LO8632 Phosphofructose kinase Chlamydomonas reinhardtii: JGI Chlre2 protein ID 1594.95: Arabidopsis thaliana; Aiellomyces GenBank: NM 001037043, capsulatus; Yarrowia lipolytica; Pichia NM 179694, NM 119066, stipitis; Dictyostelium discoidetim; NM 125551; XM OO1537193; Tetrahymena thermophila: AY142710; XM OO1382.359, Trypanosoma brucei: Plasmodium XM OO1383.014: XM 639070; falcipartin; Spinacia oleracea; XM 001017610; XM 838827; XM 001347929; DQ437575; Fructose-diphosphate Chlamydomonas reinhardtii GenBank: X69969; AF308587: aldolase chloroplast; Fragaria Xananassa NM 005165; XM OO1609195; cytoplasm; Homo Sapiens; Babesia XM 001312327, bovis; Trichomonas vaginalis; Pichia XM 001312338; stipitis; Arabidopsis thaliana XM OO1387466; NM 120057, NM 001036644 Triose phosphate Arabidopsis thaliana; Chlamydomonas GenBank: NM 127687, isomerase reinhardtii; Scierotinia Scierotiorum: AF247559; AY742323; Chlorella pyrenoidosa; Pichia XM OO1587391; AB240149; guilliermondii; Eugiena intermedia: XM 001485684; DQ459379; Eugiena longa; Spinacia oleracea; AY742325; L36387: AY438596; Soianum Chacoense; Hordeum vulgare: U83414: EF575877; Oryza sativa Glucose-1-phosphate Arabidopsis thaliana; Zea mays; GenBank: NM 127730, adenylyltransferase Chlamydia trachomatis; Soianum NM 124205, NM 121927, tuberosum (potato); Shigella flexneri: AYO59862; EF694839, Lycopersicon escientiin EF694838: AFO87165; P55242; NP 709206; TO7674 Starch synthase Chlamydomonas reinhardtii: GenBank: AF026422, AF026421, Phaseolus vulgaris; Oryza sativa; DQ019314, AF433156; Arabidopsis thaliana; Colocasia AB293998; D16202, AB115917, escienta; Amaranthus crientiis; AY2994.04: AF121673, Parachiorelia kessieri: Titicum AK226881; NM 101044: aestivain; Sorghum bicolor; Astragalus AY225862, AY142712; membranaceus; Perilla frutescens: Zea DQ178026; AB232549;Y16340; mays; Ipomoea batatas AF168786; AFO97922: AF210699; AFO 19297; AFO 68834 Alpha-amylase Hordeum vulgare aleurone cells; GenBank: JO4202; Trichomonas vaginalis; Phanerochaete XM 001319100; EF143986: chrysosporium; Chlamydomonas AY324649; NM 129551; reinhardtii; Arabidopsis thaliana; XO7896 Dictyoglomus thermophilum heat stable amylase gene; Beta-amylase Arabidopsis thaliana; Hordeum GenBank: NM 113297: D21349; vulgare; Musa actiminata DQ166026 Starch phosphorylase Citrus hybrid cultivar root; Solanum Genbank: AYO98895; P53535: tuberosum chloroplast; Arabidopsis NM 113857, NM 114564: thaliana; Tritictim aestivum; Ipomoea AF27.5551; M64362 batatas Phosphoglucomutase Oryza sativa plastid; Aiellomyces GenBank: AC105932, capsulatiis; Pichia Stipitis; AF455812; XM OO1536436; Lodderomyces elongisports; XM OO1383281; Aspergilius finigatus; Arabidopsis XM OO1527445; XM 749345; thaliana; Popitius to mentosa; Oryza NM 124561, NM 180508, Saiiva; Zea mays AY128901; AY479974; AF455812; U89342, U89341 Glucosephosphate Chlamydomonas reinhardtii: JGI Chlre3 protein ID 135202; (glucose-6-phosphate) Saccharomyces cerevisiae; Pichia GenBank: M21696; isomerase stipitis; Aiellomyces capsulatus; XM OO1385873; Spinacia oleracea cytosol; Oryza XM OO1537043; TO9154; Saiiva cytoplasm; Arabidopsis P42862; NM 123638, thaliana; Zea mayS NM 118595; U17225 US 8,986,963 B2 19 20 -continued

GenBank Accession Number, JGI Protein ID or Enzyme Source (Organism) Citation Hexokinase Aiellomyces capsulatus; Pichia Stipitis; GenBank: XM OO1541513; (glucokinase) Pichia angusta; Thermosynechococcus XM OO138.6652, AY278027; elongates; Babesia bovis; Solantin XM OO1386035; NC 004113; Chacoense; Oryza sativa; Arabidopsis XM OO1608698; DQ177440; thaiiana DQ116383; NM 112895 NADP(H) phosphatase Methanococcus jannaschii The Journal Of Biological Chemistry 280 (47): 39200-39207 (2005) NAD kinase Babesia bovis; Trichomonas vaginalis GenBank: XM OO1609395; XM OO1324239 Pyruvate-NADP Peranema trichophorum; Eugiena GenBank: EF114757: oxidoreductase gracilis AB021127, AJ278425 Pyruvate-ferredoxin Mastigamoeba balamuthi: GenBank: AY101767; YO9702; oxidoreductase Desulfovibrio africanus; Entamoeba U3O149; XM OO1582310, histolytica; Trichomonas vaginalis; XM 001313670, Cryptosporidium parvum; XM OO1321286, Cryptosporidium baileyi; Giardia XM 001307087, iambia: Entamoeba histolytica; XM 001311860, Hydrogenobacter thermophilus; XM 001314776, Clostridium pasteurianum; XM 001307250; EFO30517; EFO30516; XM 764947; XM 651927; AB042412; Y17727

Targeting the Designer Enzymes to the Stroma Region of to use nuclear-genome-encodable designer genes to confer a Chloroplasts Switchable butanol-production pathway. Consequently, Some of the designer enzymes discussed above. Such as, nucleic acids encoding for these enzymes also need to be pyruvate-ferredoxin oxidoreductase, thiolase, 3-hydroxybu 30 genetically engineered with proper sequence modification tyryl-CoA dehydrogenase, crotonase, butyryl-CoA dehydro Such that the enzymes are controllably expressed and are genase, butyraldehyde dehydrogenase, and butanol dehydro inserted into the chloroplasts to create a designer butanol genase are known to function in certain special bacteria Such production pathway. as Clostridium; but wild-type plant chloroplasts generally do According to one of the various embodiments, it is best to not possess these enzymes to function with the Calvin cycle. 35 express the designer butanol-producing-pathway enzymes Therefore, in one of the various embodiments in creating a only into chloroplasts (at the stroma region), exactly where butanol-producing eukaryotic designer organism, designer the action of the enzymes is needed to enable photosynthetic nucleic acids encoding for these enzymes are expressed in the production of butanol. If expressed without a chloroplast chloroplast(s) of a host cell. This can be accomplished by targeted insertion mechanism, the enzymes would just stay in delivery of designer butanol-production-pathway gene(s) 40 the cytosol and not be able to directly interact with the Calvin into the chloroplast genome of the eukaryotic host cell typi cycle for butanol production. Therefore, in addition to the cally using a genegun. In certain extent, the molecular genet obvious distinctive features in pathway designs and associ ics of chloroplasts are similar to that of cyanobacteria. After ated approaches, another significant distinction is that one of being delivered into the chloroplast, a designer DNA con the various embodiments innovatively employs a chloroplast struct that contains a pair of proper recombination sites as 45 targeted mechanism for genetic insertion of many designer illustrated in FIG. 2F can be incorporated into the chloroplast butanol-production-pathway enzymes into chloroplast to genome through a natural process of homologous DNA directly interact with the Calvin cycle for photobiological double recombination. butanol production. In another embodiment, nucleic acids encoding for these With a chloroplast stroma-targeted mechanism, the cells enzymes are genetically engineered Such that the enzymes 50 will not only be able to produce butanol but also to grow and expressed are inserted into the chloroplasts to operate with the regenerate themselves when they are returned to certain con Calvin cycle there. Depending on the genetic background of ditions under which the designer pathway is turned off. Such a particular host organism, some of the designer enzymes as under aerobic conditions when designer hydrogenase pro discussed above Such as phosphoglycerate mutase and eno moter-controlled butanol-production-pathway genes are lase may exist at Some background levels in its native form in 55 used. Designer algae, plants, or plant cells that contain normal a wild-type chloroplast. For various reasons including often mitochondria should be able to use the reducing power the lack of their controllability, however, some of the chloro (NADH) from organic reserves (and/or some exogenous plast background enzymes may or may not be sufficient to organic Substrate such as acetate or Sugar) to power the cells serve as a significant part of the designer butanol-production immediately after returning to aerobic conditions. Conse pathway(s). Furthermore, a number of useful inducible pro 60 quently, when the designer algae, plants, or plant cells are moters happen to function in the nuclear genome. For returned to aerobic conditions after use under anaerobic con example, both the hydrogenase (Hyd1) promoter and the ditions for photosynthetic butanol production, the cells will nitrate reductase (Nial) promoter that can be used to control stop making the butanol-producing-pathway enzymes and the expression of the designer butanol-production pathways start to restore the normal photoautotrophic capability by are located in the nuclear genome of Chlamydomonas rein 65 synthesizing new and functional chloroplasts. Therefore, it is hardtii, of which the genome has recently been sequenced. possible to use Such genetically engineered designer alga/ Therefore, in one of the various embodiments, it is preferred plant organisms for repeated cycles of photoautotrophic US 8,986,963 B2 21 22 growth under normal aerobic conditions and efficient produc Use of a Genetic Switch to Control the Expression of a tion of butanol directly from CO, and HO under certain Designer Butanol-Producing Pathway. specific designer butanol-producing conditions such as under Another key feature of the invention is the application of a anaerobic conditions and/or in the presence of nitrate when a genetic Switch to control the expression of the designer Nial promoter-controlled butanol-production pathway is butanol-producing pathway(s), as illustrated in FIG. 1. This used. switchability is accomplished through the use of an externally The targeted insertion of designer butanol-production inducible promoter so that the designer transgenes are induc pathway enzymes can be accomplished through use of a DNA ibly expressed under certain specific inducing conditions. sequence that encodes for a stroma 'signal’ peptide. A Preferably, the promoter employed to control the expression 10 of designer genes in a host is originated from the host itselfor stroma-protein signal (transit) peptide directs the transport a closely related organism. The activities and inducibility of a and insertion of a newly synthesized protein into stroma. In promoter in a host cell can be tested by placing the promoter accordance with one of the various embodiments, a specific in front of a reporting gene, introducing this reporter con targeting DNA sequence is preferably placed in between the struct into the host tissue or cells by any of the known DNA promoter and a designer butanol-production-pathway 15 delivery techniques, and assessing the expression of the enzyme sequence, as shown in a designer DNA construct reporter gene. (FIG. 2A). This targeting sequence encodes for a signal (tran In a preferred embodiment, the inducible promoter used to sit) peptide that is synthesized as part of the apoprotein of an control the expression of designer genes is a promoter that is enzyme in the cytosol. The transit peptide guides the insertion inducible by anaerobiosis, i.e., active under anaerobic condi of an apoprotein of a designer butanol-production-pathway tions but inactive under aerobic conditions. A designer alga/ enzyme from cytosol into the chloroplast. After the apopro plant organism can perform autotrophic photosynthesis using tein is inserted into the chloroplast, the transit peptide is CO as the carbon Source under aerobic conditions, and when cleaved off from the apoprotein, which then becomes an the designer organism culture is grown and ready for photo active enzyme. synthetic butanol production, anaerobic conditions will be A number of transit peptide sequences are suitable for use 25 applied to turn on the promoter and the designer genes that for the targeted insertion of the designer butanol-production encode a designer butanol-production pathway(s). pathway enzymes into chloroplast, including but not limited A number of promoters that become active under anaerobic to the transit peptide sequences of the hydrogenase apopro conditions are suitable for use in the present invention. For teins (such as HydA1 (Hyd1) and HydA2, GenBank acces example, the promoters of the hydrogenase genes (HydA1 30 (Hyd1) and HydA2, GenBank accession number: AJ308413, sion number AJ308413, AF289201, AY090770), ferredoxin AF289201, AYO90770) of Chlamydomonas reinhardtii, apoprotein (Frx1, accession numbers L10349, P07839), which is active under anaerobic conditions but inactive under thioredoxin m apoprotein (TrX2, X62335), glutamine syn aerobic conditions, can be used as an effective genetic Switch thase apoprotein (GS2, Q42689), LhcII apoproteins to control the expression of the designer genes in a host alga, (AB051210, AB051208, AB051205), PSII-T apoprotein 35 Such as Chlamydomonas reinhardtii. In fact, Chlamydomo (PsbT), PSII-S apoprotein (PsbS), PSII-W apoprotein nas cells contain several nuclear genes that are coordinately (PsbW), CFCF subunit-Yapoprotein (AtpC), CFCF sub induced under anaerobic conditions. These include the hydro unit-ö apoprotein (Atpl), U41442), CFOCF subunit-II apo genase structural gene itself (Hyd1), the Cyc6 gene encoding protein (AtpG), photosystem I (PSI) apoproteins (such as, of the apoprotein of Cytochrome C, and the CpX1 gene encod genes Psal D. PsaE, PsaF, PsaG, Psah, and Psak), Rubisco 40 ing coprogen oxidase. The regulatory regions for the latter SSU apoproteins (such as RbcS2, X04472). Throughout this two have been well characterized, and a region of about 100 specification, when reference is made to a transit peptide by proves Sufficient to confer regulation by anaerobiosis in sequence, such as, for example, any of the transit peptide synthetic gene constructs (Quinn, Barraco, Ericksson and sequence described above, it includes their functional ana Merchant (2000). “Coordinate copper- and oxygen-respon logs, modified designer sequences, and combinations thereof. 45 sive Cyc6 and Cpxl expression in Chlamydomonas is medi A “functional analog or “modified designer sequence' in ated by the same element.” J Biol Chem 275: 6080-6089). this context refers to a peptide sequence derived or modified Although the above inducible algal promoters may be suit (by, e.g., conservative Substitution, moderate deletion or addi able for use in other plant hosts, especially in plants closely tion of amino acids, or modification of side chains of amino related to algae, the promoters of the homologous genes from acids) based on a native transit peptide sequence, such as 50 these other plants, including higher plants, can be obtained those identified above, that has the same function as the native and employed to control the expression of designer genes in transit peptide sequence, i.e., effecting targeted insertion of a those plants. desired enzyme. In another embodiment, the inducible promoter used in the In certain specific embodiments, the following transit pep present invention is an algal nitrate reductase (Nial) pro tide sequences are used to guide the insertion of the designer 55 moter, which is inducible by growth in a medium containing butanol-production-pathway enzymes into the stroma region nitrate and repressed in a nitrate-deficient but ammonium of the chloroplast: the Hyd1 transit peptide (having the amino containing medium (Loppes and Radoux (2002) “Two short acid sequence: msalylkpca aysirgsscr arqvapraplaastVrvala regions of the promoter are essential for activation and repres tleaparrlgnvacaa (SEQ ID NO. 54)), the RbcS2 transit pep sion of the nitrate reductase gene in Chlamydomonas rein tides (having the amino acid sequence: maaviakSSV Saavar 60 hardtii,' Mol Genet Genomics 268: 42-48). Therefore, the pars Svrpmaalkpavkaapvaap aqanq (SEQID NO: 55)), ferre Nia1 (gene accession number AF203033) promoter can be doxin transit peptide (having the amino acid sequence: selected for use to control the expression of the designer mamamrs (SEQ ID NO. 56)), the CFCF subunit-8 transit genes in an alga according to the concentration levels of peptide (having the amino acid sequence: mlaaksiagp nitrate and ammonium in a culture medium. Additional rafkasavra apkagrrtVV Vma (SEQID NO: 57)), their analogs, 65 inducible promoters that can also be selected for use in the functional derivatives, designer sequences, and combinations present invention include, for example, the heat-shock pro thereof. tein promoter HSP70A (accession number: DQ05.9999, US 8,986,963 B2 23 24 AY456093, M98823: Schroda, Blocker, Beek (2000) The Bank: AAC17122), Synechococcus sp. WH 7805 (GenBank: HSP70A promoter as a tool for the improved expression of ZP 01124915), and Cyanothece sp. CCY0110 (GenBank: transgenes in Chlamydomonas. Plant Journal 21:121-131), ZP 01727861). the promoter of CablI-1 gene (accession number M24072), In yet another embodiment, an inducible promoter selected the promoter of Ca1 gene (accession number P20507), and for use is the light- and heat-responsive chaperone genegroE the promoter of Ca2 gene (accession number P24258). promoter, which can be induced by heat and/or light Kojima In the case of blue-green algae (oxyphotobacteria includ and Nakamoto (2007) “A novel light- and heat-responsive ing cyanobacteria and oxychlorobacteria), there are also a regulation of the groE transcription in the absence of HrcA or number of inducible promoters that can be selected for use in CIRCE in cyanobacteria.” FEBS Letters 581:1871-1880). A 10 number of groE promoters such as the groES and groEL the present invention. For example, the promoters of the (chaperones) promoters are available for use as an inducible anaerobic-responsive bidirectional hydrogenase hoX genes of promoter in controlling the expression of the designer Nostoc sp. PCC 7120 (GenBank: BAO00019), Prochlorothrix butanol-production-pathway enzymes. The groE promoter hollandica (GenBank: U884.00; hoxUYH operon promoter), sequences that can be selected and modified for use in one of Synechocystis sp. strain PCC 6803 (CyanoBase: s 111220 and 15 the various embodiments include (but not limited to) the s111223), Synechococcus elongatus PCC 6301 (CyanoBase: groES and/or groEL promoters of the following oxyphoto syc 1235c). Arthrospira platensis (GenBank: ABC26906), bacteria: Synechocystis sp. (GenBank: D12677.1), Syn Cyanothece sp. CCY0110 (GenBank: ZP 01727419) and echocystis sp. PCC 6803 (GenBank: BAO00022.2), Synecho Synechococcus sp. PCC 7002 (GenBank: AAN03566), which coccus elongatus PCC 6301 (GenBank: AP008231.1), are active under anaerobic conditions but inactive under aero Synechococcus sp (GenBank: M58751.1), Synechococcus bic conditions (Sjoholm, Oliveira, and Lindblad (2007) elongatus PCC 7942 (GenBank: CP000100.1), Nostoc sp. “Transcription and regulation of the bidirectional hydroge PCC 7120 (GenBank: BAO00019.2), Anabaena variabilis nase in the Cyanobacterium Nostoc sp. strain PCC 7120. ATCC 29413 (GenBank: CP000117.1), Anabaena sp. L-31 Applied and Environmental Microbiology, 73(17): 5435 (GenBank: AF324500); Thermosynechococcus elongatus 5446), can be used as an effective genetic switch to control the 25 BP-1 (CyanoBase: til.0185, thi0186), Synechococcus vulca expression of the designer genes in a host oxyphotobacte nus (GenBank: D78139), Oscillatoria sp. NKBG091600 rium, such as Nostoc sp. PCC 7120, Synechocystis sp. strain (GenBank: AF054630), Prochlorococcus marinus MIT9313 PCC 6803, Synechococcus elongatus PCC 6301, Cyanothece (GenBank: BX572099), Prochlorococcus marinus str. MIT sp. CCYO110, Arthrospira platensis, or Synechococcus sp. 9303 (GenBank: CP000554), Prochlorococcus marinus str. PCC 7002. 30 MIT 921 1 (GenBank: ZP 01006613), Synechococcus sp. In another embodiment in creating switchable butanol WH8102 (GenBank: BX569690), Synechococcus sp. production designer organisms such as switchable designer CC9605 (GenBank: CP000110), Prochlorococcus marinus oxyphotobacteria, the inducible promoter selected for use is a subsp. marinus str. CCMP 1375 (GenBank: AE017126), and nitrite reductase (nirA) promoter, which is inducible by Prochlorococcus marinus MED4 (GenBank: BX548174). growth in a medium containing nitrate and repressed in a 35 Additional inducible promoters that can also be selected nitrate-deficient but ammonium-containing medium (Qi. for use in the present invention include: for example, the Hao, Ng, Slater, Baszis, Weiss, and Valentin (2005) Appli metal (zinc)-inducible smt promoter of Synechococcus PCC cation of the Synechococcus nirA promoter to establish an 7942 (Erbe, Adams, Taylor and Hall (1996) “Cyanobacteria inducible expression system for engineering the Synechocys carrying an Smit-lux transcriptional fusion as biosensors for tis tocopherol pathway. Applied and Environmental Micro 40 the detection of heavy metal cations.” Journal of Industrial biology, 71 (10): 5678-5684; Maeda, Kawaguchi, Ohe, and Microbiology, 17:80-83); the iron-responsive idiA promoter Omata (1998) “cis-Acting sequences required for Ntch3-de of Synechococcus elongatus PCC 7942 (Michel, Pistorius, pendent, nitrite-responsive positive regulation of the nitrate and Golden (2001) “Unusual regulatory elements for iron assimilation operon in the Cyanobacterium Synechococcus deficiency induction of the idiA gene of Synechococcus elon sp. strain PCC 7942. Journal of Bacteriology, 180(16):4080 45 gatus PCC 7942' Journal of Bacteriology, 183(17):5015 4088). Therefore, the nirA promoter sequences can be 5024); the redox-responsive cyanobacterial crhR promoter selected for use to control the expression of the designer (Patterson-Fortin, Colvin and Owttrim (2006) “A Lex A-re genes in a number of oxyphotobacteria according to the con lated protein regulates redox-sensitive expression of the centration levels of nitrate and ammonium in a culture cyanobacterial RNA helicase, crh R', Nucleic Acids medium. The nirA promoter sequences that can be selected 50 Research, 34(12):3446-3454); the heat-shock gene hsp16.6 and modified for use include (but not limited to) the nirA promoter of Synechocystis sp. PCC 6803 (Fang and Barnum promoters of the following oxyphotobacteria: Synechococcus (2004) “Expression of the heat shock gene hsp16.6 and pro elongatus PCC 6301 (GenBank: AP008231, region 355890 moter analysis in the Cyanobacterium, Synechocystis sp. 255950), Synechococcus sp. (GenBank: X6768.0.1, PCC 6803," Current Microbiology 49:192-198); the small D16303.1, D12723.1, and D00677), Synechocystis sp. PCC 55 heat-shock protein (Hsp) promoter Such as Synechococcus 6803 (GenBank: NP 442378, BAO00022, AB001339, vulcanus gene hspA promoter (Nakamoto, Suzuki, and Roy D63999-D64006, D90899-D90917), Anabaena sp. (Gen (2000) “Constitutive expression of a small heat-shock protein Bank: X99708.1), Nostoc sp. PCC 7120 (GenBank: confers cellular thermotolerance and thermal protection to BAO00019.2 and AJ319648), Plectonema boryanum (Gen the photosynthetic apparatus in cyanobacteria.” FEBS Letters Bank: D31732.1), Synechococcus elongatus PCC 7942 (Gen 60 483:169-174); the CO-responsive promoters of oxyphoto Bank: P39661, CP000100.1), Thermosynechococcus elonga bacterial carbonic-anhydrase genes (GenBank: EAZ90903, tus BP-1 (GenBank: BAC08901, NP 682139), Phormidium EAZ90685, ZP 01624337, EAW33650, ABB17341, laminosum (GenBank: CAA79655, Q51879), Mastigocladus AAT41924, CA089711, ZP 00111671, YP 400464, laminosus (GenBank: ABD49353, ABD49351, ABD49349, AAC44830; and CyanoBase: all2929, PMT1568 slr0051, ABD49347), Anabaena variabilis ATCC 29413 (GenBank: 65 slr1347, and syco167c); the nitrate-reductase-gene (narB) YP 325032), Prochlorococcus marinus str. MIT 9303 (Gen promoters (such as GenBank accession numbers: Bank:YP 001018981), Synechococcus sp. WH 8103 (Gen BAC08907, NP 682145, AAO25121; ABI46326, US 8,986,963 B2 25 26 YP 732075, BAB72570, NP 484656); the green/red light designer butanol-production-pathway DNA construct, a responsive promoters such as the light-regulated cpcB2A2 transgenic designer alga that contains this DNA construct will promoter of Fremyella diplosiphon (Casey and Grossman be able to perform autotrophic photosynthesis using ambient (1994) “In vivo and in vitro characterization of the light air CO as the carbon Source and grows normally under aero regulated cpcB2A2 promoter of Fremyella diplosiphont' bic conditions. Such as in an open pond. When the algal Journal of Bacteriology, 176(20):6362-6374); and the UV culture is grown and ready for butanol production, the light responsive promoters of cyanobacterial genes leXA, designer transgene(s) can then be expressed by induction recA and ruvB (Domain, Houot, Chauvat, and Cassier-Chau under anaerobic conditions because of the use of the hydro vat (2004) “Function and regulation of the cyanobacterial genase promoter. The expression of designer gene(s) pro genes lex A, recA and ruvB: Lex A is critical to the survival of 10 duces a set of designer butanol-production-pathway enzymes cells facing inorganic carbon starvation. Molecular Micro to work with the Calvin cycle for photobiological butanol biology, 53(1):65-80). production (FIG. 1). Furthermore, in one of the various embodiments, certain The two PCR primers are a PCR forward primer (PCRFD “semi-inducible' or constitutive promoters can also be primer) located at the beginning (the 5' end) of the DNA selected for use in combination of an inducible promoter(s) 15 construct and a PCR reverse primer (PCRRE primer) located for construction of a designer butanol-production pathway(s) at the other end (the 3' end) as shown in FIG. 2A. This pair of as well. For example, the promoters of oxyphotobacterial PCR primers is designed to provide certain convenience Rubisco operon such as the rbcL genes (GenBank: X65960, when needed for relatively easy PCR amplification of the ZP 01728,542, Q3M674, BAF48766, NP 895035, designer DNA construct, which is helpful not only during and 0907262A; CyanoBase: PMT1205, PMMO550, Pro0551, after the designer DNA construct is synthesized in prepara tl11506, SYNW1718, glr2156, alr1524, slrO009), which have tion for gene transformation, but also after the designer DNA certain light-dependence but could be regarded almost as construct is delivered into the genome of a host alga for constitutive promoters, can also be selected for use in com Verification of the designer gene in the transformants. For bination of an inducible promoter(s) such as the nirA, hoX, example, after the transformation of the designer gene is and/or groE promoters for construction of the designer 25 accomplished in a Chlamydomonas reinhardtii-arg7 host cell butanol-production pathway(s) as well. using the techniques of electroporation and argininosuccinate Throughout this specification, when reference is made to lyase (arg7) complementation screening, the resulted trans inducible promoter, Such as, for example, any of the inducible formants can be then analyzed by a PCR DNA assay of their promoters described above, it includes their analogs, func nuclear DNA using this pair of PCR primers to verify whether tional derivatives, designer sequences, and combinations 30 the entire designer butanol-production-pathway gene (the thereof. A “functional analog or “modified designer DNA construct) is successfully incorporated into the genome sequence” in this context refers to a promoter sequence of a given transformant. When the nuclear DNA PCR assay of derived or modified (by, e.g., Substitution, moderate deletion a transformant can generate a PCR product that matches with or addition or modification of nucleotides) based on a native the predicted DNA size and sequence according to the promoter sequence, such as those identified hereinabove, that 35 designer DNA construct, the successful incorporation of the retains the function of the native promoter sequence. designer gene(s) into the genome of the transformant is veri DNA Constructs and Transformation into Host Organisms fied. DNA constructs are generated in order to introduce Therefore, the various embodiments also teach the associ designer butanol-production-pathway genes to a host alga, ated method to effectively create the designer transgenic plant, plant tissue or plant cells. That is, a nucleotide sequence 40 algae, plants, or plant cells for photobiological butanol pro encoding a designer butanol-production-pathway enzyme is duction. This method, in one of embodiments, includes the placed in a vector, in an operable linkage to a promoter, following steps: a) Selecting an appropriate host alga, plant, preferably an inducible promoter, and in an operable linkage plant tissue, or plant cells with respect to their genetic back to a nucleotide sequence coding for an appropriate chloro grounds and special features in relation to butanol produc plast-targeting transit-peptide sequence. In a preferred 45 tion; b) Introducing the nucleic acid constructs of the designer embodiment, nucleic acid constructs are made to have the genes into the genome of said hostalga, plant, plant tissue, or elements placed in the following 5' (upstream) to 3' (down plant cells; c) Verifying the incorporation of the designer stream) orientation: an externally inducible promoter, a tran genes in the transformed alga, plant, plant tissue, or plant cells sit targeting sequence, and a nucleic acid encoding a designer with DNA PCR assays using the said PCR primers of the butanol-production-pathway enzyme, and preferably an 50 designer DNA construct; d) Measuring and Verifying the appropriate transcription termination sequence. One or more designer organism features such as the inducible expression designer genes (DNA constructs) can be placed into one of the designer butanol-pathway genes for photosynthetic genetic vector. An example of Such a construct is depicted in butanol production from carbon dioxide and water by assays FIG. 2A. As shown in the embodiment illustrated in FIG. 2A, of mRNA, protein, and butanol-production characteristics a designer butanol-production-pathway transgene is a nucleic 55 according to the specific designer features of the DNA con acid construct comprising: a) a PCR forward primer; b) an struct(s) (FIG. 2A). externally inducible promoter; c) a transit targeting sequence; The above embodiment of the method for creating the d) a designer butanol-production-pathway-enzyme-encoding designer transgenic organism for photobiological butanol sequence with an appropriate transcription termination production can also be repeatedly applied for a plurality of sequence; and e) a PCR reverse primer. 60 operational cycles to achieve more desirable results. In vari In accordance with various embodiments, any of the com ous embodiments, any of the steps a) through d) of this ponents a) through e) of this DNA construct are adjusted to method described above are adjusted to suit for certain spe Suit for certain specific conditions. In practice, any of the cific conditions. In various embodiments, any of the steps a) components a) through e) of this DNA construct are applied through d) of the method are applied in full or in part, and/or in full or in part, and/or in any adjusted combination to 65 in any adjusted combination. achieve more desirable results. For example, when an algal Examples of designer butanol-production-pathway genes hydrogenase promoter is used as an inducible promoter in the (DNA constructs) are shown in the sequence listings. SEQID US 8,986,963 B2 27 28 NO: 1 presents a detailed DNA construct of a designer example 1, SEQID NO: 1, except that a Butyryl-CoA Dehy Butanol Dehydrogenase gene (1809 bp) that includes a PCR drogenase encoding sequence (427-1563) selected/modified FD primer (sequence 1-20), a 262-bp nitrate reductase Nial from the sequences of a Clostridium beijerinckii Butyryl promoter (21-282), a 135-bp RbcS2 transit peptide (283 CoA Dehydrogenase (AF494018) is used and restriction sites 417), an enzyme-encoding sequence (418-1566) selected and of Xho I Ndel and Xbal are added to make the key compo modified from a Clostridium saccharoperbutylacetonicum nents such as the targeting sequence (292-426) and the Butanol Dehydrogenase sequence (AB257.439), a 223-bp designer enzyme sequence (427-1563) as a modular unit that RbcS2 terminator (1567-1789), and a PCR RE primer (1790 can be flexible replaced when necessary to save cost of gene 1809). The 262-bp Nial promoter (DNA sequence 21-282) is synthesis and enhance work productivity. Please note, the used as an example of an inducible promoter to control the 10 expression of a designer butanol-production-pathway enzyme does not have to be Clostridium beijerinckii Butyryl Butanol Dehydrogenase gene (DNA sequence 418-1566). CoA Dehydrogenase; a number of butyryl-CoA dehydroge The 135-bp RbcS2 transit peptide (DNA sequence 283-417) nase enzymes (such as those listed in Table 1) including their is used as an example to guide the insertion of the designer isozymes, designer modified enzymes, and functional ana enzyme (DNA sequence 418-1566) into the chloroplast of the 15 logs from other sources such as Butyrivibrio fibrisolvens, host organism. The RbcS2 terminator (DNA sequence 1567 Butyrate producing bacterium L2-50, Thermoanaerobacte 1789) is employed so that the transcription and translation of rium thermosaccharolyticum, can also be selected for use. the designer gene is properly terminated to produce the SEQID NO. 4 presents example 4 for a designer Crotonase designer apoprotein (RbcS2 transit peptide-Butanol Dehy DNA construct (1482 bp) that includes a PCR FD primer drogenase) as desired. Because the Nial promoter is a nuclear (sequence 1-20), a 262-bp nitrate reductase promoter (21 DNA that can control the expression only for nuclear genes, 282), a 9-bp XhoI Ndel site (283-291) a 135-bp RbcS2 transit the synthetic butanol-production-pathway gene in this peptide (292-426), a Crotonase-encoding sequence (427 example is designed according to the codon usage of Chlamy 1209) selected/modified from the sequences of a Clostridium domonas nuclear genome. Therefore, in this case, the beijerinckii Crotonase (Genbank: AF494018), a 21-bp designer enzyme gene is transcribed in nucleus. Its mRNA is 25 Lumio-tag-encoding sequence (1210-1230), a 9-bp Xbal site naturally translocated into cytosol, where the mRNA is trans (1231-1239) containing a stop codon, a 223-bp RbcS2 termi lated to an apoprotein that consists of the RbcS2 transit pep nator (1240-1462), and a PCR RE primer (1463-1482) at the tide (corresponding to DNA sequence 283-417) with its 3' end. This DNA construct is similar to example 3, SEQID C-terminal end linked together with the N-terminal end of the NO: 3, except that a Crotonase-encoding sequence (427 Butanol Dehydrogenase protein (corresponding to DNA 30 1209) selected/modified from the sequences of a Clostridium sequence 418-1566). The transit peptide of the apoprotein beijerinckii Crotonase (Genbank: AF494018) is used and a guides its transportation across the chloroplast membranes 21-bp Lumio-tag-encoding sequence (1210-1230) is added at and into the stroma area, where the transit peptide is cut off the C-terminal end of the enolase sequence. The 21-bp from the apoprotein. The resulting Butanol Dehydrogenase Lumio-tag sequence (1210-1230) is employed here to encode then resumes its function as an enzyme for the designer 35 a Lumio peptide sequence Gly-Cys-Cys-Pro-Gly-Cys-Cys, butanol-production pathway in chloroplast. The two PCR which can become fluorescent when treated with a Lumio primers (sequences 1-20 and 1790-1809) are selected and reagent that is now commercially available from Invitrogen. modified from the sequence of a Human actingene and can be Lumio molecular tagging technology is based on an EDT paired with each other. Blasting the sequences against (1.2-ethanedithiol) coupled biarsenical derivative (the Lumio Chlamydomonas GenBank found no homologous sequences 40 reagent) of fluorescein that binds to an engineered tetracys of them. Therefore, they can be used as appropriate PCR teine sequence (Keppetipola, Coffman, and et al (2003). primers in DNA PCR assays for verification of the designer Rapid detection of in vitro expressed proteins using Lumi gene in the transformed alga. oTM technology, Gene Expression, 25.3: 7-11). The tetracys SEQID NO: 2 presents example 2 for a designer Butyral teine sequence consists of Cys-Cys-Xaa-Xaa-Cys-Cys, dehyde Dehydrogenase DNA construct (2067 bp) that 45 where Xaa is any non-cysteine amino acid such as Pro or Gly includes a PCRFD primer (sequence 1-20), a 262-bp nitrate in this example. The EDT-linked Lumio reagent allows free reductase Nial promoter (21-282), a 135-bp RbcS2 transit rotation of the arsenic atoms that quenches the fluorescence peptide (283-417), a Butyraldehyde Dehydrogenase-encod of fluorescein. Covalent bond formation between the thiols of ing sequence (418-1824) selected and modified from a the Lumio's arsenic groups and the tetracysteines prevents Clostridium saccharoperbutylacetonicum Butyraldehyde 50 free rotation of arsenic atoms that releases the fluorescence of Dehydrogenase sequence (AY251646), a 223-bp RbcS2 ter fluorescein (Griffin, Adams, and Tsien (1998), “Specific minator (1825-2047), and a PCR RE primer (2048-2067). covalent labeling of recombinant protein molecules inside This DNA construct is similar to example 1, SEQID NO: 1. live cells’, Science, 281:269-272). This also permits the visu except that a Butyraldehyde Dehydrogenase-encoding alization of the tetracysteine-tagged proteins by fluorescent sequence (418-1824) selected and modified from a 55 molecular imaging. Therefore, use of the Lumio tag in this Clostridium saccharoperbutylacetonicum Butyraldehyde manner enables monitoring and/or tracking of the designer Dehydrogenase sequence (AY251646) is used. Crotonase when expressed to verify whether the designer SEQID NO: 3 presents example 3 for a designer Butyryl butanol-production pathway enzyme is indeed delivered into CoA Dehydrogenase construct (1815bp) that includes a PCR the chloroplast of a host organism as designed. The Lumio tag FD primer (sequence 1-20), a 262-bp nitrate reductase pro 60 (a short 7 amino acid peptide) that is linked to the C-terminal moter (21-282), a 9-bp Xho I Ndel site (283-291), a 135-bp end of the Crotonase protein in this example should have RbcS2 transit peptide (292-426), a Butyryl-CoA Dehydroge minimal effect on the function of the designer enzyme, but nase encoding sequence (427-1563) selected/modified from enable the designer enzyme molecule to be visualized when the sequences of a Clostridium beijerinckii Butyryl-CoA treated with the Lumio reagent. Use of the Lumio tag is Dehydrogenase (AF494018), a 9-bp Xbal site (1564-1572), a 65 entirely optional. If the Lumio tag somehow affects the 223-bp RbcS2 terminator (1573-1795), and a PCRRE primer designer enzyme function, this tag can be deleted in the DNA (1796-1815) at the 3' end. This DNA construct is similar to sequence design. US 8,986,963 B2 29 30 SEQ ID NO: 5 presents example 5 for a designer 3-Hy sequence which is more active than the native Nial promoter droxybutyryl-CoA Dehydrogenase DNA construct (1367 bp) (Loppes and Radoux (2002) “Two short regions of the pro that includes a PCR FD primer (sequence 1-20), a 84-bp moter are essential for activation and repression of the nitrate nitrate reductase promoter (21-104), a 9-bp Xho I Ndel site reductase gene in Chlamydomonas reinhardtii,' Mol Genet (105-113) a 135-bp RbcS2 transit peptide (114-248), a 3-Hy Genomics 268: 42-48). Use of this type of inducible promoter droxybutyryl-CoA Dehydrogenase-encoding sequence (249 sequences with various promoter strengths can also help in 1094) selected/modified from a Clostridium beijerinckii adjusting the expression levels of the designer enzymes for 3-Hydroxybutyryl-CoA Dehydrogenase sequence (Gen the butanol-production pathway(s). bank: AF494018), a 21-bp Lumio-tag sequence (1095-1115), SEQID NO: 8 presents example 8 for a designer Pyruvate a 9-bp Xbal site (1116-1124), a 223-bp RbcS2 terminator 10 Kinase DNA construct (2021 bp) that includes a PCR FD (1125-1347), and a PCR RE primer (1348-1367). This DNA primer (sequence 1-20), a 84-bp nitrate reductase promoter construct is similar to example 4, SEQID NO: 4, except that (21-104), a 9-bp Xho I Ndel site (105-113) a 135-bp RbcS2 an 84-bp nitrate reductase promoter (21-104) and a 3-Hy transit peptide (114-248), a pyruvate kinase-encoding droxybutyryl-CoA Dehydrogenase-encoding sequence (249 sequence (249-1748) selected/modified from a Saccharomy 1094) selected/modified from a Clostridium beijerinckii 15 ces cerevisiae Pyruvate Kinase sequence (GenBank: 3-Hydroxybutyryl-CoA Dehydrogenase sequence (Gen AY949876), a 21-bp Lumio-tag sequence (1749-1769), a bank: AF494018) are used. The 84-bp nitrate-reductase pro 9-bp Xbal site (1770-1778), a 223-bp RbcS2 terminator moter is artificially created by joining two partially homolo (1779-2001), and a PCR RE primer (2002-2021). This DNA gous sequence regions (-231 to -201 and -77 to -25 with construct is similar to example 6, SEQID NO: 6, except that respect to the start site of transcription) of the native Chlamy a pyruvate kinase-encoding sequence (249-1748) is used. domonas reinhardtii Nial promoter. Experimental studies SEQID NO: 9 presents example 9 for a designer Enolase have demonstrated that the 84-bp sequence is more active gene (1815 bp) consisting of a PCR FD primer (sequence than the native Nial promoter (Loppes and Radoux (2002) 1-20), a 262-bp nitrate reductase promoter (21-282), a 9-bp “Two short regions of the promoter are essential for activation Xho I Ndel site (283-291) a 135-bp RbcS2 transit peptide and repression of the nitrate reductase gene in Chlamydomo 25 (292-426), a enolase-encoding sequence (427-1542) nas reinhardtii,' Mol Genet Genomics 268: 42-48). There selected/modified from the sequences of a Chlamydomonas fore, this is also an example where functional synthetic reinhardtii cytosolic enolase (Genbank: X66412, P31683), a sequences, analogs, functional derivatives and/or designer 21-bp Lumio-tag-encoding sequence (1507-1527), a 9-bp modified sequences such as the synthetic 84-bp sequence can Xbal site (1543-1551) containing a stop codon, a 223-bp be selected for use according to various embodiments in this 30 RbcS2 terminator (1552-1795), and a PCR RE primer (1796 invention. 1815) at the 3' end. This DNA construct is similar to example SEQID NO: 6 presents example 6 for a designer Thiolase 3, SEQID NO: 3, except that an enolase-encoding sequence DNA construct (1721 bp) that includes a PCR FD primer (427-1542) selected/modified from the sequences of a (sequence 1-20), a 84-bp nitrate reductase promoter (21-104), Chlamydomonas reinhardtii cytosolic enolase is used. a 9-bp Xho I Ndel site (105-113) a 135-bp RbcS2 transit 35 SEQID NO: 10 presents example 10 for a designer Phos peptide (114-248), a Thiolase-encoding sequence (248-1448) phoglycerate-Mutase DNA construct (2349 bp) that includes selected/modified from a Butyrivibriofibrisolvens Thiolase a PCRFD primer (sequence 1-20), a 262-bp nitrate reductase sequence (AB190764), a 21-bp Lumio-tag sequence (1449 promoter (21-282), a 9-bp Xho I Ndel site (283-291), a 135 1469), a 9-bp Xbal site (1470-1478), a 223-bp RbcS2 termi bp RbcS2 transit peptide (292-426), a phosphoglycerate-mu nator (1479-1701), and a PCR RE primer (1702-1721). This 40 tase encoding sequence (427-2097) selected/modified from DNA construct is also similar to example 4, SEQID NO: 4, the sequences of a Chlamydomonas reinhardtii cytosolic except that a Thiolase-encoding-encoding sequence (249 phosphoglycerate mutase (JGI Chlre2 protein ID 161689, 1448) and an 84-bp synthetic Nial promoter (21-104) are Genbank: AF268078), a 9-bp Xbal site (2098-2106), a 223 used. This is another example that functional synthetic bp RbcS2 terminator (2107-2329), and a PCR RE primer sequences can also be selected for use in designer DNA 45 (2330-2349) at the 3' end. This DNA construct is similar to COnStructS. example 3, SEQ ID NO: 3, except that a phosphoglycerate SEQID NO: 7 presents example 7 for a designer Pyruvate mutase encoding sequence (427-2097) selected/modified Ferredoxin Oxidoreductase DNA construct (4211 bp) that from the sequences of a Chlamydomonas reinhardtii cytoso includes a PCRFD primer (sequence 1-20), a 2x84-bp nitrate lic phosphoglycerate mutase is used. reductase promoter (21-188), a 9-bp Xho I Ndel site (189 50 SEQID NO: 11 presents example 11 for a designer Phos 197) a 135-bp RbcS2 transit peptide (198-332), a Pyruvate phoglycerate Kinase DNA construct (1908bp) that includes a Ferredoxin Oxidoreductase-encoding sequence (333-3938) PCRFD primer (sequence 1-20), a 262-bp nitrate reductase selected/modified from the sequences of a Mastigamoeba Nial promoter (21-282), a phosphoglycerate-kinase-encod balamuthi Pyruvate-ferredoxin oxidoreductase (GenBank: ing sequence (283-1665) selected from a Chlamydomonas AY101767), a 21-bp Lumio-tag sequence (3939-3959), a 55 reinhardtii chloroplast phosphoglycerate-kinase sequence 9-bp Xbal site (3960-3968), a 223-bp RbcS2 terminator including its chloroplast signal peptide and mature enzyme (3969-4191), and a PCR RE primer (4192-4211). This DNA sequence (GenBank: U14912), a 223-bp RbcS2 terminator construct is also similar to example 4, SEQID NO: 4, except (1666-1888), and a PCR RE primer (1889-1908). This DNA a designer 2x84-bp Nial promoter and a Pyruvate-Ferre construct is similar to example 1, SEQ ID NO: 1, except a doxin Oxidoreductase-encoding sequence (333-3938) 60 phosphoglycerate-kinase-encoding sequence (283-1665) selected/modified from the sequences of a Mastigamoeba selected from a Chlamydomonas reinhardtii chloroplast balamuthi Pyruvate-ferredoxin oxidoreductase (GenBank: phosphoglycerate-kinase sequence including its chloroplast AY101767) are used. The 2x84-bp Nial promoter is con signal peptide and mature enzyme sequence is used. There structed as a tandem duplication of the 84-bp synthetic Nial fore, this is also an example where the sequence of a nuclear promoter sequence presented in SEQ ID NO: 6 above. 65 encoded chloroplast enzyme such as the Chlamydomonas Experimental tests have shown that the 2x84-bp synthetic reinhardtii chloroplast phosphoglycerate kinase can also be Nial promoter is even more powerful than the 84-bp used in design and construction of a designer butanol-produc US 8,986,963 B2 31 32 tion pathway gene when appropriate with a proper inducible SEQID NO:17 presents example 17 for a designer HydA1 promoter such as the Nial promoter (DNA sequence 21-282). promoter-linked Pyruvate-NADP" oxidoreductase DNA SEQID NO: 12 presents example 12 for a designer Glyc construct (6092 bp) that includes a PCRFD primer (sequence eraldehyde-3-Phosphate Dehydrogenase gene (1677 bp) that 1-20), a 282-bp HydA1 promoter (21-302), a 135-bp RbcS2 includes a PCRFD primer (sequence 1-20), a 262-bp nitrate transit peptide (303-437), a Pyruvate-NADP" oxidoreduc reductase Nial promoter (21-282), a 135-bp RbcS2 transit tase-encoding sequence (438-5849) selected/modified from a peptide (283-417), an enzyme-encoding sequence (418 Euglena gracilis Pyruvate-NADP oxidoreductase sequence 1434) selected and modified from a Mesostigma viride cyto (GenBank Accession Number AB021127), a 223-bp RbcS2 Solic glyceraldehyde-3-phosphate dehydrogenase (mRNA) terminator (5850-6072), and a PCR RE primer (6073-6092). sequence (GenBank accession number DQ873404), a 223-bp 10 SEQID NO:18 presents example 18 for a designer HydA1 RbcS2 terminator (1435-1657), and a PCR RE primer (1658 promoter-linked Thiolase DNA construct (1856 bp) that 1677). This DNA construct is similar to example 1, SEQID includes a PCRFD primer (sequence 1-20), a 282-bp HydA1 NO: 1, except that an enzyme-encoding sequence (418-1434) promoter (21-302), a 135-bp RbcS2 transit peptide (303 selected and modified from a Mesostigma viride cytosolic 15 437), a Thiolase-encoding sequence (438-1613) selected/ glyceraldehyde-3-phosphate dehydrogenase (mRNA) modified from the sequences of a Thermoanaerobacterium sequence (GenBank accession number DQ873404) is used. thermosaccharolyticum Thiolase (GenBank Z92974), a 223 SEQ ID NO: 13 presents example 13 for a designer bp RbcS2 terminator (1614-1836), and a PCR RE primer HydA1-promoter-linked Phosphoglycerate Mutase DNA (1837-1856). construct (2351 bp) that includes a PCRFD primer (sequence SEQID NO:19 presents example 19 for a designer HydA1 1-20), a 282-bp HydA1 promoter (21-302), a 135-bp RbcS2 promoter-linked 3-Hydroxybutyryl-CoA dehydrogenase transit peptide (303-437), a phosphoglycerate-mutase encod DNA construct (1550 bp) that includes a PCR FD primer ing sequence (438-2108) selected/modified from the (sequence 1-20), a 282-bp HydA1 promoter (21-302), a 135 sequences of a Chlamydomonas reinhardtii cytosolic phos bp RbcS2 transit peptide (303-437), a 3-Hydroxybutyryl phoglycerate mutase (JGI Chlre2 protein ID 161689, Gen 25 CoA dehydrogenase-encoding sequence (438-1307) bank: AF268078), a 223-bp RbcS2 terminator (2109-2331), selected/modified from the sequences of a Thermoanaero and a PCR RE primer (2332-2351). This designer DNA con bacterium thermosaccharolyticum 3-Hydroxybutyryl-CoA struct is quite similar to example 1, SEQID NO:1, except that dehydrogenase (GenBank Z92974), a 223-bp RbcS2 termi a 282-bp HydA1 promoter (21-302) and a phosphoglycerate nator (1308-1530), and a PCR RE primer (1531-1550). mutase encoding sequence (438-2108) selected/modified 30 SEQID NO:20 presents example 20 for a designer HydA1 from the sequences of a Chlamydomonas reinhardtii cytoso promoter-linked Crotonase DNA construct (1457 bp) that lic phosphoglycerate mutase are used. The 282-bp HydA1 includes a PCRFD primer (sequence 1-20), a 282-bp HydA1 promoter (21-302) has been proven active by experimental promoter (21-302), a 135-bp RbcS2 transit peptide (303 assays at the inventor's laboratory. Use of the HydA1 pro 437), a Crotonase-encoding sequence (438-1214) selected/ moter (21-302) enables activation of designer enzyme 35 modified from the sequences of a Thermoanaerobacterium expression by using anaerobic culture-medium conditions. thermosaccharolyticum Crotonase (GenBank Z92974), a With the same principle of using an inducible anaerobic 223-bpRbcS2 terminator (1215-1437), and a PCR RE primer promoter and a chloroplast-targeting sequence as that shown (1438-1457). in SEQID NO: 13 (example 13), SEQID NOS: 14-23 show SEQID NO:21 presents example 21 for a designer HydA1 designer-gene examples 14-23. Briefly, SEQID NO: 14 pre 40 promoter-linked Butyryl-CoA dehydrogenase DNA con sents example 14 for a designer HydAl-promoter-linked struct (1817 bp) that includes a PCR FD primer (sequence Enolase DNA construct (1796 bp) that includes a PCR FD 1-20), a 282-bp HydA1 promoter (21-302), a 135-bp RbcS2 primer (sequence 1-20), a 282-bp HydA1 promoter (21-302), transit peptide (303-437), a Butyryl-CoA dehydrogenase-en a 135-bp RbcS2 transit peptide (303-437), a Enolase-encod coding sequence (438-1574) selected/modified from the ing sequence (438-1553) selected/modified from the 45 sequences of a Thermoanaerobacterium thermosaccharolyti sequences of a Chlamydomonas reinhardtii cytosolic enolase cum Butyryl-CoA dehydrogenase (GenBank Z92974), a 223 (Genbank: X66412, P31683), a 223-bp RbcS2 terminator bp RbcS2 terminator (1575-1797), and a PCR RE primer (1554-1776), and a PCR RE primer (1777-1796). (1798-1817). SEQ ID NO: 15 presents example 15 for a designer SEQ ID NO: 22 presents example 22 for a designer HydA1-promoter-controlled Pyruvate-Kinase DNA con 50 HydA1-promoter-linked Butyraldehyde dehydrogenase struct that includes a PCR FD primer (sequence 1-20), a DNA construct (2084 bp) that includes a PCR FD primer 282-bp HydA1 promoter (21-302), a 135-bp RbcS2 transit (sequence 1-20), a 282-bp HydA1 promoter (21-302), a 135 peptide (303-437), a Pyruvate Kinase-encoding sequence bp RbcS2 transit peptide (303-437), a Butyraldehyde dehy (438-1589) selected/modified from a Chlamydomonas rein drogenase-encoding sequence (438-1841) selected/modified hardtii cytosolic pyruvate kinase sequence (JGI Chlre3 pro 55 from the sequences of a Clostridium saccharoperbutylac tein ID 138105), a 223-bp RbcS2 terminator (1590-1812), etonicum Butyraldehyde dehydrogenase (GenBank and a PCR RE primer (1813-1832). AY251646), a 223-bp RbcS2 terminator (1842-2064), and a SEQID NO:16 presents example 16 for a designer HydA1 PCR RE primer (2065-2084). promoter-linked Pyruvate-ferredoxin oxidoreductase DNA SEQ ID NO: 23 presents example 23 for a designer construct (4376 bp) that includes a PCRFD primer (sequence 60 HydA1-promoter-linked Butanol dehydrogenase DNA con 1-20), a 282-bp HydA1 promoter (21-302), a 135-bp RbcS2 struct (1733 bp) that includes a PCR FD primer (sequence transit peptide (303-437), a Pyruvate-ferredoxin oxidoreduc 1-20), a 282-bp HydA1 promoter (21-302), a 135-bp RbcS2 tase-encoding sequence (438-4133) selected/modified from a transit peptide (303-437), a Butanol dehydrogenase-encod Desulfovibrio africanus Pyruvate-ferredoxin oxidoreductase ing sequence (438-1490) selected/modified from the sequence (GenBank Accession Number Y09702), a 223-bp 65 sequences of a Clostridium beijerinckii Butanol dehydroge RbcS2 terminator (4134-4356), and a PCR RE primer (4357 nase (GenBank AF157307), a 223-bp RbcS2 terminator 4376). (1491-1713), and a PCR RE primer (1714-1733). US 8,986,963 B2 33 34 With the same principle of using a 2x84 synthetic Nial phoglycerate-branched butanol-production pathway (03-12 promoter and a chloroplast-targeting mechanism as men in FIG. 1). When necessary, a transformant containing the ten tioned previously, SEQID NOS:24-26 show more examples DNA constructs can be further transformed to get more of designer-enzyme DNA-constructs. Briefly, SEQ ID NO: designer genes into its genomic DNA with an additional 24 presents example 24 for a designer Fructose-Diphosphate selection marker Such as streptomycin. By using combina Aldolase DNA construct that includes a PCR FD primer tions of various designer-enzymes DNA constructs such as (sequence 1-20), a 2x84-bp NR promoter (21-188), a Fruc those presented in SEQID NO: 1-26 in genetic transforma tose-Diphosphate Aldolase-encoding sequence (189-1313) tion with an appropriate host organism, various butanol-pro selected/modified from a C. reinhardtii chloroplast fructose duction pathways such as those illustrated in FIG. 1 can be 1,6-bisphosphate aldolase sequence (GenBank: X69969), a 10 constructed. For example, the designer DNA constructs of 223-bpRbcS2 terminator (1314-1536), and a PCR RE primer SEQ ID NO: 1-12 can be selected for construction of the (1537-1556). glyceraldehydes-3-phosphate-branched butanol-production SEQID NO: 25 presents example 24 for a designer Triose pathway (01-12 in FIG. 1); The designer DNA constructs of Phosphate-Isomerase DNA construct that includes a PCRFD SEQID NO: 1-12, 24, and 25 can be selected for construction primer (sequence 1-20), a 2x84-bp NR promoter (21-188), a 15 of the fructose-1,6-diphosphate-branched butanol-produc Triose-Phosphate Isomerase-encoding sequence (189-1136) tion pathway (20-33); and the designer DNA constructs of selected and modified from a Arabidopsis thaliana chloro SEQID NO: 1-12 and 24-26 can be selected for construction plast triosephosphate-isomerase sequence (GenBank: of the fructose-6-phosphate-branched butanol-production AF247559), a 223-bp RbcS2 terminator (1137-1359), and a pathway (19-33). PCR RE primer (1360-1379). Additional Host Modifications to Enhance Photosynthetic SEQID NO: 26 presents example 26 for a designer Phos Butanol Production phofructose-Kinase DNA construct that includes a PCR FD An NADPH/NADH Conversion Mechanism primer (sequence 1-20), a 2x84-bp NR promoter (21-188), a According to the photosynthetic butanol production path 135-bp RbcS2 transit peptide (189-323), a Phosphofructose way(s), to produce one molecule ofbutanol from 4CO and Kinase-encoding sequence (324-1913) selected/modified 25 5HO is likely to require 14 ATP and 12 NADPH, both of from Arabidopsis thaliana 6-phosphofructokinase sequence which are generated by photosynthetic water splitting and (GenBank: NM 001037043), a 223-bp RbcS2 terminator photophosphorylation across the thylakoid membrane. In (1914-2136), and a PCR RE primer (2137-2156). order for the 3-phosphoglycerate-branched butanol-produc The nucleic acid constructs, such as those presented in the tion pathway (03-12 in FIG. 1) to operate, it is a preferred examples above, may include additional appropriate 30 practice to use abutanol-production-pathway enzyme(s) that sequences, for example, a selection marker gene, and an can use NADPH that is generated by the photo-driven elec optional biomolecular tag sequence (such as the Lumio tag trontransport process. Clostridium saccharoperbutylacetoni described in example 4, SEQID NO: 4). Selectable markers cum butanol dehydrogenase (GenBank accession number: that can be selected for use in the constructs include markers AB257.439) and butyaldehyde dehydrogenase (GenBank: conferring resistances to kanamycin, hygromycin, spectino 35 AY251646) are examples of a butanol-production-pathway mycin, Streptomycin, Sulfonyl urea, gentamycin, chloram enzyme that is capable of accepting either NADP(H) or NAD phenicol, among others, all of which have been cloned and are (H). Such abutanol-production-pathway enzyme that can use available to those skilled in the art. Alternatively, the selective both NADPH and NADH (i.e., NAD(P)H) can also be marker is a nutrition marker gene that can complement a selected for use in this 3-phosphoglycerate-branched and any deficiency in the host organism. For example, the gene encod 40 of the other designer butanol-production pathway(s) (FIG. 1) ing argininosuccinate lyase (arg7) can be used as a selection as well. Clostridium beijerinckii Butyryl-CoA dehydroge marker gene in the designer construct, which permits identi nase (GenBank: AF494018) and 3-Hydroxybutyryl-CoA fication of transformants when Chlamydomonas reinhardtii dehydrogenase (GenBank: AF494018) are examples of a arg7-(minus) cells are used as host cells. butanol-production-pathway enzyme that can accept only Nucleic acid constructs carrying designer genes can be 45 NAD(H). When a butanol-production-pathway enzyme that delivered into a host alga, blue-green alga, plant, or plant can only use NADH is employed, it may require an NADPH/ tissue or cells using the available gene-transformation tech NADH conversion mechanism in order for this 3-phospho niques. Such as electroporation, PEG induced uptake, and glycerate-branched butanol-production pathway to operate ballistic delivery of DNA, and Agrobacterium-mediated well. However, depending on the genetic backgrounds of a transformation. For the purpose of delivering a designer con 50 host organism, a conversion mechanism between NADPH struct into algal cells, the techniques of electroporation, glass and NADH may exist in the host so that NADPH and NADH bead, and biolistic genegun can be selected for use as pre may be interchangeably used in the organism. In addition, it is ferred methods; and an alga with single cells or simplethallus known that NADPH could be converted into NADH by a structure is preferred for use in transformation. Transfor NADPH-phosphatase activity (Pattanayak and Chatterjee mants can be identified and tested based on routine tech 55 (1998) “Nicotinamide adenine dinucleotide phosphate phos niques. phatase facilitates dark reduction of nitrate: regulation by The various designer genes can be introduced into host nitrate and ammonia. Biologia Plantarium 41(1):75-84) and cells sequentially in a step-wise manner, or simultaneously that NAD can be converted to NADP by a NAD kinase activ using one construct or in one transformation. For example, ity (Muto, Miyachi, Usuda, Edwards and Bassham (1981) the ten DNA constructs shown in SEQID NO: 13-16 (or 17) 60 "Light-induced conversion of nicotinamide adenine dinucle and 18-23 for the ten-enzyme 3-phosphoglycerate-branched otide to nicotinamide adenine dinucleotide phosphate in butanol-production pathway can be placed into a genetic vec higher plant leaves. Plant Physiology 68(2):324-328; Mat tor such as p389-Arg7 with a single selection marker (Arg7). sumura-Kadota, Muto, Miyachi (1982) “Light-induced con Therefore, by use of a plasmid in this manner, it is possible to version of NAD' to NADP" in Chlorella cells. Biochimica deliver all the ten DNA constructs (designer genes) into an 65 Biophysica Acta 679(2):300-300). Therefore, when enhanced arginine-requiring Chlamydomonas reinhardtii-arg7 host NADPH/NADH conversion is desirable, the host may be (CC-48) in one transformation for expression of the 3-phos genetically modified to enhance the NADPH phosphatase US 8,986,963 B2 35 36 and NAD kinase activities. Thus, in one of the various tase 15, and hexose-phosphate-isomerase 16 of the starch embodiments, the photosynthetic butanol-producing synthesis pathway which connects with the Calvin cycle designer plant, designer alga or plant cell further contains (FIG. 1). additional designer transgenes (FIG.2B) to inducibly express Introduction of a genetically transmittable factor that can one or more enzymes to facilitate the NADPH/NADH inter inhibit the starch-synthesis activity that is in competition with conversion, such as the NADPH phosphatase and NAD designer butanol-production pathway(s) for the Calvin-cycle kinase (GenBank: XM 001.609395, XM 001324239), in products can further enhance photosynthetic butanol produc the stroma region of the algal chloroplast. tion. In a specific embodiment, a genetically encoded-able Another embodiment that can provide an NADPH/NADH inhibitor (FIG. 2C) to the competitive starch-synthesis path 10 way is an interfering RNA (iRNA) molecule that specifically conversion mechanism is by properly selecting an appropri inhibits the synthesis of a starch-synthesis-pathway enzyme, ate branching point at the Calvin cycle for a designer butanol for example, starch synthase 16, glucose-1-phosphate (G-1- production pathway to branch from. To confer this NADPH/ P) adenylyltransferase 15, phosphoglucomutase 14, and/or NADH conversion mechanism by pathway design according hexose-phosphate-isomerase 13 as shown with numerical to this embodiment, it is a preferred practice to branch a 15 labels 13-16 in FIG.1. The DNA sequences encoding starch designer butanol-production pathway at or after the point of synthase iRNA, glucose-1-phosphate (G-1-P) adenylyltrans glyceraldehydes-3-phosphate of the Calvin cycle as shown in ferase iRNA, a phosphoglucomutase iRNA and/or a G-P- FIG. 1. In these pathway designs, the NADPH/NADH con isomerase iRNA, respectively, can be designed and synthe version is achieved essentially by a two-step mechanism: 1) sized based on RNA interference techniques known to those Use of the step with the Calvin-cycle's glyceraldehyde-3- skilled in the art (Liszewski (Jun. 1, 2003) Progress in RNA phosphate dehydrogenase, which uses NADPH in reducing interference, Genetic Engineering News, Vol. 23, number 11, 1,3-diphosphoglycerate to glyceraldehydes-3-phosphate: pp. 1-59). Generally speaking, an interfering RNA (iRNA) and 2) use of the step with the designer pathway's NAD"- molecule is anti-sense but complementary to a normal mRNA dependent glyceraldehyde-3-phosphate dehydrogenase 01. of a particular protein (gene) so that such iPNA molecule can which produces NADH in oxidizing glyceraldehyde-3-phos 25 specifically bind with the normal mRNA of the particular phate to 1,3-diphosphoglycerate. The net result of the two gene, thus inhibiting (blocking) the translation of the gene steps described above is the conversion of NADPH to NADH, specific mRNA to protein (Fire, Xu, Montgomery, Kostas, which can Supply the needed reducing power in the form of Driver, Mello (1998) “Potent and specific genetic interfer NADH for the designer butanol-production pathway(s). For ence by double-stranded RNA in Caenorhabditis elegans'. step 1), use of the Calvin-cycle's NADPH-dependent glycer- 30 Nature 391 (6669):806-11; Dykxhoorn, Novina, Sharp aldehyde-3-phosphate dehydrogenase naturally in the host (2003) “Killing the messenger: short RNAs that silence gene organism is usually sufficient. Consequently, introduction of expression”, Nat Rev Mol Cell Biol. 4(6):457-67). a designer NAD"-dependent glyceraldehyde-3-phosphate Examples of a designer starch-synthesis iRNA DNA con dehydrogenase 01 to work with the Calvin-cycle's NADPH struct (FIG. 2C) are shown in SEQID NO: 27 and 28 listed. dependent glyceraldehyde-3-phosphate dehydrogenase may 35 Briefly, SEQID NO: 27 presents example 27 for a designer confer the function of an NADPH/NADH conversion mecha Nial-promoter-controlled Starch-Synthase-iRNA DNA con nism, which is needed for the 3-phosphoglycerate-branched struct (860 bp) that includes a PCR FD primer (sequence butanol-production pathway (03-12 in FIG. 1) to operate 1-20), a 262-bp Nial promoter (21-282), a Starch-Synthase well. For this reason, the designer NAD-dependent glycer iRNA sequence (283-617) consisting of start codon atg and a aldehyde-3-phosphate-dehydrogenase DNA construct (ex 40 reverse complement sequence of two unique sequence frag ample 12, SEQIDNO:12) is used also as an NADPH/NADH ments of a Chlamydomonas reinhardtii Starch-synthase conversion designer gene (FIG. 2B) to Support the mRNA sequence (GenBank: AF026422), a 223-bp RbcS2 3-phosphoglycerate-branched butanol-production pathway terminator (618-850), and a PCR RE primer (851-860). (03-12 in FIG.1) in one of the various embodiments. This also Because of the use of a Nial promoter (21-282), this designer explains why it is important to use a NAD-dependent glyc 45 starch-synthesis iRNA gene is designed to be expressed only eraldehyde-3-phosphate dehydrogenase 01 to confer this when needed to enhance photobiological butanol production two-step NADPH/NADH conversion mechanism for the in the presence of its specific inducer, nitrate (NO), which designer butanol-production pathway(s). Therefore, in one of can be added into the culture medium as a fertilizer for induc the various embodiments, it is also a preferred practice to use tion of the designer organisms. The Starch-Synthase iRNA a NAD-dependent glyceraldehyde-3-phosphate dehydroge 50 sequence (283-617) is designed to bind with the normal nase, its isozymes, functional derivatives, analogs, designer mRNA of the starch synthase gene, thus blocking its transla modified enzymes and/or combinations thereof in the tion into a functional starch synthase. The inhibition of the designer butanol-production pathway(s) as illustrated in FIG. starch/glycogen synthase activity at 16 in this manner is to 1. channel more photosynthetic products of the Calvin cycle iRNA Techniques to Further Tame Photosynthesis Regula 55 into the Calvin-cycle-branched butanol-production path tion Mechanism way(s) such as the glyceraldehydes-3-phosphate-branched In another embodiment of the present invention, the host butanol-production pathway 01-12 as illustrated in FIG. 1. plant or cell is further modified to tame the Calvin cycle so SEQ ID NO: 28 presents example 28 for a designer that the host can directly produce liquid fuel butanol instead HydA1-promoter-controlled Starch-Synthase-iRNA DNA of synthesizing starch (glycogen in the case of oxyphotobac 60 construct (1328bp) that includes a PCRFD primer (sequence teria), celluloses and lignocelluloses that are often inefficient 1-20), a 282-bp HydA1 promoter (21-302), a designer Starch and hard for the biorefinery industry to use. According to the Synthase iRNA sequence (303-1085), a 223-bp RbcS2 termi one of the various embodiments, inactivation of starch-syn nator (1086-1308), and a PCR RE primer (1309-1328). The thesis activity is achieved by Suppressing the expression of designer Starch-Synthase-iRNA sequence (303-1085) com any of the key enzymes, such as, starch synthase (glycogen 65 prises of: a 300-bp sense fragment (303-602) selected from synthase in the case of oxyphotobacteria) 13, glucose-1- the first 300-bp unique coding sequence of a Chlamydomonas phosphate (G-1-P)adenylyltransferase 14, phosphoglucomu reinhardtii starch synthase mRNA sequence (GenBank: US 8,986,963 B2 37 38 AF026422), a 183-bp designer intron-like loop (603-785), FIG. 1 also illustrates the use of a designer starch/glyco and a 300-bp antisense sequence (786-1085) complement to gen-to-butanol pathway with designer enzymes (as labeled the first 300-bp coding sequence of a Chlamydomonas rein from 17 to 33) in combination with a Calvin-cycle-branched hardtii starch-synthase-mRNA sequence (GenBank: designer butanol-production pathway(s) such as the glycer AF026422). This designer Starch-Synthase-iRNA sequence 5 aldehydes-3-phosphate-branched butanol-production path (303-1085) is designed to inhibit the synthesis of starch syn way 01-12 for enhanced photobiological butanol production. thase by the following two mechanisms. First, the 300-bp Similar to the benefits of using the Calvin-cycle-branched antisense complement iRNA sequence (corresponding to designer butanol-production pathways, the use of the DNA sequence 786-1085) binds with the normal mRNA of designer starch/glycogen-to-butanol pathway (17-33) can 10 also help to convert the photosynthetic products to butanol the starch synthase gene, thus blocking its translation into a before the sugars could be converted into other complicated functional starch synthase. Second, the 300-bp antisense biomolecules such as lignocellulosic biomasses which can complement iRNA sequence (corresponding to DNA not be readily used by the biorefinery industries. Therefore, sequence 786-1085) can also bind with the 300-bp sense appropriate use of the Calvin-cycle-branched designer counterpart (corresponding to DNA sequence 303-602) in the 15 butanol-production pathway(s) (such as 01-12,03-12, and/or same designer iRNA molecule, forming a hairpin-like 20-33) and/or the designer starch/glycogen-to-butanol path double-stranded RNA structure with the 183-bp designer way (17-33) may represent revolutionary interalia technolo intron-like sequence (603-785) as a loop. Experimental stud gies that can effectively bypass the bottleneck problems of the ies have shown that this type of hairpin-like double-stranded current biomass technology including the “lignocellulosic RNA can also trigger post-transcriptional gene silencing (Fu recalcitrance' problem. hrmann, Stahlberg, Govorunova, Rank and Hegemann (2001) Another feature is that a Calvin-cycle-branched designer Journal of Cell Science 1 14:3857-3863). Because of the use butanol-production pathway activity (such as 01-12. 03-12, of a HydA1 promoter (21-302), this designer starch-synthe and/or 20-33) can occur predominantly during the days when sis-iRNA gene is designed to be expressed only under anaero there is light because it uses an intermediate product of the bic conditions when needed to enhance photobiological 25 Calvin cycle which requires Supplies of reducing power butanol production by channeling more photosynthetic (NADPH) and energy (ATP) generated by the photosynthetic products of the Calvin cycle into the butanol-production path water splitting and the light-driven proton-translocation way(s) such as 01-12,03-12, and/or 20-33 as illustrated in coupled electron transport process through the thylakoid FIG 1. membrane system. The designer starch/glycogen-to-butanol Designer Starch-Degradation and Glycolysis Genes 30 pathway (17-33) which can use the Surplus Sugar that has In yet another embodiment of the present invention, the been stored as starch/glycogen during photosynthesis can photobiological butanol production is enhanced by incorpo operate not only during the days, but also at nights. Conse rating an additional set of designer genes (FIG. 2D) that can quently, the use of a Calvin-cycle-branched designer butanol facilitate starch/glycogen degradation and glycolysis in com production pathway (such as 01-12. 03-12, and/or 20-33) bination with the designer butanol-production gene(s) (FIG. 35 together with a designer starch/glycogen-to-butanol path 2A). Such additional designer genes for starch degradation way(s) (17-33) as illustrated in FIG. 1 enables production of include, for example, genes coding for 17: amylase, starch butanol both during the days and at nights. phosphorylase, hexokinase, phosphoglucomutase, and for Because the expression for both the designer starch/glyco 18: glucose-phosphate-isomerase (G-P-isomerase) as illus gen-to-butanol pathway(s) and the Calvin-cycle-branched trated in FIG.1. The designer glycolysis genes encode chlo 40 designer butanol-production pathway(s) is controlled by the roplast-targeted glycolysis enzymes: glucosephosphate use of an inducible promoter Such as an anaerobic hydroge isomerase 18, phosphofructose kinase 19, aldolase 20, triose nase promoter, this type of designer organisms is also able to phosphate isomerase 21, glyceraldehyde-3-phosphate dehy grow photoautotrophically under aerobic (normal) condi drogenase 22, phosphoglycerate kinase 23, phosphoglycerate tions. When the designer photosynthetic organisms are grown mutase 24, enolase 25, and pyruvate kinase 26. The designer 45 and ready for photobiological butanol production, the cells starch-degradation and glycolysis genes in combination with are then placed under the specific inducing conditions such as any of the butanol-production pathways shown in FIG. 1 can under anaerobic conditions or an ammonium-to-nitrate fer form additional pathway(s) from starch/glycogen to butanol tilizer use shift, if designer Nial/nirA promoter-controlled (17-33). Consequently, co-expression of the designer starch butanol-production pathway(s) is used for enhanced butanol degradation and glycolysis genes with the butanol-produc 50 production, as shown in FIGS. 1 and 3. tion-pathway genes can enhance photobiological production Examples of designer starch (glycogen)-degradation genes of butanol as well. Therefore, this embodiment represents are shown in SEQ ID NO: 29-33 listed. Briefly, SEQ ID another approach to tame the Calvin cycle for enhanced pho NO:29 presents example 29 for a designer Amylase DNA tobiological production ofbutanol. In this case, some of the construct (1889 bp) that includes a PCRFD primer (sequence Calvin-cycle products flow through the starch synthesis path 55 1-20), a 2x84-bp NR promoter (21-188), a 9-bp Xho I NdeI way (13-16) followed by the starch/glycogen-to-butanol site (189-197), a 135-bp RbcS2 transit peptide (198-332), an pathway (17-33) as shown in FIG. 1. In this case, starch/ Amylase-encoding sequence (333-1616) selected and modi glycogen acts as a transient storage pool of the Calvin-cycle fied from a Barley alpha-amylase (GenBank: J04202A my46 products before they can be converted to butanol. This mecha expression tested in aleurone cells), a 21-bp Lumio-tag nism can be quite useful in maximizing the butanol-produc 60 sequence (1617-1637), a 9-bp Xbal site (1638-1646), a 223 tion yield in certain cases. For example, at high Sunlight bp RbcS2 terminator (1647-1869), and a PCR RE primer intensity Such as around noon, the rate of Calvin-cycle pho (1870-1889). tosynthetic CO fixation can be so high that may exceed the SEQID NO:30 presents example 30 for a designer Starch maximal rate capacity of a butanol-production pathway(s): Phosphorylase DNA construct (3089 bp) that includes a PCR use of the starch-synthesis mechanism allows temporary Stor 65 FD primer (sequence 1-20), a 2x84-bp NR promoter (21 age of the excess photosynthetic products to be used later for 188), a 135-bp RbcS2 transit peptide (189-323), a Starch butanol production as well. Phosphorylase-encoding sequence (324-2846) selected and US 8,986,963 B2 39 40 modified from a Citrus root starch-phosphorylase sequence the chloroplast and cytoplasm parts of the designer pathway is (GenBank: AY098895, expression tested in citrus root), a accomplished by use of the triose phosphate-phosphate trans 223-bp RbcS2 terminator (2847-3069), and a PCRRE primer locator, which facilitates translocation of dihydroxyacetone (3070-3089). across the chloroplast membrane. By use of the triose phos SEQID NO: 31 presents example 31 for a designer Hex phate-phosphate translocator, it also enables the glyceralde ose-Kinase DNA construct (1949 bp) that includes a PCRFD hyde-3-phospahite-branched designer butanol-production primer (sequence 1-20), a 2x84-bp NR promoter (21-188), a pathway to operate not only in chloroplast, but also in cyto 135-bp RbcS2 transit peptide (189-323), a Hexose Kinase plasm as well. The cytoplasm part of the designer butanol encoding sequence (324-1706) selected and modified from production pathway can be constructed by use of designer Ajellomyces capsulatus hexokinase mRNA sequence (Gen 10 butanol-production pathway genes (DNA constructs of FIG. bank: XM 001541513), a 223-bp RbcS2 terminator (1707 2A) with their chloroplast-targeting sequence omitted as 1929), and a PCR RE primer (1930-1949). shown in FIG. 2E. SEQID NO: 32 presents example 32 for a designer Phos Designer Oxyphotobacteria with Designer Butanol-Produc phoglucomutase DNA construct (2249 bp) that includes a tion Pathways in Cytoplasm PCR FD primer (sequence 1-20), a 2x84-bp NR promoter 15 In prokaryotic photosynthetic organisms such as blue (21-188), a 135-bp RbcS2 transit peptide (189-323), a Phos green algae (oxyphotobacteria including cyanobacteria and phoglucomutase-encoding sequence (324-2006) selected and oxychlorobacteria), which typically contain photosynthetic modified from Pichia stipitis phosphoglucomutase sequence thylakoid membrane but no chloroplast structure, the Calvin (GenBank: XM 001383281), a 223-bp RbcS2 terminator cycle is located in the cytoplasm. In this special case, the (2007-2229), and a PCR RE primer (2230-2249). entire designer butanol-production pathway(s) (FIG. 1) SEQID NO: 33 presents example 33 for a designer Glu including (but not limited to) the glyceraldehyde-3-phos cosephosphate-Isomerase DNA construct (2231 bp) that phate branched butanol-production pathway (01-12), the includes a PCR FD primer (sequence 1-20), a 2x84-bp NR 3-phosphpglycerate-branched butanol-production pathway promoter (21-188), a 135-bp RbcS2 transit peptide (189 (03-12), the fructose-1,6-diphosphate-branched pathway 323), a Glucosephosphate Isomerase-encoding sequence 25 (20-33), the fructose-6-phosphate-branched pathway (324-1988) selected and modified from a S. cerevisiae phos (19-33), and the starch (or glycogen)-to-butanol pathways phoglucoisomerase sequence (GenBank: M21696), a 223-bp (17-33) are adjusted in design to operate with the Calvin cycle RbcS2 terminator (1989-2211), and a PCR RE primer (2212 in the cytoplasm of a blue-green alga. The construction of the 2231). cytoplasm designer butanol-production pathways can be The designer starch-degradation genes Such as those 30 accomplished by use of designer butanol-production pathway shown in SEQ ID NO: 29-33 can be selected for use in genes (DNA construct of FIG. 2A) with their chloroplast combination with various designer butanol-production-path targeting sequence all omitted. When the chloroplast-target way genes for construction of various designer starch-degra ing sequence is omitted in the designer DNA construct(s) as dation butanol-production pathways such as the pathways illustrated in FIG. 2E, the designer gene(s) is transcribed and shown in FIG. 1. For example, the designer genes shown in 35 translated into designer enzymes in the cytoplasm whereby SEQ ID NOS: 1-12, 24-26, and 29-33 can be selected for conferring the designer butanol-production pathway(s). The construction of a Nial promoter-controlled starch-to-butanol designer gene(s) can be incorporated into the chromosomal production pathway that comprises of the following designer and/or plasmid DNA in host blue-green algae (oxyphotobac enzymes: amylase, starch phosphorylase, hexokinase, phos teria including cyanobacteria and oxychlorobacteria) by phoglucomutase, glucosephosphate isomerase, phosphofruc 40 using the techniques of gene transformation known to those tose kinase, fructose diphosphate aldolase, triose phosphate skilled in the art. It is a preferred practice to integrate the isomerase, glyceraldehyde-3-phosphate dehydrogenase, designer genes through an integrative transformation into the phosphoglycerate kinase, phosphoglycerate mutase, enolase, chromosomal DNA that can usually provide better genetic pyruvate kinase, pyruvate-NADP" oxidoreductase (or pyru stability for the designer genes. In oxyphotobacteria Such as vate-ferredoxin oxidoreductase), thiolase, 3-hydroxybutyryl 45 cyanobacteria, integrative transformation can be achieved CoA dehydrogenase, crotonase, butyryl-CoA dehydroge through a process of homologous DNA double recombina nase, butyraldehyde dehydrogenase, and butanol tion into the hosts chromosomal DNA using a designer DNA dehydrogenase. This starch/glycogen-to-butanol pathway construct as illustrated in FIG. 2F, which typically, from the 5' 17-33 may be used alone and/or in combinations with other upstream to the 3' downstream, consists of recombination butanol-production pathway(s) such as the 3-phosphoglycer 50 site 1, a designer butanol-production-pathway gene(s), and ate-branched butanol-production pathway 03-12 as illus recombination site 2. This type of DNA constructs (FIG.2F) trated in FIG. 1. can be delivered into oxyphotobacteria (blue-green algae) Distribution of Designer Butanol-Production Pathways with a number of available genetic transformation techniques Between Chloroplast and Cytoplasm including electroporation, natural transformation, and/or In yet another embodiment of the present invention, pho 55 conjugation. The transgenic designer organisms created from tobiological butanol productivity is enhanced by a selected blue-green algae are also called designer blue-green algae distribution of the designer butanol-production pathway(s) (designeroxyphotobacteria including designer cyanobacteria between chloroplast and cytoplasm in a eukaryotic plant cell. and designer oxychlorobacteria). That is, not all the designer butanol-production pathway(s) Examples of designer oxyphotobacterial butanol-produc (FIG. 1) have to operate in the chloroplast; when needed, part 60 tion-pathway genes are shown in SEQID NO. 34-45 listed. of the designer butanol-production pathway(s) can operate in Briefly, SEQ ID NO:34 presents example 34 for a designer cytoplasmas well. For example, in one of the various embodi oxyphotobacterial Butanol Dehydrogenase DNA construct ments, a significant part of the designer starch-to-butanol (1709 bp) that includes a PCRFD primer (sequence 1-20), a pathway activity from dihydroxyacetone phosphate to 400-bp nitrite reductase (nirA) promoter from Thermosyn butanol (21-33) is designed to occur at the cytoplasm while 65 echococcus elongatus BP-1 (21-420), an enzyme-encoding the steps from starch to dihydroxyacetone phosphate (17-20) sequence (421-1569) selected and modified from a are in the chloroplast. In this example, the linkage between Clostridium saccharoperbutylacetonicum Butanol Dehydro US 8,986,963 B2 41 42 genase sequence (AB257439), a 120-bp rbcS terminator from kinase (GenBank: AF065890), a 120-bp Thermosynechococ Thermosynechococcus elongatus BP-1 (1570-1689), and a cus elongatus BP-1 rbcS terminator (1667-1786), and a PCR PCR RE primer (1690-1709) at the 3' end. RE primer (1787-1806) at the 3' end. SEQ ID NO:35 presents example 35 for a designer oxy SEQ ID NO:42 presents example 42 for a designer oxy photobacterial Butyraldehyde Dehydrogenase DNA con photobacterial Enolase DNA construct (1696 bp) that struct (1967 bp) that includes a PCR FD primer (sequence includes a PCR FD primer (sequence 1-20), a 231-bp nirA 1-20), a 400-bp Thermosynechococcus elongatus BP-1 nitrite promoter from Thermosynechococcus elongatus BP-1 (21 reductase nirA promoter (21-420), an enzyme-encoding 251), a enolase-encoding sequence (252-1556) selected/ sequence (421-1827) selected and modified from a modified from the sequences of a Chlamydomonas rein Clostridium saccharoperbutylacetonicum Butyraldehyde 10 hardtii cytosolic enolase (GenBank: X66412, P31683), a Dehydrogenase sequence (AY251646), a 120-bp rbcS termi 120-bp rbcS terminator from Thermosynechococcus elonga nator from Thermosynechococcus elongatus BP-1 (1828 tus BP-1 (1557-1676), and a PCR RE primer (1677-1696) at 1947), and a PCR RE primer (1948-1967) at the 3' end. the 3' end. SEQ ID NO:36 presents example 36 for a designer oxy SEQ ID NO:43 presents example 43 for a designer oxy photobacterial Butyryl-CoA Dehydrogenase DNA construct 15 photobacterial Phosphoglycerate-Mutase DNA construct (1602 bp) that includes a PCRFD primer (sequence 1-20), a (2029 bp) that includes a PCRFD primer (sequence 1-20), a 305-bp Thermosynechococcus elongatus BP-1 nitrate reduc 231-bp nirA promoter from Thermosynechococcus elongatus tase promoter (21-325), a Butyryl-CoA Dehydrogenase BP-1 (21-251), a phosphoglycerate-mutase encoding encoding sequence (326-1422) selected/modified from the sequence (252-1889) selected/modified from the sequences sequences of a Clostridium beijerinckii Butyryl-CoA Dehy of a Pelotomaculum thermopropionicum SI phosphoglycer drogenase (AF494018), a 120-bp Thermosynechococcus ate mutase (GenBank: YP 001213270), a 120-bp Thermo elongatus BP-1 rbcS terminator (1423-1582), and a PCR RE synechococcus elongatus BP-1 rbcS terminator (1890-2009), primer (1583-1602) at the 3' end. and a PCR RE primer (2010-2029) at the 3' end. SEQ ID NO:37 presents example 37 for a designer oxy SEQ ID NO:44 presents example 44 for a designer oxy photobacterial Crotonase DNA construct (1248 bp) that 25 photobacterial Phosphoglycerate Kinase DNA construct includes a PCRFD primer (sequence 1-20), a 305-bp Ther (1687 bp) that includes a PCRFD primer (sequence 1-20), a mosynechococcus elongatus BP-1 nitrate reductase promoter 231-bp nirA promoter from Thermosynechococcus elongatus (21-325), a Crotonase-encoding sequence (326-1108) BP-1 (21-251), a phosphoglycerate-kinase-encoding selected/modified from the sequences of a Clostridium sequence (252-1433) selected from Pelotomaculum thermo beijerinckii Crotonase (GenBank: AF494018), 120-bp Ther 30 propionicum SI phosphoglycerate kinase (BAF60903), a mosynechococcus elongatus BP-1 rbcS terminator (1109– 234-bp Thermosynechococcus elongatus BP-1 rbcS termina 1228), and a PCR RE primer (1229-1248). tor (1434-1667), and a PCR RE primer (1668-1687). SEQ ID NO:38 presents example 38 for a designer oxy SEQ ID NO:45 presents example 45 for a designer oxy photobacterial 3-Hydroxybutyryl-CoADehydrogenase DNA photobacterial Glyceraldehyde-3-Phosphate Dehydrogenase construct (1311 bp) that include of a PCR FD primer (se 35 DNA construct (1514 bp) that includes a PCR FD primer quence 1-20), a 305-bp nirA promoter from (21-325), a 3-Hy (sequence 1-20), a 305-bp Thermosynechococcus elongatus droxybutyryl-CoA Dehydrogenase-encoding sequence (326 BP-1 nirA promoter (21-325), an enzyme-encoding sequence 1171) selected/modified from a Clostridium beijerinckii (326-1260) selected and modified from Blastochloris viridis 3-Hydroxybutyryl-CoA Dehydrogenase sequence Crotonase NAD-dependent Glyceraldehyde-3-phosphate dehydroge (GenBank: AF494018), a 120-bp Thermosynechococcus 40 nase (CAC80993), a 234-bp rbcS terminator from Thermo elongatus BP-1 rbcS terminator (1172-1291), and a PCR RE synechococcus elongatus BP-1 (1261-1494), and a PCR RE primer (1292-1311). primer (1495-1514). SEQ ID NO:39 presents example 39 for a designer oxy The designer oxyphotobacterial genes such as those shown photobacterial Thiolase DNA construct (1665 bp) that in SEQID NO:34-45 can be selected for use in full or in part, includes a PCR FD primer (sequence 1-20), a 305-bp nirA 45 and/or in combination with various other designer butanol promoter from Thermosynechococcus elongatus BP-1 (21 production-pathway genes for construction of various 325), a Thiolase-encoding sequence (326-1525) selected/ designer oxyphotobacterial butanol-production pathways modified from a Butyrivibriofibrisolvens Thiolase sequence such as the pathways shown in FIG. 1. For example, the (AB190764), a 120-bp rbcS terminator from Thermosyn designer genes shown in SEQID NOS: 34-45 can be selected echococcus elongatus BP-1 (1526-1645), and a PCR RE 50 for construction of an oxyphotobacterial nirA promoter-con primer (1646-1665). trolled and glyceraldehyde-3-phosphate-branched butanol SEQ ID NO:40 presents example 40 for a designer oxy production pathway (01-12) that comprises of the following photobacterial Pyruvate-Ferredoxin Oxidoreductase DNA designer enzymes: NAD-dependent glyceraldehyde-3-phos construct (4071 bp) that includes a PCRFD primer (sequence phate dehydrogenase 01, phosphoglycerate kinase 02, phos 1-20), a 305-bp nirA promoter from Thermosynechococcus 55 phoglycerate mutase 03, enolase 04, pyruvate kinase 05, elongatus BP-1 (21-325), a Pyruvate-Ferredoxin Oxi pyruvate-ferredoxin oxidoreductase (or pyruvate-NADP" doreductase-encoding sequence (326-3931) selected/modi oxidoreductase) 06, thiolase 07, 3-hydroxybutyryl-CoA fied from the sequences of a Mastigamoeba balamuthi Pyru dehydrogenase 08, crotonase 09, butyryl-CoA dehydroge vate-ferredoxin oxidoreductase (GenBank: AY101767), a nase 10, butyraldehyde dehydrogenase 11, and butanol dehy 120-bp rbcS terminator from Thermosynechococcus elonga 60 drogenase 12. Use of these designer oxyphotobacterial tus BP-1 (3932-4051), and a PCR RE primer (4052-4071). butanol-production-pathway genes (SEQID NOS: 34-45) in SEQ ID NO:41 presents example 41 for a designer oxy a thermophilic and/or thermotolerant cyanobacterium may photobacterial Pyruvate Kinase DNA construct (1806 bp) represent a thermophilic and/or thermotolerant butanol-pro that includes a PCR FD primer (sequence 1-20), a 305-bp ducing oxyphotobacterium. Fox example, use of these nirA promoter from Thermosynechococcus elongatus BP-1 65 designer genes (SEQ ID NOS: 34-45) in a thermophilic/ (21-325), a pyruvate kinase-encoding sequence (326-1666) thermotolerant cyanobacterium Such as Thermosynechococ selected/modified from a Thermoproteus tenax pyruvate cus elongatus BP-1 may represent a designer thermophilic/ US 8,986,963 B2 43 44 thermotolerant butanol-producing cyanobacterium such as a sion of the proton-channel gene is designed to occur through designer butanol-producing Thermosynechococcus. its transcription in the nucleus and its translation in the cyto Further Host Modifications to Help Ensure Biosafety Sol. Because of the specific molecular design, the expressed The present invention also provides biosafety-guarded proton channels are automatically inserted into the cytoplasm photosynthetic biofuel (e.g., butanol and/or related higher membrane, but leave the photosynthetic thylakoid membrane alcohols) production methods based on cell-division-control intact. The insertion of the designer proton channels into lable designer transgenic plants (such as algae and oxypho cytoplasm membrane collapses the proton gradient across the tobacteria) or plant cells. For example, the cell-division-con cytoplasm membrane so that the cell division and mating trollable designer photosynthetic organisms (FIG. 3) are function are permanently disabled. However, the photosyn created through use of a designer biosafety-control gene(s) 10 (FIG. 2G) in conjunction with the designer butanol-produc thetic thylakoid membrane inside the chloroplast is kept tion-pathway gene(s) (FIGS. 2A-2F) such that their cell divi intact (functional) So that the designer biofuel-production sion and mating function can be controllably stopped to pro pathway enzymes expressed into the stroma region can work vide better biosafety features. with the Calvin cycle for photobiological production of bio In one of the various embodiments, a fundamental feature 15 fuels from CO, and HO. That is, when both the designer is that a designer cell-division-controllable photosynthetic proton-channel gene and the designer biofuel-production organism (such as an alga, plant cell, or oxyphotobacterium) pathway gene(s) are turned on, the designer organism contains two key functions (FIG. 3A): a designer biosafety becomes a non-reproducible cell for dedicated photosyn mechanism(s) and a designer biofuel-production pathway(s). thetic production of biofuels. Because the cell division and As shown in FIG. 3B, the designer biosafety feature(s) is mating function are permanently disabled (killed) at this conferred by a number of mechanisms including: (1) the stage, the designer-organism culture is no longer a living inducible insertion of designer proton-channels into cyto matter except its catalytic function for photochemical conver plasm membrane to permanently disable any cell division and sion of CO and HO into a biofuel. It will no longer be able mating capability, (2) the selective application of designer to mate or exchange any genetic materials with any other cell-division-cycle regulatory protein or interference RNA 25 cells, even if it somehow comes in contact with a wild-type (iRNA) to permanently inhibit the cell division cycle and cell as it would be the case of an accidental release into the preferably keep the cell at the G phase or Go state, and (3) the environments. innovative use of a high-CO-requiring host photosynthetic According to one of the various embodiments, the nitrate organism for expression of the designer biofuel-production reductase (Nial) promoter or nitrite reductase (nirA) pro pathway(s). Examples of the designer biofuel-production 30 moter is a preferred inducible promoter for use to control the pathway(s) include the designer butanol-production path expression of the designer genes. In the presence of ammo way(s), which work with the Calvin cycle to synthesize bio nium (but not nitrate) in culture medium, for example, a fuel such as butanol directly from carbon dioxide (CO) and designer organism with Nial-promoter-controlled designer water (H2O). The designer cell-division-control technology proton-channel gene and biofuel-production-pathway can help ensure biosafety in using the designer organisms for 35 photosynthetic biofuel production. Accordingly, this embodi gene(s) can grow photoauotrophically using CO as the car ment provides, interalia, biosafety-guarded methods for pro bon source and H2O as the source of electrons just like a ducing biofuel (e.g., butanol and/or related higher alcohols) wild-type organism. When the designer organism culture is based on a cell-division-controllable designer biofuel-pro grown and ready for photobiological production of biofuels, ducing alga, cyanobacterium, oxychlorobacterium, plant or 40 the expression of both the designer proton-channel gene and plant cells. the designer biofuel-production-pathway gene(s) can then be In one of the various embodiments, a cell-division-control induced by adding some nitrate fertilizer into the culture lable designer butanol-producing eukaryotic alga or plant cell medium. Nitrate is widely present in soils and nearly all is created by introducing a designer proton-channel gene surface water on Earth. Therefore, even if a Nial-promoter (FIG.2H) into a hostalga or plant cell (FIG.3B). SEQID NO: 45 controlled designer organism is accidentally released into the 46 presents example 46 for a detailed DNA construct of a natural environment, it will soon die since the nitrate in the designer Nial-promoter-controlled proton-channel gene environment will trig the expression of a Nial-promoter (609 bp) that includes a PCR FD primer (sequence 1-20), a controlled designer proton-channel gene which inserts pro 262-bp nitrate reductase Nial promoter (21-282), a Melittin ton-channels into the cytoplasm membrane thereby killing proton-channel encoding sequence (283-366), a 223-bp 50 the cell. That is, a designer photosynthetic organism with RbcS2 terminator (367-589), and a PCR RE primer (590 Nial-promoter-controlled proton-channel gene is pro 609). grammed to die as soon as it sees nitrate in the environment. The expression of the designer proton-channel gene (FIG. This characteristic of cell-division-controllable designer 2H) is controlled by an inducible promoter such as the nitrate organisms with Nial-promoter-controlled proton-channel reductase (Nial) promoter, which can also be used to control 55 gene provides an added biosafety feature. the expression of a designer biofuel-production-pathway The artin constructing proton-channel gene (FIG.2H) with gene(s). Therefore, before the expression of the designer a thylakoid-membrane targeting sequence has recently been gene(s) is induced, the designer organism can grow photoau disclosed James W. Lee (2007). Designer proton-channel totrophically using CO as the carbon Source and H2O as the transgenic algae for photobiological hydrogen production, source of electrons just like wild-type organism. When the 60 PCT International Publication Number: WO 2007/134340 designer organism culture is grown and ready for photobio A2. In the present invention of creating a cell-division-con logical production of biofuels, the cell culture is then placed trollable designer organism, the thylakoid-membrane-target under a specific inducing condition (such as by adding nitrate ing sequence must be omitted in the proton-channel gene into the culture medium if the nitrate reductase (Nial) pro design. For example, the essential components of a Nial moter is used as an inducible promoter) to induce the expres 65 promoter-controlled designer proton-channel gene can sim sion of both the designer proton-channel gene and the ply be a Nial promoter linked with a proton-channel-encod designer biofuel-production-pathway gene(s). The expres ing sequence (without any thylakoid-membrane-targeting US 8,986,963 B2 45 46 sequence) so that the proton channel will insert into the cyto creating the envisioned cell-division-controllable designer plasm membrane but not into the photosynthetic thylakoid organisms for biosafety-guarded photobiological production membrane. of biofuels from CO and H2O. No designer proton-channel According to one of the various embodiments, it is a pre gene is required here. ferred practice to use the same inducible promoter Such as the In another embodiment, a cell-division-controllable Nial promoter to control the expression of both the designer designer organism (FIG. 3B) is created by use of a designer proton-channel gene and the designer biofuel-production cell-division-cycle regulatory gene as a biosafety-control pathway genes. In this way, the designer biofuel-production gene (FIG. 2G) that can control the expression of the cell pathway(s) can be inducibly expressed simultaneously with division-cycle (cdc) genes in the host organism so that it can the expression of the designer proton-channel gene that ter 10 inducibly turn off its reproductive functions such as perma minates certain cellular functions including cell division and nently shutting off the cell division and mating capability mating. upon specific induction of the designer gene. In one of the various embodiments, an inducible promoter Biologically, it is the expression of the natural cdc genes that can be used in this designer biosafety embodiment is that controls the cell growth and cell division cycle in cyano selected from the group consisting of the hydrogenase pro 15 bacteria, algae, and higher plant cells. The most basic func moters HydA1 (Hyd1) and HydA2, accession number: tion of the cell cycle is to duplicate accurately the vast amount AJ308413, AF289201, AYO90770), the Cyc6 gene promoter, of DNA in the chromosomes during the S phase (S for syn the Cpxl gene promoter, the heat-shock protein promoter thesis) and then segregate the copies precisely into two HSP70A, the CablI-1 gene (accession number M24072) pro genetically identical daughter cells during the Mphase (M for moter, the Ca1 gene (accession number P20507) promoter, mitosis). Mitosis begins typically with chromosome conden the Ca2 gene (accession number P24258) promoter, the sation: the duplicated DNA strands, packaged into elongated nitrate reductase (Nial) promoter, the nitrite-reductase-gene chromosomes, condense into the much-more compact chro (nirA) promoters, the bidirectional-hydrogenase-gene hoX mosomes required for their segregation. The nuclear enve promoters, the light- and heat-responsive groE promoters, the lope then breaks down, and the replicated chromosomes, each Rubisco-operon rbcL promoters, the metal (zinc)-inducible 25 consisting of a pair of sister chromatids, become attached to Smt promoter, the iron-responsive idiA promoter, the redox the microtubules of the mitotic spindle. As mitosis proceeds, responsive crh R promoter, the heat-shock-gene hsp16.6 pro the cell pauses briefly in a state called metaphase, when the moter, the small heat-shock protein (Hsp) promoter, the CO chromosomes are aligned at the equator of the mitotic responsive carbonic-anhydrase-gene promoters, the green/ spindle, poised for segregation. The Sudden segregation of red light responsive cpcB2A2 promoter, the UV-light 30 sister chromatids marks the beginning of anaphase during responsive leXA, recA and ruvB promoters, the nitrate-reduc which the chromosomes move to opposite poles of the tase-gene (narE) promoters, and combinations thereof. spindle, where they decondense and reform intact nuclei. The In another embodiment, a cell-division-controllable cell is then pinched into two by cytoplasmic division (cytoki designer photosynthetic organism is created by use of a car nesis) and the cell division is then complete. Note, most cells bonic anhydrase deficient mutant or a high-CO-requiring 35 require much more time to grow and double their mass of mutant as a host organism to create the designer biofuel proteins and organelles than they require to replicate their production organism. High-CO-requiring mutants that can DNA (the S phase) and divide (the Mphase). Therefore, there be selected for use in this invention include (but not limited are two gap phases: a G phase between Mphase and S phase, to): Chlamydomonas reinhardtii carbonic-anhydrase-defi and a G2 phase between S phase and mitosis. As a result, the cient mutantl2-1C (CC-1219 calmt-). Chlamydomonas rein 40 eukaryotic cell cycle is traditionally divided into four sequen hardtiicia3 mutant (Plant Physiology 2003, 132:2267-2275), tial phases: G. S. G., and M. Physiologically, the two gap the high-CO-requiring mutant M3 of Synechococcus sp. phases also provide time for the cell to monitor the internal Strain PCC 7942, or the carboxysome-deficient cells of Syn and external environment to ensure that conditions are Suit echocystis sp. PCC 6803 (Plant biol (Stuttg) 2005, 7:342 able and preparation are complete before the cell commits 347) that lacks the CO-concentrating mechanism can grow 45 itself to the major upheavals of S phase and mitosis. The G photoautotrophically only under elevated CO concentration phase is especially important in this aspect. Its length can vary level such as 0.2-3% CO. greatly depending on external conditions and extracellular Under atmospheric CO concentration level (380 ppm), the signals from other cells. If extracellular conditions are unfa carbonic anhydrase deficient or high-CO-requiring mutants vorable, for example, cells delay progress through G and commonly cannot Survive. Therefore, the key concept here is 50 may even enter a specialized resting state known as Go (G that a high-CO2-requiring designer biofuel-production Zero), in which they remain for days, weeks, or even for years organism that lacks the CO concentrating mechanism will be before resuming proliferation. Indeed, many cells remain grown and used for photobiological production of biofuels permanently in Go State until they die. always under an elevated CO, concentration level (0.2-5% In one of the various embodiments, a designer gene(s) that CO) in a sealed bioreactor with CO feeding. Such a designer 55 encodes a designer cdc-regulatory protein or a specific calc transgenic organism cannot survive when it is exposed to an iRNA is used to inducibly inhibit the expression of certain cdc atmospheric CO concentration level (380 ppm=0.038% gene(s) to stop cell division and disable the mating capability CO) because its CO-concetrating mechanism (CCM) for when the designer gene(s) is trigged by a specific inducing effective photosynthetic CO fixation has been impaired by condition. When the cell-division-controllable designer cul the mutation. Even if such a designer organism is accidentally 60 ture is grown and ready for photosynthetic production of released into the natural environment, its cell will soon not be biofuels, for example, it is a preferred practice to induce the able to divide or mate, but die quickly of carbon starvation expression of a specific designer cdc-iRNA gene(s) along since it cannot effectively perform photosynthetic CO, fixa with induction of the designer biofuel-production-pathway tion at the atmospheric CO concentration (380 ppm). There gene(s) so that the cells will permanently halt at the G phase fore, use of such a high-CO-requiring mutant as a host organ 65 or Go State. In this way, the grown designer-organism cells ism for the genetic transformation of the designer biofuel become perfect catalysts for photosynthetic production of production-pathway gene(s) represents another way in biofuels from CO and HO while their functions of cell US 8,986,963 B2 47 48 division and mating are permanently shut off at the G phase bp Synechococcus sp. strain PCC 7942 rbcS terminator or Go State to help ensure biosafety. (1663-1970), and a PCR RE primer (1971-1990). Use of the biosafety embodiments with various designer SEQID NO: 50 presents example 50 for a designer nirA biofuel-production-pathways genes listed in SEQ ID NOS: promoter-controlled Enolase DNA construct (1765 bp) that 1-45 (and 58-165) can create various biosafety-guarded pho includes a PCRFD primer (sequence 1-20), a 88-bp Synecho tobiological biofuel producers (FIGS. 3A, 3B, and 3C). Note, coccus sp. strain PCC 7942 nitrite reductase nirA promoter SEQ ID NOS: 46 and 1-12 (examples 1-12) represent an (21-108), a 9-bp Xho I Ndel site (109-117), an enolase example for a cell-division-controllable designer eukaryotic encoding sequence (118-1407) selected from the sequence of organism Such as a cell-division-controllable designer alga a Cyanothece sp. CCYO110 enolase (GenBank: (e.g., Chlamydomonas) that contains a designer Nial-pro 10 ZP 01727912), a 21-bp Lumio-tag-encoding sequence (1408-1428), a 9-bp Xbal site (1429-1437) containing a stop moter-controlled proton-channel gene (SEQID NO: 46) and codon, a 308-bp Synechococcus sp. strain PCC 7942 rbcS a set of designer Nial-promoter-controlled butanol-produc terminator (1438-1745), and a PCR RE primer (1746-1765) tion-pathway genes (SEQ ID NOS: 1-12). Because the at the 3' end. designer proton-channel gene and the designer biofuel-pro 15 SEQID NO: 51 presents example 51 for a designer nirA duction-pathway gene(s) are all controlled by the same Nial promoter-controlled Pyruvate Kinase DNA construct (1888 promoter sequences, they can be simultaneously expressed bp) that includes a PCRFD primer (sequence 1-20), a 88-bp upon induction by adding nitrate fertilizer into the culture Synechococcus sp. strain PCC 7942 nitrite reductase nirA medium to provide the biosafety-guarded photosynthetic bio promoter (21-108), a 9-bp XhoI Ndel site (109-117), a Pyru fuel-producing capability as illustrated in FIG.3B. Use of the vate-Kinase-encoding sequence (118-1530) selected from a designer Nial-promoter-controlled butanol-production Selenomonas ruminantium Pyruvate Kinase sequence (Gen pathway genes (SEQID NOS: 1-12) in a high CO-requiring Bank: AB037182), a 21-bp Lumio-tag sequence (1531 host photosynthetic organism, such as Chlamydomonas rein 1551), a 9-bp Xbal site (1552-1560), a 308-bp Synechococ hardtii carbonic-anhydrase-deficient mutant 12-1C cus sp. strain PCC 7942 rbcS terminator (1561-1868), and a (CC-1219 cal mt-) or Chlamydomonas reinhardtii cia3 25 PCR RE primer (1869-1888). mutant, represents another example in creating a designer SEQID NO: 52 presents example 52 for a designer nirA cell-division-controllable photosynthetic organism to help promoter-controlled Pyruvate Decarboxylase DNA construct ensure biosafety. (2188 bp) that includes a PCRFD primer (sequence 1-20), a This designer biosafety feature may be useful to the pro 88-bp Synechococcus sp. strain PCC 7942 nitrite reductase duction of other biofuels such as biooils, biohydrogen, etha 30 nirA promoter (21-108), a 9-bp Xho I Ndel site (109-117), a nol, and intermediate products as well. For example, this Pyruvate-Decarboxylase-encoding sequence (118-1830) biosafety embodiment in combination with a set of designer selected from the sequences of a Pichia stipitis pyruvate ethanol-production-pathway genes such as those shown SEQ decarboxylase sequence (GenBank: XM 001387668), a ID NOS: 47-53 can represent a cell-division-controllable 21-bp Lumio-tag sequence (1831-1851), a 9-bp Xbal site ethanol producer (FIG.3C). Briefly, SEQID NO.47 presents 35 (1852-1860), a 308-bp Synechococcus sp. strain PCC 7942 example 47 for a detailed DNA construct (1360 base pairs rbcS terminator (1861-2168), and a PCR RE primer (2169 (bp)) of a nirA-promoter-controlled designer NAD-depen 2188) at the 3' end. dent Glyceraldehyde-3-Phosphate-Dehydrogenase gene SEQID NO: 53 presents example 53 for a nirA-promoter including: a PCR FD primer (sequence 1-20), a 88-bp nirA controlled designer NAD(P)H-dependent Alcohol Dehydro promoter (21-108) selected from the Synechococcus sp. strain 40 genase DNA construct (1510 bp) that includes a PCR FD PCC 7942 (freshwater cyanobacterium) nitrite-reductase primer (sequence 1-20), a 88-bp Synechococcus sp. Strain gene promoter sequence, an enzyme-encoding sequence PCC 7942 nitrite-reductase nirA promoter (21-108), a NAD (109-1032) selected and modified from a Cyanidium cal (P)H dependent Alcohol-Dehydrogenase-encoding sequence darium cytosolic NAD-dependent glyceraldehyde-3-phos (109-1161) selected/modified (its mitochondrial signal pep phate-dehydrogenase sequence (GenBank accession num 45 tide sequence removed) from the sequence of a Kluyveromy ber: CAC85917), a 308-bp Synechococcus sp. strain PCC ces lactis alcohol dehydrogenase (ADH3) gene (GenBank: 7942 rbcS terminator (1033-1340), and a PCR RE primer X62766), a 21-bp Lumio-tag sequence (1162-1182), a 308 (1341-1360) at the 3' end. bp Synechococcus sp. strain PCC 7942 rbcS terminator SEQID NO: 48 presents example 48 for a designer nirA (1183-1490), and a PCRRE primer (1491-1510) at the 3' end. promoter-controlled Phosphoglycerate Kinase DNA con 50 Note, SEQ ID NOS: 47-53 (DNA-construct examples struct (1621 bp) that includes a PCR FD primer (sequence 47-53) represent a set of designer nirA-promoter-controlled 1-20), a 88-bp Synechococcus sp. strain PCC 7942 nitrite ethanol-production-pathway genes that can be used in oxy reductase nirA promoter (21-108), a phosphoglycerate-ki photobacteria such as Synechococcus sp. strain PCC 7942. nase-encoding sequence (109-1293) selected from a Geoba Use of this set of designer ethanol-production-pathway genes cillus kaustophilus HTA426 phosphoglycerate-kinase 55 in a high-CO2-requiring cyanobacterium such as the Syn sequence (GenBank: BAD77342), a 308-bp Synechococcus echococcus sp. Strain PCC 7942 mutant M3 represents sp. strain PCC 7942 rbcS terminator (1294-1601), and a PCR another example of cell-division-controllable designer RE primer (1602-1621). cyanobacterium for biosafety-guarded photosynthetic pro SEQID NO: 49 presents example 49 for a designer nirA duction of biofuels from CO and H2O. promoter-controlled Phosphoglycerate-Mutase DNA con 60 More on Designer Calvin-Cycle-Channeled Production of struct (1990 bp) that includes a PCR FD primer (sequence Butanol and Related Higher Alcohols 1-20), a 88-bp Synechococcus sp. strain PCC 7942 nitrite The present invention further discloses designer Calvin reductase nirA promoter (21-108), a 9-bp Xho I Ndel site cycle-channeled and photosynthetic-NADPH (reduced nico (109-117), a phosphoglycerate-mutase encoding sequence tinamide adenine dinucleotide phosphate)-enhanced path (118-1653) selected from the sequences of a Caldicellulosir 65 ways, associated designer DNA constructs (designer genes) uptor saccharolyticus DSM 8903 phosphoglycerate mutase and designer transgenic photosynthetic organisms for photo (GenBank: ABP67536), a 9-bp Xbal site (1654-1662), a 308 biological production of butanol and related higher alcohols US 8,986,963 B2 49 50 from carbon dioxide and water. In this context throughout this the net results of the designer Calvin-cycle-channeled and specification as mentioned before, a “higher alcohol or photosynthetic NADPH-enhanced pathways in working with “related higher alcohol refers to an alcohol that comprises at the Calvin cycle are production ofbutanol and related higher least four carbonatoms, including both straight and branched alcohols from carbon dioxide (CO) and water (HO) using higher alcohols such as 1-butanol and 2-methyl-1-butanol. photosynthetically generated ATP (Adenosine triphosphate) The Calvin-cycle-channeled and photosynthetic-NADPH and NADPH (reduced nicotinamide adenine dinucleotide enhanced pathways are constructed with designer enzymes phosphate). A significant feature is the innovative utilization expressed through use of designer genes in host photosyn ofan NADPH-dependent glyceraldehyde-3-phosphate dehy thetic organisms such as algae and oxyphotobacteria (includ drogenase 34 and a nicotinamide adenine dinucleotide ing cyanobacteria and oxychlorobacteria) organisms for pho 10 (NAD)-dependent glyceraldehyde-3-phosphate dehydroge tobiological production of butanol and related higher nase 35 to serve as a NADPH/NADH conversion mechanism alcohols. The said butanol and related higher alcohols are that can convert certain amount of photosynthetically gener selected from the group consisting of 1-butanol, 2-methyl ated NADPH to NADH which can then be used by NADH 1-butanol, isobutanol, 3-methyl-1-butanol, 1-hexanol, 1-oc requiring pathway enzymes such as an NADH-dependent tanol. 1-pentanol, 1-heptanol, 3-methyl-1-pentanol, 4-me 15 alcohol dehydrogenase 43 (examples of its encoding gene thyl-1-hexanol, 5-methyl-1-heptanol, 4-methyl-1-pentanol, with GenBank accession numbers are: BAB59540, 5-methyl-1-hexanol, and 6-methyl-1-heptanol. The designer CAA89136, NP 148480) for production of butanol and photosynthetic organisms such as designer transgenic algae higher alcohols. and oxyphotobacteria (including cyanobacteria and oxychlo More specifically, an NADPH-dependent glyceraldehyde robacteria) comprise designer Calvin-cycle-channeled and 3-phosphate dehydrogenase 34 (e.g., GenBank accession photosynthetic NADPH-enhanced pathway gene(s) and bio numbers: ADC37857, ADC87332, YP 003471459, safety-guarding technology for enhanced photobiological ZP 04395517, YP 003287699, ZP 07004478, production of butanol and related higher alcohols from car ZP 043996.16) catalyzes the following reaction that uses bon dioxide and water. NADPH in reducing 1,3-Diphosphoglycerate (1,3-DiPGA) Photosynthetic water splitting and its associated proton 25 to 3-Phosphoglyaldehyde (3-PGAld) and inorganic phos gradient-coupled electron transport process generates chemi phate (Pi): cal energy intermediate in the form of adenosine triphosphate (ATP) and reducing power in the form of reduced nicotina mide adenine dinucleotide phosphate (NADPH). However, Meanwhile, an NAD-dependent glyceraldehyde-3-phos certain butanol-related metabolic pathway enzymes Such as 30 phate dehydrogenase 35 (e.g., GenBank: ADM41489, the NADH-dependent butanol dehydrogenase (GenBank YP 003095198, ADC36961, ZP 07003925, ACQ61431, accession numbers: YP 148778, NP 561774, AAG23613, YP 002285269, ADN80469, ACI60574) catalyzes the oxi ZP 05082669, AD012118, ADC48983) can use only dation of 3-PGAld by oxidized nicotinamide adenine reduced nicotinamide adenine dinucleotide (NADH) but not dinucleotide (NAD") back to 1,3-DiPGA: NADPH. Therefore, to achieve a true coupling of a designer 35 pathway with the Calvin cycle for photosynthetic production of butanol and related higher alcohols, it is a preferred prac The net result of the enzymatic reactions 3 and 4 is the tice to use an effective NADPH/NADH conversion mecha conversion of photosynthetically generated NADPH to nism and/or NADPH-using enzyme(s) (such as NADPH NADH, which various NADH-requiring designer pathway dependent enzymes) in construction of a compatible designer 40 enzymes such as NADH-dependent alcohol dehydrogenase pathway(s) to couple with the photosynthesis/Calvin-cycle 43 can use in producing butanol and related higher alcohols. process in accordance with the present invention. When there is too much NADH, this NADPH/NADH con According to one of the various embodiments, a number of version system can run also reversely to balance the Supply of various designer Calvin-cycle-channeled pathways can be NADH and NADPH. Therefore, it is a preferred practice to created by use of an NADPH/NADH conversion mechanism 45 innovatively utilize this NADPH/NADH conversion system in combination with certainamino-acids-metabolic pathways under control of a designer Switchable promoter Such as nirA for production of butanol and higher alcohols from carbon (or Nial for eukaryotic system) promoter when/if needed to dioxide and water. The Calvin-cycle-channeled and photo achieve robust production of butanol and related higher alco synthetic-NADPH-enhanced pathways are constructed typi hols. Various designer Calvin-cycle-channeled pathways in cally with designer enzymes that are selectively expressed 50 combination of a NADPH/NADH conversion mechanism through use of designer genes in a host photosynthetic organ with certain amino-acids-metabolism-related pathways for ism Such as a host alga or oxyphotobacterium for production photobiological production of butanol and related higher ofbutanol and higher alcohols. A list of exemplary enzymes alcohols are further described hereinbelow. that can be selected for use in construction of the Calvin Table 2 lists examples of enzymes for construction of cycle-channeled and photosynthetic-NADPH-enhanced 55 designer Calvin-cycle-linked pathways for production of pathways are presented in Table 2. As shown in FIGS. 4-10, butanol and related higher alcohols.

GenBank Accession Number, JGI Protein ID or Enzyme? callout number Source (Organism) Citation Oceanithermus profundus DSM ADR35708; Phosphoglycerate 14977; ADI65627, YP 003722750; mutase Nostoc azoliae 0708; YP OO1470593, ABV33529; (phosphoglyceromutase) Thermotoga lettingae TMO; ADIO2216, YP 003702781; Syntrophothermus lipocalidus DSM YP 001212148: 12680; YP OO14098.91; US 8,986,963 B2 51 52 -continued

GenBank Accession Number, JGI Protein ID or Enzyme? calloutnumber Source (Organism) Citation Pelotomaculum thermopropionicum YP 002573254, SI: YP 002573195; Fervidobacterium nodosum Rt 17-B1; ABS60234: Caldicellulosinuptor bes.cii DSM 6725: ABQ47079,YP 001244998: Fervidobacterium nodosum Rt 17-B1; YP OO3496402, BAI80646; Thermotoga petrophila RKU-1; ZP 05046421; Deferribacter desulfuricans SSM1; YP 003.138980, Cyanobium sp. PCC 7001: YP 0.03138979; Cyanothece sp. PCC 8802: JGI Chlre2 protein ID 161689, Chlamydomonas reinhardtii GenBank: AF268078: cytoplasm; Aspergiiitisfiinigatus; XM 747847; XM 749597; Coccidioides immitis: Leishmania XM OO1248115; braziliensis; Aiellomyces capsulatus; XM 001569263; Monocercomonoides sp.: Aspergilius XM 00153.9892; DQ665859; clavatus; Arabidopsis thaliana; Zea XM OO1270940; NM 117020; mayS M80912 O4: Syntrophothermus lipocalidus DSM ADIO2602, YP 003703167; Enolase 12680; Nostoc azoliae 0708; ADI63801; Thermotoga petrophila RKU-1; ABQ46079; Spirochaeta thermophila DSM 6192; YP OO3875216, ADNO2943; Cyanothece sp. PCC 7822; YP 003886899, ADN13624; Hydrogenobacter thermophilus TK-6: YP 0034.32637, BAI69436; Thermosynechococcus elongatus BP BACO8209; 1, ABO16851: Prochiorococcus marinus str. MIT ZP 01083626; 9301; Synechococcus sp. WH 5701: ABG51970; Trichodesmium erythraeum IMS101; ABA23124; Anabaena variabilis ATCC 29413: BAB75237; Nostoc sp. PCC 7120; GenBank: X66412, P31683; Chlamydomonas reinhardtii AK222035; DQ221745; cytoplasm; Arabidopsis thaliana; XM OO1528071; Leishmania Mexicana: Lodderomyces XM OO1611873; elongisportis; Babesia bovis; XM OO1594215; Scierotinia Scierotiorum; Pichia XM 001483612; AB221057; guilliermondii; Spirotrichonympha EF122486, UO9450; DQ845796; leidyi; Oryza sativa; Timastix ABO88633; U82438; D64113: pyriformis; Leticonostoc U13799; AY307449; U17973 mesenteroides; Davidiella tassiana; Aspergilius oryzae; Schizosaccharomyces pombe; Brassica naptis; Zea mays 05: Syntrophothermus lipocalidus DSM DIO2459, YP 003703024; Pyruvate kinase 12680; Cyanothece sp. PCC 8802: P 002372431; Thermotoga lettingae TMO; P OO1471580, ABV34516; Caldicellulosinuptor bes.cii DSM 6725: P 002573139; Geobacilius kaustophilus HTA426; P 148872: Thermosynechococcus elongatus BP P 681306, BACO8068; 1: P OO1306168, ABR30783; Thermosipho melanesiensis BI429; P 001244.312, ABQ46736; Thermotoga petrophila RKU-1; BP67416, YP 001180607; Caldicellulosinuptor saccharolyticus CL43749, YP 002482578; DSM 8903; Cyanothece sp. PCC 7425; P 001514814; Acaryochloris marina MBIC11017; P OO3138017; Cyanothece sp. PCC 8801; P OO1655408; Microcystis aeruginosa NIES-843; P 003890281; Cyanothece sp. PCC 7822; P 003422225; cyanobacterium UCYN-A: P 03273505; Arthrospira maxima CS-328; P 05035056; Synechococcus sp. PCC 7335; JGI Chlre3 protein ID 138105; Chlamydomonas reinhardtii GenBank: AK229638; cytoplasm; Arabidopsis thaliana; AY949876, AY949890, Saccharomyces cerevisiae; Babesia AY949888; XM OO1612087; bovis; Scierotinia Scierotiorum: XM OO1594.710; Trichomonas vaginalis; Pichia XM OO1329865; guilliermondii; Pichia stipitis; XM 001487289; Lodderomyces elongisports; XM OO1384591; Coccidioides immitis; Timastix XM OO1528210; pyriformis; Glycine max (soybean) XM 001240868; DQ845797; LO8632 Peranema trichophorum; Eugiena GenBank: EF114757: Pyruvate-NADP gracilis AB021127, AJ278425 oxidoreductase US 8,986,963 B2 53 54 -continued

GenBank Accession Number, JGI Protein ID or Enzyme? calloutnumber Source (Organism) Citation O6b: Mastigamoeba balamuthi: GenBank: AY101767; YO9702; Pyruvate-ferredoxin Desulfovibrio africanus; Entamoeba U3O149; XM OO1582310, oxidoreductase histolytica; Trichomonas vaginalis; XM 001313670, Cryptosporidium parvum; XM OO1321286, Cryptosporidium baileyi; Giardia XM 001307087, iambia: Entamoeba histolytica; XM 001311860, Hydrogenobacter thermophilus; XM 001314776, Clostridium pasteurianum; XM 001307250; EFO30517; EFO30516; XM 764947; XM 651927; AB042412; Y17727 07: Butyrivibriofibrisolvens; butyrate GenBank: AB190764: Thiolase producing bacterium L2-50; DQ987697; Z92974; Thermoanaerobacterium thermosaccharolyticum; O8: Clostridium beijerinckii; Butyrivibrio GenBank: AF4940.18: 3-Hydroxybutyryl-CoA fibrisolvens; Aiellomyces capsulatus; AB190764; XM OO1537366; dehydrogenase Aspergilius finigatus; Aspergilius XM 741533; XM OO1274776: clavatus; Neosartorya fischeri: XM 001262361; DQ987697; Butyrate-producing bacterium L2-50; BTOO1208; Z92974; Arabidopsis thaliana; Thermoanaerobacterium thermosaccharolyticum; 09: Clostridium beijerinckii:Butyrivibrio GenBank: AF4940.18: Crotonase fibrisolvens; Butyrate-producing AB190764; DQ987697; Z92974 bacterium L2-50: Thermoanaerobacterium thermosaccharolyticum; 10: Clostridium beijerinckii; Butyrivibrio GenBank: AF4940.18: Butyryl-CoA fibrisolvens; Butyrate-producing AB190764; DQ987697; Z92974 dehydrogenase bacterium L2-50: Thermoanaerobacterium thermosaccharolyticum; 11: Cliostridium GenBank: AY251646 Butyraldehyde Saccharoperbativiacetonictim dehydrogenase 12a: Geobacilius kaustophilus HTA426; YP 148778, BAD77210; NADH-dependent Clostridium perfingens str. 13; NP 561774, BAB80564: Butanol dehydrogenase Carboxydothermus AAG23613; hydrogenoformans; P 05082669, EEA96294; Pseudovibrio sp. JE062; DO12118: Ciostridium carboxidivorans P7: DC48983, YP 003425875; Bacilius pseudofirmus OF4; P 693981, BAC15O15; Oceanobacillusiheyensis HTE831; P 06159969, EEZ61452: Slackia exigua ATCC 700122; P 05633940; Fusobacterium ulcerans ATCC 49185: P O5388801; Listeria monocytogenes FSLJ1-175; BB28961: Chiorobium chiorochromatii CalD3; P O2952811; Clostridium perfingens D str. P 02641897; JGS1721; Clostridium perfingens P 02638.128; NCTC 8239; Clostridium perfingens P 02634798: CPE str. F4969; Clostridium T24774; perfingens B str. ATCC 3626; P 026.14964, ZP 02614746; Clostridium botulinum NCTC 2916: P 488606, BAB76265; Nostoc sp. PCC 7120; 12b: Clostridium perfingens str. 13; P 562172, BAB80962; NADPH-dependent Clostridium saccharobutyllicum; AAA83520; Butanol dehydrogenase Subdoigranulum variabile DSM FB77036; 15176; Butyrivibrio crossotus DSM FF67629, ZP 05792927; 2876; Oribacterium sp. oral taxon 078 P 06597730, EFE92592; str. FO262; Clostridium sp. M62/1; FE12215, ZP 06346636; Clostridium hathewayi DSM 13479; FC98086, ZP 06115415; Subdoigranulum variabile DSM P 05979,561; 15176; Faecalibacterium prausnitzi P 05615704, EEU95840; A2-165; Blautia hansenii DSM 20583; P 05853.889, EEX22072; Roseburia intestinais L1-82, P 04745071, EEU99657; Bacilius cereus Rock3-28; P 04236939, EEL31374; Eubacterium rectale ATCC 33656; P 002938098, ACR75964; Clostridium sp. HGF2: FR36834; Atopobium rimae ATCC 496.26; P 03568088; Clostridium perfingens D str. P O2952006; JGS1721; Clostridium perfingens P 02642725; NCTC 8239; Clostridium butyricum P O2950013, ZP O2950012: 5521: Cliostridium carboxidivorans P 06856327; P7; P 001922606, US 8,986,963 B2 55 56 -continued

GenBank Accession Number, JGI Protein ID or Enzyme? calloutnumber Source (Organism) Citation Cliostridium bointinum E3 str. Alaska P 001922335, E43: Clostridium novyi NT: CD52989; YP 878.939; Cliostridium bointinum B str. Eklund P 001887401; 17B: Thermococcus sp. AM4; EB74113; Fusobacterium sp. D11; FD81183; Anaerococcus vaginalis ATCC 51170; P 05473100, EEU12061; Clostridium perfingens CPE str. DT27639; F4969; Clostridium perfingens B str. DT24389; ATCC 3626; 13: Chlamydomonas reinhardtii: enBank: AF026422, Starch synthase Phaseolus vulgaris; Oryza sativa; FO26421, DQ019314, Arabidopsis thaliana; Colocasia F433156; AB293998; D16202, escienta; Amaranthus crientiis; B115917, AY2994.04; Parachiorelia kessieri: Titicum F121673, AK226881: aestivum; Sorghum bicolor; M 101044: AY225862, Astragalus membranaceus; Perilla Y142712; DQ178026; finitesCens; Zea mays; Ipomoea batatas B232549; Y16340; AF168786; FO97922: AF210699; FO19297: AFO68834 14: Arabidopsis thaliana; Zea mays; enBank: NM 127730, Glucose-1-phosphate Chlamydia trachomatis; Soianum M 124205, NM 121927, adenylyltransferase tuberosum (potato); Shigella flexneri: YO59862; EF694839, Lycopersicon escientiin F694838: AFO87165; P55242; P 709206; TO7674 15: Oryza sativa plastid; Aiellomyces enBank: AC105932, Phosphoglucomutase capsulatiis; Pichia Stipitis; F455812; XM OO1536436; Lodderomyces elongisports; XM OO1383281; Aspergilius finigatus; Arabidopsis XM OO1527445; XM 749345; thaliana; Popitius to mentosa; Oryza NM 124561, NM 180508, Saiiva; Zea mays AY128901; AY479974; AF455812; U89342, U89341 16: Staphylococciis Carnostis Subsp. YP 002633806, CAL27621; Hexose-phosphate cannosus TM300; isomerase 17: Hordeum vulgare aleuron cells; GenBank: JO4202; Alpha-amylase; Trichomonas vaginalis; XM 001319100; EF143986: Phanerochaete chrysosporium; AY324649; NM 129551; Chlamydomonas reinhardtii: XO7896; Arabidopsis thaliana; Dictyoglomits thermophilum heat-stable amylase gene; Beta-amylase; Arabidopsis thaliana; Hordeum GenBank: NM 113297: vulgare; Musa actiminate; D21349; DQ166026; Starch phosphorylase; Citrus hybrid cultivar root; Solanum GenBank: AYO98895; P53535: tuberosum chloroplast; Arabidopsis NM 113857, NM 114564: thaliana; Tritictim aestivum; Ipomoea AF27.5551; M64362 batatas; 18: Chlamydomonas reinhardtii: JGI Chlre3 protein ID 135202; Glucose-phosphate Saccharomyces cerevisiae; Pichia GenBank: M21696; (glucose-6-phosphate) stipitis; Aiellomyces capsulatus; XM OO1385873; isomerase Spinacia oleracea cytosol; Oryza XM OO1537043; TO9154; Saiiva cytoplasm; Arabidopsis P42862; NM 123638, thaliana; Zea mayS NM 118595; U17225 19: Chlamydomonas reinhardtii: JGI Chlre2 protein ID 1594.95: Phosphofructose kinase Arabidopsis thaliana; Aiellomyces GenBank: NM 001037043, capsulatiis; Yarrowia lipolytica: NM 179694, NM 119066, Pichia stipitis; Dictyostelium NM 125551; XM OO1537193; discoideum; Tetrahymena AY142710; XM OO1382.359, thermophila: Trypanosoma brucei. XM OO1383.014: XM 639070; Plasmodium falciparum; Spinacia XM 001017610; XM 838827; oleracea; XM 001347929; DQ437575; 2O: Chlamydomonas reinhardtii GenBank: X69969; AF308587: Fructose-diphosphate chloroplast; Fragaria Xananassa NM 005165; XM OO1609195; aldolase cytoplasm; Homo Sapiens; Babesia XM 001312327, bovis; Trichomonas vaginalis; Pichia XM 001312338; stipitis; Arabidopsis thaliana XM OO1387466; NM 120057, NM 001036644 21: Arabidopsis thaliana; Chlamydomonas GenBank: NM 127687, Triose phosphate reinhardtii; Scierotinia Scierotiorum: AF247559; AY742323; isomerase Chlorella pyrenoidosa; Pichia XM OO1587391; AB240149; guilliermondii; Eugiena intermedia: XM 001485684; DQ459379; Eugiena longa; Spinacia oleracea; AY742325; L36387: Soianum chacoense: Hordeum AY438596; U83414: EF575877; vulgare; Oryza Saiiva US 8,986,963 B2 57 58 -continued

GenBank Accession Number, JGI Protein ID or Enzyme? calloutnumber Source (Organism) Citation 34: Staphylococcus aureus 04-02981; DC37.857: NADPH-dependent Staphylococci is lugdunensis; DC87332; Glyceraldehyde-3- Staphylococcus lugdunensis HKU09; P OO3471459; phosphate Vibrio cholerae BX 330286; P 04395517; dehydrogenase Vibrio sp. Ex25; P 003287699; Pseudomonas Savastanoi pv.; P 07004478, EFIOO 105; Vibrio cholerae B33; P 0439.9616 Grimonitia hollisae CIP 101886; P 06052.988, EEY71738; Vibrio mimicus MB-451, P 06041160; Vibrio coralliilyticus ATCC BAA-450; P 0588.6203; Vibrio cholerae MJ-1236; C 002876243; Zea mays cytosolic NADP dependent; C 001105589; Apium graveolens; AAFO8296; Vibrio cholerae B33; EO17521; Vibrio cholerae TMA 21; EO13209; Vibrio choierae bv. aibensis VL426: EOO1829; Vibrio orientalis CIP 102891; C 05943395; Vibrio cholerae MJ-1236; C Q62447; Vibrio cholerae CT 5369-93; 06049761; Vibrio sp. RC586: 06079970; Vibrio furnissii CIP 102972: 05878983; Vibrio metschnikovii CIP 69.14; 05883 187; 35: Edwardsielia tarda FL6-60; DM41489; NAD-dependent Flavobacteriaceae bacterium 3519-10; P 003095198; Glyceraldehyde-3- Staphylococcus aureus 04-02981; DC36961: phosphate Pseudomonas Savastanoi pv. P 07003925; dehydrogenase savastanoi NCPPB 3335; CQ61431, YP 002878104; Vibrio cholerae MJ-1236; P 002285269; Streptococci is pyogenes NZ131; DN80469; Helicobacter pylori 908; C I 6 O 5 74; Streptococci is pyogenes NZ131; DC88142: Staphylococcus lugdunensis HKU09; CY51070; Vibrio sp. Ex25; DK67090; Stenotrophomonas Chelatiphaga: DK67075; Pseudoxanthomonas doikaonensis; DK67085, ACH90636; Stenotrophomonas maliophia; P 044O1333; Vibrio choierae B33; Photobacterium P 06155532; damselae subsp. damselae CIP P 06080908: 102761; Vibrio sp. RC586: P 06052393; Grimonitia hollisae CIP 101886; X42220; Vibrio furnissii CIP 102972. 05292346; Acidithiobacilius caldus ATCC 51756; AC41000; Nostoc sp. PCC 7120; EO22474; Vibrio cholerae BX 330286; EO13042; Vibrio cholerae TMA 21; CAC41000; Nostoc sp. PCC 7120; CAA04942: Pinus Sylvestris; ACO58643, ACO58642: Cheilanthes avapensis; ACO58624, ACO58623; Cheiianthes wootonii: CBH41484, CBH41483; Astrolepis laevis; 36: Hydrogenobacter thermophilus TK-6: P 003433013, ADO45737, (R)-Citramalate Geobacter hemidjiensis Bem; A. 69812; Synthase Geobacter sulfurreducens KN400; CH38284; (EC 2.3.1.182) Meihanobrevibacter ruminantium M1; 84633; Leptospira bifiexa Serovar Patoc OO1719; strain Patoc 1 (Paris); Leptospira K13757; biflexia serovar Monteraierio; K13756; Leptospira interrogans Serovar K13755; Australis; Leptospira interrogans K13753; Serovar Pomona; Leptospira K13754; interrogans Serovar Autumnais; K13752; Leptospira interrogans Serovar K13751: Pyrogenes; Leptospira interrogans K13750; Serovar Canicola; Leptospira K13749; interrogans Serovar Lai; L11763, Acetohaiobium arabaticum DSM 003998693; 5501; Leadbetterella byssophila DSM K66631; 17132; Bacteroides xylamisolvens Q72644; XB1A; Mucilaginibacter paludis DSM DE82919; 18603; Prevoteiia ruminicola 23: BQ04337; Flavobacterium johnsoniae UW 101; P 06244204, Victivais vadensis ATCC BAA-548: A99692; Prevotella copri DSM 18205; B36404, ZP 06251228; US 8,986,963 B2 59 60 -continued

GenBank Accession Number, JGI Protein ID or Enzyme? calloutnumber Source (Organism) Citation Alistipes shahi WAL 8301; CBK64953; Methyliobacter tundripaludum SV96; ZP 07654184: Meihanosarcina mazei Go 1: NP 632695; 37: Eubacterium eligens ATCC 27750 YP 00293O810, (R)-2-Methylmalate Methanocaldococcus jannaschii: YP 00293O809; dehydratase (large and Sebaidella termitidis ATCC 33386; P81291; Small subunits) Eubacterium eligens ATCC 27750; ACZ06998: (EC 4.2.1.35) ACR72362, ACR72361, ACR72363, YP 00293O808; 38: Thermotoga petrophila RKU-1; BQ46641, ABQ46640; 3-Isopropylmalate Cyanothece sp. PCC 7822; P 003886427, dehydratase (large + Syntrophothermus lipocalidus DSM P 003889452; Small subunits) 12680; DIO2900, ADIO2899, (EC 4.2.1.33) Caldicellulosinuptor saccharolyticus P 003703465, ADIO1294; DSM 8903; BP66933, ABP66934; Pelotomaculum thermopropionicum 001211082, SI, 001211083: Caldicellulosinuptor bes.cii DSM 6725: OO2573950, Caldicellulosinuptor saccharolyticus OO2573949; DSM 8903; 00118O124, E. coi: 00118.0125; Spirochaeta thermophila DSM 6192; euC, ECKO074, JWOO71; Pelotomaculum thermopropionicum uD, ECKO073, JWO070; SI: P OO3875294, Hydrogenobacter thermophilus TK-6: OO3873373; Deferribacter desulfuricans SSM1; OO1213069, Anoxybacilius flavithermus WK1; OO1213068; Thermosynechococcus elongatus BP 003433547, 1: OO3432351; Geobacilius kaustophilus HTA426; OO34955.05, Synechocystis sp. PCC 6803; P OO3495504; Chlamydomonas reinhardtii, ACJ32977, ACJ32978; BAC08461, BAC08786; BAD76941, BAD76940; BAA18738, BAA18298; P 001 702135, P OO1696402; 39: Thermotoga petrophila RKU-1; BQ46392, YP 001243968; 3-Isopropylmalate Cyanothece sp. PCC 7822; P 003888480, ADN15205; dehydrogenase Thermosynechococcus elongatus BP ACO9152, NP 682390; (EC 1.1.1.85) 1: DIO2898, YP 003703463: Syntrophothermus lipocalidus DSM DQ78220; 12680; P 002573948: Caldicellulosinuptor bes.cii DSM 6725: P 003998692; Paludibacter propionicigenes WB4: BP66935; Leadbetterella byssophila DSM AAA16706, YP 00118O126; 17132: 001211084; Caldicellulosinuptor saccharolyticus 148510, BAD76942: DSM 8903; Thermus thermophilus: 003433176; Pelotomaculum thermopropionicum OO3873639; SI: OO3495917: Geobacilius kaustophilus HTA426; 002314961; Hydrogenobacter thermophilus TK-6: 002955.062, EFJ43816; Spirochaeta thermophila DSM 6192; OO1701074, Deferribacter desulfuricans SSM1; OO1701073; Anoxybacilius flavithermus WK1; OO3O83133; Voivox Carterif nagariensis; Chlamydomonas reinhardtii: Ostreococcii Statiri; 40: Thermotoga petrophila RKU-1; BQ46395,YP 001243971; 2-Isopropylmalate Cyanothece sp. PCC 7822; P 00389.0122, ADN16847: Synthase Cyanothece sp. PCC 8802: CU99797; (EC 2.3.3.13) Nostoc punctiforme PCC 73102; CC82459: Pelotomaculum thermopropionicum P 001211081; SI: P 0034.32474, BAI69273; Hydrogenobacter thermophilus TK-6: P 414616, AAC73185: E. coli: Caldicellulosinuptor BP66753, YP 001179944; saccharolyticus DSM 8903; P 003703466, ADIO2901; Syntrophothermus lipocalidus DSM P 148511, BAD76943; 12680; Geobacilius kaustophilus P 002572404; HTA426; Caldicellulosinuptor bes.cii P 002314960, ACJ32975; DSM 6725; Anoxybacilius P 003496874, BAI81118; flavithermus WK1: Deferribacter P 682187, BACO8949; desulfuricans SSM1; DNO3009, YP OO3875282; Thermosynechococcus elongatus BP P 001469896, ABV32832; US 8,986,963 B2 61 62 -continued

GenBank Accession Number, JGI Protein ID or Enzyme? calloutnumber Source (Organism) Citation 1: Spirochaeta thermophila DSM P 002.945733, 6192: Thermotoga lettingae TMO: FJS2728; Voivox Carterif nagariensis; CO69978, XP OO2508720; Micromonas sp. RCC299; P 003 063010, EEH52949; Micromonas pusilla CCMP1545; P OO1696603, EDP08580; Chlamydomonas reinhardtii: 41: Geobacilius kaustophilus HTA426; P 148509, YP 148508; isopropylmalate Anabaena variabilis ATCC 29413: P 324.467, YP 324.466; isomerase largeismall Synechocystis sp. PCC 6803; P 442926, NP 441618: Subunits Anoxybacilius flavithermus WK1; P 002314962, (EC 4.2.1.33) Thermosynechococcus elongatus BP P 002314963; 1: P 682024, NP 681699; Spirochaeta thermophila DSM 6192; P 0.03873372; Salmonella enterica Subsp. enterica G23133, CBG23132: serovar Typhimurium str. D23580; P 05702396; Staphylococcus aureus A5937; ET20545; Francisella philomiragia subsp. AAA53236; philomiragia ATCC 25015: BK88.972: Neisseria lactanica; Franciseiia EV86047; novicida U112; Staphylococcus P 05607839; aureus A5937; Staphylococcus aureus EO38992; subsp. aureus 68-397: Fusobacterium DN35429; sp. 2 1 31; Francisella novicida DP98363, ADP98362; GA99-3549; marine bacterium HP15; P 092517, YP 092516; Bacilius licheniformis ATCC 14580; P 353947, YP 353945; Rhodobacter sphaeroides 2.4.1; P OO1631647, Bordeteila petri DSM 12804: P OO1631646; Agrobacterium vitis S4: C OO2551071, C OO2551071; 42: Lactococci is lactis; AAS491.66: 2-keto acid Lactococci is lactis Subsp. lactis ADA65057, YP 003353820; decarboxylase KF147; CAG34226; (EC 4.1.1.72, etc) Lactococcus lactis subsp. Lactis; AAA35267; Kluyveromyces marxianus; CAA59953; Kluyveromyces lactis; AOQBE6; Mycobacterium avium 104; AOPL16: Mycobacterium ulcerans Agy99; Q7U140; Mycobacterium bovis; Q9CBD6; Mycobacterium leprae; YP 00215.0004: Proteus mirabilis HI4320; ADC36400; Staphylococcus aureus 04-02981; AAM21208; Acetobacter pasteuriants; CAA39398: Saccharomyces cerevisiae: AAA27696; Zymomonas mobilis Subsp. mobilis O53865; CP4; Mycobacterium tuberculosis: AOR480; Mycobacterium smegmatis str. MC2 A1 KGY5; 155: Mycobacterium bovis BCG str. Pasteur 1173P2: 43: Thermoplasma volcanium GSS1; BABS954O Alcohol dehydrogenase Giuconacetobacter hanseni ATCC P 06834544: (NAD dependent) 23769; Saccharomyces cerevisiae: 4.AA89136: (EC 1.1.1.1); Aeropyrim pernix K1; P 1484.80; Rhodobacieraies bacterium P 05073895; HTCC2083: Bradyrhizobium P 769420; japonicum USDA 110; DIO1021; Syntrophothermus lipocalidus DSM OO1411173; 12680; Fervidobacterium nodosum O65604; Rt17-B1: Desulfotaiea psychrophila A.IO3878: LSv54: Acetobacter pasteurianus IFO P 192500; 3283-03: Gluconobacter oxydans BK38651: 621H: Aeromonas hydrophila subsp. O O 8 3 O hydrophila ATCC 7966: Acetobacter pasteurianus IFO 3283-01; Streptomyces hygroscopicnis ATCC 53653; 44: Pelotomaculum thermopropionicum P 001211038, BAF58669; Alcohol dehydrogenase SI: P 04573952, EEO43462; (NADPH dependent) Fusobacterium sp. 7 1: P 002494.014, (EC 1.1.1.2): Pichia pastoris GS115; P 002490014: Pichia pastoris GS115; AY71835, XP 002492217, Escherichia coi str. K-12 substr. AY67733; MG1655; hD, NP 417484, AAC76047: Clostridium hathewayi DSM 13479; FC99049; Clostridium butyricum 5521; P O294.8287 Fusobacterium ulcerans ATCC 49185: P 05632371; US 8,986,963 B2 63 64 -continued

GenBank Accession Number, JGI Protein ID or Enzyme? calloutnumber Source (Organism) Citation Fusobacterium sp. D11: Desulfovibrio Z. P 05440863; desulfitricans subsp. destifiricans str. P 389756; G20; Clostridium novyi NT: P 878957; Ciostridium tetani E88: P 782735; Aureobasidium pitilitians; DG56699; Schefersomyces stipitis CBS 6054, BN66271, XP OO1384.300: Thermotoga lettingae TMO; P OO1471424; Thermotoga petrophila RKU-1; P OO1244106; Coprinopsis cinerea okayama 7#130; P 001834460; Saccharomyces cerevisiae EC1118; AY82157; Saccharomyces cerevisiae JAY291; EU07 174; 45: Thermaerobacter subterraneus DSM FR61439; Phosphoenolpyruvate 13965; Cyanothece sp. PCC 7822; P 003887888; carboxylase Thermus sp.: Rhodothermus marinus; AAO7723; CAA67760; (EC 4.1.1.31) Thermosynechococcus elongatus BP P 682702, BACO9464; 1: P 003998059, ADQ17706; Leadbetterella byssophila DSM DQ81501, YP 004.045007; 17132: FQ77722; Riemerella anatipestifer DSM 15868; P 003706036; Mucilaginibacterpaludis DSM 18603; P OO3911597, ADN74523; Truepera radio victrix DSM 17093: P 003.685046; Ferrimonas balearica DSM 9799; P 00368 1843; Meiothermus Silvanus DSM 9946: P 07594.313, ZP 07565817; Nocardiopsis dassonvillei Subsp. DD27759; dassonviiiei DSM 43111: E. coi, P 0038O1346, ADK68466; Meiothermus ruber DSM 1279; P 06967036, EFH90147; Olsenella uli DSM 7084; P 866412, CAD78193; Ktedonobacter racemifer DSM 44963: DR36285: Rhodopinellula baitica SH 1: DP96559; Oceanithermus profundus DSM DR23252: 14977; P 07746438: marine bacterium HP15: P 627344; Marivirga tractuosa DSM 4126; BX34873; Mucilaginibacterpaludis DSM 18603; P 07544559; Streptomyces coelicolor A3(2): BO18389; Delfia acidovorans SPH-1; BM76577; Actinobacilitis pietiropneumoniae BM72969; serovar 13 str. N273; P OO3842669, ADL50905; Prochiorococcus marinus str. MIT AMO7667; 9301: Prochiorococcus marinus str. BF44963; NATL1A P 06399624; Prochiorococcus marinus str. MIT BL64615; 9515; Cliostridium ceilutiovorans P 83.0113; 743B; P 004.01.0507: Neisseria meningitidis Z2491; C OO32735O2: Deinococcus geothermalis DSM 03496.338: 11300; Micromonospora sp. L5; 02894.226; Chlorobiumphaeobacteroides DSM 266; Arthrobacter sp. FB24; Rhodomicrobium vanniei ATCC 17100; Gordonia bronchialis DSM 43247: Thermus aquaticus Y51MC23; Burkholderia ambifaria IOP40-10; 46: Thermotoga lettingae TMO; P OO14701.26; Aspartate Synechococcus elongatus PCC 6301; P 172275; aminotransferase Synechococcus elongatus PCC 7942: P 401562; (EC 2.6.1.1) Thermosipho melanesiensis BI429; P OO1306480; Thermotoga petrophila RKU-1; P OO124.4588; Thermus thermophilus; Anoxybacilius flavithermus WK1; P 002315494; Bacilius sp.: E. coli, AAA22250; aspC: BAB34434; Pelotomaculum thermopropionicum P 001211971; SI: A.B86290; Phormidium lapideum; P 001410686, Fervidobacterium nodosum Rt 17-B1; P OO1409589; Geobacilius kaustophilus HTA426; P 148025,YP 147632, Thermosynechococcus elongatus BP P 146225; NP 683147; 1: CJ34747; Anoxybacilius flavithermus WK1; AD77213, BAD76064; Geobacilius kaustophilus HTA426; P OO3874653; Spirochaeta thermophila DSM 6192; P 002572445; Caldicellulosinuptor bes.cii DSM 6725: s P 001179582; Caldicellulosinuptor saccharolyticus AAA79371; DSM 8903; AAA33942: Arabidopsis thaliana; CAA42430; US 8,986,963 B2 65 66 -continued

GenBank Accession Number, JGI Protein ID or Enzyme? calloutnumber Source (Organism) Citation Glycine max; XP OO1696609; Lupinus anguistifolius; XP 003 060871; Chlamydomonas reinhardtii: Micromonas pusilla CCMP1545; 47: Thermotoga lettingae TMO; P OO1470361, ABV33297; Aspartokinase Cyanothece sp. PCC 8802: P OO3136939; (EC = 2.7.2.4) Thermotoga petrophila RKU-1 P OO1244864, Hydrogenobacter thermophilus TK-6: C OO1243977; Anoxybacilius flavithermus WK1; C 003432105, BAI68904; Bacilius sp.: CJ35001; Spirochaeta thermophila DSM 6192; AAA22251: Anoxybacilius flavithermus WK1; P OO3873788, ADNO1515; Geobacilius kaustophilus HTA426; CJ34043, YP 002316986: Syntrophothermus lipocalidus DSM AD77480, YP 149048; 12680: E. coli: DIO2230,YP 003702795; Thermosynechococcus elongatus BP P 07594328, ZP 07565832; 1: P 682623, BACO9385; Fervidobacterium nodosum Rt 17-B1; BS59942, YP 001410786; Spirochaeta thermophila DSM 6192; P OO3873302, ADNO1029; Pelotomaculum thermopropionicum P 001212149, SI: P 001211837; Caldicellulosinuptor saccharolyticus BP66605; DSM 8903; Caldicellulosinuptor besci P 002573821; DSM 6725; Thermosipho melanesiensis P 001307097, ABR31712; BI429: Thermotoga lettingae TMO; C O O 470985, ABV33921; Arabidopsis thaliana; 3 7 6: Chlamydomonas reinhardtii: AA o 698,576, EDP08069, C OO1695.256;O O 48: Thermotoga lettingae TMO; P OO1470981, ABV33917: Aspartate-semialdehyde Trichodesmium erythraeum IMS101; BG50031: dehydrogenase Prochiorococcus marinus str. MIT BM76828; 93.03; BQ47283, YP 001244859; Thermotoga petrophila RKU-1; BP67176, YP 001180367; Caldicellulosinuptor saccharolyticus DIO1804, YP 003702369; DSM 8903: Syntrophothermus P 00146O230, lipocalidus DSM 12680; E. coli: P 001464895; Fervidobacterium nodosum Rt17-B1 P OO1409594, ABS59937; Caldicellulosinuptor bes.cii DSM 6725: P 002573009; Thermosipho melanesiensis BI429; P 001307092, ABR31707; Spirochaeta thermophila DSM 6192; P OO3875128, ADNO2855; Pelotomaculum thermopropionicum P 001211836, BAF59467; SI: P OO3432252, BAI69051; Hydrogenobacter thermophilus TK-6: P 002316029, ACJ34044: Anoxybacilius flavithermus WK1; P 147128, BAD75560; Geobacilius kaustophilus HTA426; P OO3496635, BAI80879; Deferribacter desulfuricans SSM1; P 680860, BACO7622; Thermosynechococcus elongatus BP AA G23574, AAG23573; 1: P OO1695059, EDP02211; Carboxydothermus BH11018; hydrogenoformans; CU30050; Chlamydomonas reinhardtii: CG41594; Polytomella parva; BR26065; Glycine max; Zea mays; Oryza sativa Indica Group: 49: Syntrophothermus lipocalidus DSM DIO2231, YP 003702796; Homoserine 12680; Cyanothece sp. PCC 7822; P 003887242; dehydrogenase Caldicellulosinuptor bes.cii DSM 6725: P 002573819; Caldicellulosinuptor saccharolyticus BP66607, YP 001179798: DSM 8903: E. coli: FJ980O2: Spirochaeta thermophila DSM 6192; P OO3873441, ADNO1168; Pelotomaculum thermopropionicum P 001212151, BAF59782; SI: P 00343 1981, BAI68780; Hydrogenobacter thermophilus TK-6; P 00231.6756, ACJ34771; Anoxybacilius flavithermus WKI. P 148817, BAD77249; Geobacilius kaustophilus HTA426; P 003496401, BAI80645; Deferribacter desulfuricans SSM1; P 681068, BACO7830; Thermosynechococcus elongatus BP BG78600, AAZ98830; 1: P OO1699712, EDP07408; Glycine max; ACO69662, XP 0025.08404; Chlamydomonas reinhardtii: Micromonas sp. RCC299; US 8,986,963 B2 67 68 -continued

GenBank Accession Number, JGI Protein ID or Enzyme? calloutnumber Source (Organism) Citation 50: Thermotoga petrophila RKU-1; YP 001243979, ABQ46403; Homoserine kinase Cyanothece sp. PCC 7822; YP 003886645; (EC 2.7.1.39) Caldicellulosinuptor bes.cii DSM 6725: YP 002573820; Caldicellulosinuptor saccharolyticus ABP66606, YP 001179797; DSM 8903: E. coli: AP OOO667, BAB96580; Anoxybacilius flavithermus WK1; YP 00231.6754, ACJ34769; Geobacilius kaustophilus HTA426; YP 148815, BAD77247; Thermosynechococcus elongatus BP NP 682555, BACO9317; 1: YP 001212150, BAF59781; Pelotomaculum thermopropionicum YP 003433124, BAI69923; SI: XP 001701899, EDP06874; Hydrogenobacter thermophilus TK-6: ABC24954; Chlamydomonas reinhardtii: NP 179318, AAD33097; Prototheca wickerhamii; ACU26535: Arabidopsis thaliana; ACG46592: Glycine max; Zea mays; 51: Thermotoga petrophila RKU-1; P 001243978, ABQ46402; Threonine synthase Cyanothece sp. PCC 7425; P 002485009; (EC 4.2.99.2) Thermosipho melanesiensis BI429; P OO1306558, ABR31173; Syntrophothermus lipocalidus DSM DIO2519, YP 003703O84; 12680: E. coli: P 000668, NP 414545; Pelotomaculum thermopropionicum P 001213220; SI: P 00231.6755, ACJ34770; Anoxybacilius flavithermus WK1; P 002572552; Caldicellulosinuptor bes.cii DSM 6725: P 00118.0015, ABP66824; Caldicellulosinuptor saccharolyticus P 003433070, YP 003433019, DSM 8903; Hydrogenobacter A.I69869, BAI698.18: thermophilus TK-6; Geobacilius 148816, YP 147614; kaustophilus HTA426; 682017, NP 681772, Thermosynechococcus elongatus BP ACO8534, BAC08779; 1: OO3873303, ADNO1030; Spirochaeta thermophila DSM 6 192; 003495358, BAIT9602; Deferribacter desulfuricans SSM1; Geobacilius kaustophilus HTA426. 52: Geobacilius kaustophilus HTA426; AD76058, BAD75876, Threonine ammonia Prochiorococcus marinus str. MIT P 147626, YP 147444; lyase 9202; Synechococcus sp. PCC 7335; P 05137562; ZP 05035047; (EC 4.3.1.19) Thermotoga petrophila RKU-1; BQ46585, YP 001244.161; Pelotomaculum thermopropionicum P 001210652, BAF58283; SI: P 002315804, Anoxybacilius flavithermus WK1; P 002315746; Deferribacter desulfuricans SSM1; P OO3497384, BAI81628; E. coi: P OO1746093, ZP 07690697: Neisseria lactanica ATCC 23970; EZ76650, ZP 0598.6317; Citrobacter youngae ATCC 29220; FE07783, ZP 06571237; Neisseria polysaccharea ATCC 43768; FH23894, ZP 06863451; Providencia retigeri DSM 1131; FE52186, ZP 06127162; Neisseria subflava NJ9703; FC51529, ZP 0598.5502; Mannheimia haemolytica PHL213; P 04978734; Achromobacter piechaudi ATCC P 06687730, ZP 0668.4811; 43553; Neisseria meningitidis ATCC P 07369980, EFMO4207; 13091; Synechococcus sp. CC9902; BB26032: Synechococcus sp. PCC 7002: CA99606: Synechococcus sp. WH 8109: P 05790446, EEXO7646; Cyanobium sp. PCC 7001: DY39077, ZP 05045768; Anabaena variabilis ATCC 29413: BA20300; Microcoleus chthonoplastes PCC P 05029756; 7420; P OO1701816, EDP06791; Chlamydomonas reinhardtii, 53: Caldicellulosinuptor saccharolyticus BP66750, ABP66751, Acetolactate synthase DSM 8903; P 001179942, ABP66455, (EC 2.2.1.6) Thermotoga petrophila RKU-1; P 001179941, Thermosynechococcus elongatus BP P 001179646; 1: P 001243976, YP 003345845, Syntrophothermus lipocalidus DSM DA66432, ADA66431, 12680; BQ46399, YP 001243975, Pelotomaculum thermopropionicum BQ46400,YP 003345846; SI: P 682614, BACO9376, Geobacilius kaustophilus HTA426; P 681670, BACO8432, Caldicellulosinuptor bes.cii DSM 6725: P 682086; Hydrogenobacter thermophilus TK-6: DIO2904, YP 003703469, DIO2903, YP 003703468; A.F58709, BAF58917, P 001211286, US 8,986,963 B2 69 70 -continued

GenBank Accession Number, JGI Protein ID or Enzyme? calloutnumber Source (Organism) Citation YP 001211078; BAD76946, YP 148514, BAD76945, YP 148513; ACM59790, ACM59628, ACM59629, YP 002572563, P 002572401, P 002572402; P OO34322.99, YP 034323.00, A.I69099, BAI69098: Spirochaeta thermophila DSM 6192; P OO3874926, YP 003874927, Anoxybacilius flavithermus WK1; DN02654, ADNO2653, Deferribacter desulfuricans SSM1; CJ33615, YP 002314957, Escherichia coi str. K-12 substr. CJ32972, ACJ32973, W3110; P 002314958; Saccharomyces cerevisiae, OO3496879, BAI81123, Thermits aquaticals; OO3496878, BAI81122; Synechococcus sp. PCC 7002: 004121, BAE77622, Cyanothece sp. PCC 7424; 004122, BAE77623, Anabaena variabilis ATCC 29413: AE77528, AP 004027, Nostoc sp. PCC 7120; AB96646, AP 000741; Microcystis aeruginosa NIES-843; AA12700; Synechocystis sp. PCC 6803; DN64495, CAA89744, Synechococcus sp. JA-2-3B'a(2-13); DVO9697: Synechococcus sp. JA-3-3Ab: P OO1735999, ACBOO744; Chlamydomonas reinhardtii: P 002376012: Voivox carteri: P 324035: Bacilius subtilis subsp. subtilis str. P 487595, BAB75254; 168; Bacilius licheniformis ATCC P OO1655615; 14580; P 441297, BAA17984, 66718, NP 441304, P 442206, BAA10276: P 478353; P 475372, ABD00213, BDOO270, YP 475476, P 475533; AACO3784, AAB88292, XP 001 700185, EDO983.00, XP 001695168, EDPO1876; AAC04854, AAB88296; CAB07802 (AlsS); AAU42663 (AlsS); 54: Syntrophothermus lipocalidus DSM DIO2902, YP 003703467; Ketol-acid 12680; Caldicellulosinuptor BP66752, YP 001179943; reductoisomerase saccharolyticus DSM 8903: E. coli: AAA67577, YP 001460567; (EC 1.1.1.86) Thermotoga petrophila RKU-1; BQ46398,YP 001243974; Caiditerrivibrio nitroreducens DSM P 004.050904; 19672; OO3874858, ADNO2585; Spirochaeta thermophila DSM 6192; 001211079, BAF58710; Pelotomaculum thermopropionicum 003885458; SI: 003433279, BAIT0078: Cyanothece sp. PCC 7822; 002314959, ACJ32974; Hydrogenobacter thermophilus TK-6: OO2572403; Anoxybacilius flavithermus WK1; 1485.12, BAD76944; Caldicellulosinuptor bes.cii DSM 6725: OO3496877, BAI81121: Geobacilius kaustophilus HTA426; 683044, BACO9806; Deferribacter desulfuricans SSM1; 0.02482078: Thermosynechococcus elongatus BP CC82013; 1: BG53327: Cyanothece sp. PCC 7425; P 05036558; Nostoc punctiforme PCC 73102; P 05026584; Trichodesmium erythraeum IMS101; BO18124; Synechococcus sp. PCC 7335; DY39000; Microcoleus chthonoplastes PCC P 07166132: 7420; Prochiorococcus marinus str. AA48253, NP 001078309; MIT 9301: Cyanobium sp. PCC 7001: AA76854; Arthrospira sp. PCC 8005; CG35752; Arabidopsis thaliana; P OO1702649, EDP06428; Pistin Saiivum (pea); BH11013: Zea mays; Chlamydomonas reinhardtii: Polytomella parva; 55: Thermotoga petrophila RKU-1; P 001243973, ABQ46397; Dihydroxy-acid Cyanothece sp. PCC 7822; P 003887466; dehydratase Marivirga tractuosa DSM 4126; P 004.053736; (EC 4.2.1.9) Geobacilius kaustophilus HTA426; s P 147899, BAD76331, US 8,986,963 B2 71 72 -continued

GenBank Accession Number, JGI Protein ID or Enzyme? calloutnumber Source (Organism) Citation Syntrophothermus lipocalidus DSM P 147822, BAD76254; 12680; DIO2905, YP 003703470; Spirochaeta thermophila DSM 6192; P OO3874669, ADNO2396; Anoxybacilius flavithermus WK1; P 002315593; Caldicellulosinuptor bes.cii DSM 6725: P 002572562; Caldicellulosinuptor saccharolyticus P 001179645, ABP66454; DSM 8903: E. coli: DR29155, YP 001460564: Deferribacter desulfuricans SSM1; P 003496880, BAI81124; Thermosynechococcus elongatus BP P 68.1848, BACO8610; 1: P 0034.31766, BAI68565; Hydrogenobacter thermophilus TK-6: CC82168, ADN14191; Nostoc punctiforme PCC 73102; DI62939; Nostoc azoliae 0708; DZ97146; Arthrospira maxima CS-328; BO17457: Prochiorococcus marinus str. MIT 05044537, EDY37846; 9301; Cyanobium sp. PCC 7001: 05037932; Synechococcus sp. PCC 7335; 06383646; Arthrospira platensis str. Paraca; AGO2689; Microcystis aeruginosa NIES-843; P OO16931 79, EDPO3205; Chlamydomonas reinhardtii: ABO3O11; Arabidopsis thaliana; BR25557: Oryza sativa Indica Group: CU26534: Glycine max; 56: Schizosaccharomyces japonicus P 002173231, EEBO6938; 2-Methylbutyraldehyde yFS275; P 002490018, CAY67737, reductase Pichia pastoris GS115; M OO2489973; (EC 1.1.1.265) Saccharomyces cerevisiae S288c: AA12209, NP O10656, Aspergilius finigatus Af293; M 001180676; Debaryomyces hansenii CBS767; P 752003; Debaryomyces hansenii P 00277O138; Kluyveromyces lactis; AR65507: Lachancea thermotoierans CBS 6340: A. H O 2 5 7 9: Lachancea thermotoierans; P 002554884; Saccharomyces cerevisiae EC1118; AR24447, CAR23718; Saccharomyces cerevisiae JAY291; AY78868; EUO8013; 57: Saccharomyces cerevisiae S288c: AA10635, NM 001183405, 3-Methylbutanal Saccharomyces cerevisiae EC1118; P O14490; reductase Saccharomyces cerevisiae JAY291; AY86141; (EC 1.1.1.265) EU07090; 07: Geobacilius kaustophilus HTA426; P 147173, BAD75605; 3-Ketothiolase Azohydromonas lata; P 523526; (reversible) Rhodoferax ferrireducens T118; AAO1849, CAAO1846; Aliochromatium vinosum: P 28.6222; Dechioromonas aromatica RCB; P 001041914; Rhodobacter sphaeroides ATCC P 001166229; 17029; Rhodobacter sphaeroides BX11181: ATCC 17025; Bacillus sp. 256; P 05785678; Silicibacteriacuscaeruiensis ITI-1157: P 752635; Aspergilius finigatus Af293; AAK21958: Rhizobium etii: ZP 05784120, ZP 05781517; Citreicella sp. SE45; ZP 05742998: Silicibacter sp. TrichCH4B; AAC83659, AAD10275; Azohydromonas lata; AAC69616: Chronobacterium violiaceum: ABV95064; Dinoroseobactershibae DFL 12: AAP41838: Alcaligenes sp. SH-69; AX43351, XP 002418052; Candida dubiniensis CD36: AK18903; Pseudomonas sp. 14-3: P 002375989; Aspergillus flavus NRRL3357: AT37298, EAT37297, Aedes aegypti; P OO1654752, Schefersomyces stipitis CBS 6054; P OO1654751: Cyanothece sp. PCC 7424; BN68380, XP OO1386409; Cyanothece sp. PCC 7822; P 002375827, ACK68959; Microcystis aeruginosa NIES-843; P 003886602, ADN13327; G O 48 2 8 08: Syntrophothermus lipocalidus DSM P 003702743, ADIO2178, 3-Hydroxyacyl-CoA 12680; DIO1287, ADIO 1071; dehydrogenase Oceanithermus profundus DSM DR36325: 14977; P 002317076, Anoxybacilius flavithermus WK1; P 002315864; Pelotomaculum thermopropionicum P 001210823, BAF58454; SI: P 149248,YP 147889; US 8,986,963 B2 73 74 -continued

GenBank Accession Number, JGI Protein ID or Enzyme? calloutnumber Source (Organism) Citation Geobacilius kaustophilus HTA426; YP OO3497047, BAI81291; Deferribacter desulfuricans SSM1; EFQ32520, EFQ35765; Glomerella graminicola M1.001; P 001250712, ABQ55366; Legionella pneumophia str. Corby; P 748706, XP 748351; Aspergilius finigatus Af293; AU80763; Coprinopsis cinerea okayama 7#130; P 001559519; Botryotinia fuckeiana B05.10; BH10642;YP 001462756; Coccidioidesposadasii; E. coli: P 675197; Chelativorans sp. BNC1; CC81853, YP OO1866796; Nostoc punctiforme PCC 73102; P 07114022, CBN59220; Oscillatoria sp. PCC 6506; 09": Bordeteila petrii; CAP41574; Enoyl-CoA dehydratase Bordeteila petri DSM 12804: C OO1629844; Anoxybacilius flavithermus WK1; C 002315700, Geobacilius kaustophilus HTA426; C 002314932; Geobacilius kaatstophiluts; C 148541, YP 147845, Syntrophothermus lipocalidus DSM B D76199: BAD18341; 12680; O2939, ADIO2740, Acinetobacter sp. SE19; 02007, ADIO1364; Schefersomyces stipitis CBS 6054; G10018: Laccaria bicolor S238N-H82; BN64617, XP OO1382646; Alternaria alternate; DRO9131, XP 001888 157: Aiellomyces dermatitidis ER-3: AH83503, Aspergilius finigatus Af293; EQ91989; Cryptococci is neoformans var. sAL93360, XP 755398: neoformans JEC21; E. Coi: 572730; Aspergillus flavus NRRL3357: 5N73405, YP OO1458194; Laccaria bicolor S238N-H82; 002377859; Neosartorya fischeri NRRL 181; RO1115; Nostoc sp. Peltigera membranacea AW18645; cyanobiont': DA69246: 1O': Xanthomonas Campestris pv. A. P53709; 2-Enoyl-CoA reductase Campestris; Xanthomonas Campestris P 001905744; pv. campestris str. B100; P 06489037; Xanthomonas Campestris pv. P 06487845; musacearum NCPPB4381: P 07718056, EFQ82338; Xanthomonas Campestris pv. P 05074461, EDZ42121: vasculorum NCPPB702: P 07049092, EFI69525; Aeronicrobium marinum DSM 15272: P 886510, ABK76225; Rhodobacieraies bacterium P OO16994.17, ACA41287: HTCC2083: P 002910885, EFI27391; Lysinibacilius fusiformis ZC1; FRO5506; Mycobacterium smegmatis str. MC2 002796528, EEH39074; 155; 43955; Lysinibacilius sphaericus C3-41: O3439; Coprinopsis cinerea okayama 7#130; OO3O83795, CAL57762; Arthroderma gypseum CBS 118893; ACS323O2; Paracoccidioides brasiiensis PbO1: Paracoccidioides brasiiensis Pb18: Aiellomyces capsulatus G186AR; Ostreococcii Statiri; Jatropha circas; 11": Ciostridium cellulovorans 743B; YP OO3845606, ADL53842; Acyl-CoA reductase Thermosphaera aggregains DSM YP 003649571, ADG90619; (EC 1.2.1.50) 11486; YP 001565543, ABX37158: Delfia acidovorans SPH-1; ZP 03543536; Comamonas testosteroni KF-1: YP 002321654, ACJ51276; Bifidobacterium longum Subsp. ZP 05497968, EEU57047; infantis ATCC 15697: ZP 06211782, EFA392.09: Clostridium papyrosolvens DSM 2782; EED67822; Acidovorax avenae Subsp. avenae ZP 07740542, EFQ24431; ATCC 19860; ABXO7240, YP OO1547368; Comamonas testosteroni KF-1: ABR34265, YP OO1309221; Aminomonas paucivorans DSM ZP 03148237, EDYO5596; 12260: ZP 06885967, EFG96716; Herpetosiphon aurantiacus ATCC YP 00399.7212, ADQ16859; 23779; YP 00310.1455, ACU37609; Clostridium beijerinckii NCIMB 8052: ACY16972, YP 003268865; Geobacilius sp. G11MC16: AAT00788: Ciostridium ientocelium DSM 5427: AAD38039; Leadbetterella byssophila DSM AAR88762: 17132: ABE65991; Actinosynnema mirum DSM 43827: Haliangium ochraceum DSM 14365; Photobacterium phosphoreum; US 8,986,963 B2 75 76 -continued

GenBank Accession Number, JGI Protein ID or Enzyme? calloutnumber Source (Organism) Citation Simmondsia Chinensis: Hevea brasiliensis: Arabidopsis thaliana; 12': Mycobacterium chubuense NBB4: ACZ56328; Hexanol dehydrogenase 12": Drosophila subobscura: ABO61862, ABO65263, Octanol dehydrogenase CAD43362, CAD43361, EC 1.1.1.73 CAD54410, CAD43360, CAD43359, CAD43358 CAD43357, CAD43356; 43: Pyrococcus furiosus DSM3638; AAC25556; Short chain alcohol Burkhoideria vietnamiensis G4: ABO56626; dehydrogenase Geobacilius thermoleovorans; BAA940.92; Geobacilius kaustophilus HTA426; YP 146837, BAD75269; Anoxybacilius flavithermus WK1; YP 002314715, ACJ32730; Helicobacter pylori PeCana: YP 003927327, ADO07277; Mycobacterium chubuense NBB4: ACZ56328; Mycobacterium avium subsp. avium ZP 05215778: ATCC 25291; Aspergilius oryzae; BAE71320; cyanobacterium UCYN-A: YP 003421738, ADB953.57; Anabaena circinalis AWQC131C: ABI/5134; Cylindrospermopsis raciborskii T3: ABI/5108; Helicobacter pylori SatA64; ADOO5766; Helicobacter pylori Cuz20; ADOO4259; Mycobacterium intracellulare ATCC ZP 05228,059, ZP 05228058; 13950; Mycobacterium avium subsp. ZP 05215779; avium ATCC 25291; ZP 06834730, EFG83978; Giuconacetobacter hanseni ATCC YP OO1910563, ACD48533; 23769; Helicobacter pylori Shi470; YP 88.0627, ABK67217; Mycobacterium avium 104; ADH821.18: Citrus Sinensis; ABD65462: Gossypium hirsutim; ABZ02361, ABZO2360; Arabidopsis halleri; XP 002792148, EEH34889; Paracoccidioides brasiiensis PbO1: XP OO1940779, EDU43498: Pyrenophora tritici-repentis Pt-1C- EER38733; BFP; Aiellomyces capsulatus H143: XP OO1382930, ABN64901; Schefersomyces stipitis CBS 6054;

Designer Calvin-Cycle-Channeled 1-Butanol Producing reductant so that it can help alleviate the requirement of Pathways NADH supply for enhanced photobiological production of According to one of the various embodiments, a designer 40 butanol and other alcohols. As listed in Table 2, examples of Calvin-cycle-channeled pathway is created that takes the NADPH-dependent alcohol dehydrogenase 44 include (but Calvin-cycle intermediate product, 3-phosphoglycerate, and not limited to) the enzyme with any of the following GenBank accession numbers: YP 001211038, ZP 04573952, converts it into 1-butanol by using, for example, a set of XP 002494.014, CAY71835, NP 417484, EFC99049, and enzymes consisting of (as shown with the numerical labels 45 ZP 0294.8287. 34, 35, 03-05, 36-43 in FIG. 4): NADPH-dependent glycer Note, the 2-keto acid decarboxylase 42 (e.g., AAS49166, aldehyde-3-phosphate dehydrogenase 34, NAD-dependent ADA65057, CAG34226, AAA35267, CAA59953, glyceraldehyde-3-phosphate dehydrogenase 35, phospho AOOBE6, AOPL16) and alcohol dehydrogenase 43 (and/or glycerate mutase 03, enolase 04, pyruvate kinase 05, citra 44) have quite broad Substrate specificity. Consequently, their malate synthase 36, 2-methylmalate dehydratase 37, 3-iso 50 use can result in production of not only 1-butanol but also propylmalate dehydratase 38, 3-isopropylmalate other alcohols such as propanol depending on the genetic and dehydrogenase 39, 2-isopropylmalate synthase 40, isopropy metabolic background of the host photosynthetic organisms. lmalate isomerase 41, 2-keto acid decarboxylase 42, and This is because all 2-keto acids can be converted to alcohols alcoholdehydrogenase (NAD dependent) 43. In this pathway by the 2-keto acid decarboxylase 42 and alcohol dehydroge design, as mentioned above, the NADPH-dependent glycer 55 nase 43 (and/or 44) owning to their broad Substrate specific aldehyde-3-phosphate dehydrogenase 34 and NAD-depen ity. Therefore, according to another embodiment, it is a pre dent glyceraldehyde-3-phosphate dehydrogenase 35 serve as ferred practice to use a Substrate-specific enzyme Such as a NADPH/NADH conversion mechanism that can covert cer butanol dehydrogenase 12 when/if production of 1-butanol is tain amount of photosynthetically generated NADPH to desirable. As listed in Table 2, examples of butanol dehydro NADH which can be used by the NADH-requiring alcohol 60 genase 12 are NADH-dependent butanol dehydrogenase dehydrogenase 43 (examples of its encoding gene with the (e.g., GenBank: YP 148778, NP 561774, AAG23613, following GenBank accession numbers: BAB59540, ZP 05082669, AD012118) and/or NAD(P)H-dependent CAA89136, NP 1484.80) for production of 1-butanol by butanol dehydrogenase (e.g., NP 562172, AAA83520, reduction of butyraldehyde. EFB77036, EFF67629, ZP 06597730, EFE12215, According to one of the various embodiments, it is a pre 65 EFC98086, ZP 05979,561). ferred practice to also use an NADPH-dependent alcohol In one of the various embodiments, another designer dehydrogenase 44 that can use NADPH as the source of Calvin-cycle-channeled 1-butanol production pathway is cre US 8,986,963 B2 77 78 ated that takes the Calvin-cycle intermediate product,3-phos that includes a PCR FD primer (sequence 1-20), a 231-bp phoglycerate, and converts it into 1-butanol by using, for nirA promoter from Thermosynechococcus elongatus BP1 example, a set of enzymes consisting of (as shown with the (21-251), an enzyme-encoding sequence (252-1538) numerical labels 34, 35, 03, 04, 45-52 and 40-43 (44/12) in selected/modified from the sequences of a Syntrophothermus FIG. 4): NADPH-dependent glyceraldehyde-3-phosphate lipocalidus DSM 12680 Enolase (GenBank: ADIO2602), a dehydrogenase 34, NAD-dependent glyceraldehyde-3-phos 120-bp rbcS terminator from BP1 (1539-1658), and a PCR phate dehydrogenase 35, phosphoglycerate mutase 03, eno RE primer (1659-1678) at the 3' end. lase 04, phosphoenolpyruvate carboxylase 45, aspartate ami SEQ ID NO: 62 presents example 62 of a designer nirA notransferase 46, aspartokinase 47, aspartate-semialdehyde promoter-controlled Pyruvate Kinase (05) DNA construct dehydrogenase 48, homoserine dehydrogenase 49, 10 (2137 bp) that includes a PCRFD primer (sequence 1-20), a homoserine kinase 50, threonine synthase 51, threonine 231-bp nirA promoter from Thermosynechococcus elongatus ammonia-lyase 52.2-isopropylmalate synthase 40, isopropy BP1 (21-251), an enzyme-encoding sequence (252-1997) lmalate isomerase 41, 3-isopropylmalate dehydrogenase 39, selected/modified from the sequences of a Syntrophothermus 2-keto acid decarboxylase 42, and NAD-dependent alcohol lipocalidus DSM 12680 pyruvate kinase (GenBank: dehydrogenase 43 (and/or NADPH-dependentalcohol dehy 15 ADIO2459), a 120-bp rbcS terminator from BP1 (1998 drogenase 44, or butanol dehydrogenase 12). 2117), and a PCR RE primer (21 18-2137) at the 3' end. According to another embodiment, the amino-acids-me SEQ ID NO: 63 presents example 63 of a designer nirA tabolism-related 1-butanol production pathways numerical promoter-controlled Citramalate Synthase (36) DNA con labels 03-05, 36-43; and/or 03,04, 45-52 and 39-43 (44/12) struct (2163 bp) that includes a PCR FD primer (sequence can operate in combination and/or in parallel with other pho 1-20), a 305-bp nirA promoter (21-325), an enzyme-encod tobiological butanol production pathways. For example, as ing sequence (326-1909) selected and modified from Hydro shown also in FIG. 4, the Frctose-6-photophate-branched genobacter thermophilus TK-6 citramalate synthase (YP 1-butanol production pathway (numerical labels 13-32 and 003433013), a 234-bp rbcS terminator from BP1 (1910 44/12) can operate with the parts of amino-acids-metabolism 2143), and a PCR RE primer (2144-2163). related pathways numerical labels 36-42, and/or 45-52 and 25 SEQ ID NO: 64 presents example 64 of a designer nirA 40-42) with pyruvate and/or phosphoenolpyruvate as their promoter-controlled 3-Isopropylmalate/(R)-2-Methylmalate joining points. Dehydratase (37) DNA construct (2878 bp) consisting of a Examples of designer Calvin-cycle-channeled 1-butanol PCR FD primer (sequence 1-20), a 231-bp nirA promoter production pathway genes (DNA constructs) are shown in the from Thermosynechococcus elongatus BP1 (21-251), a DNA sequence listings. SEQID NOS: 58-70 represent a set 30 3-isopropylmalate? (R)-2-methylmalate dehydratase large of designer genes for a designer nirA-promoter-controlled subunit-encoding sequence (252-2012) selected/modified Calvin-cycle-channeled 1-butanol production pathway (as from the sequences of an Eubacterium eligens ATCC 27750 shown with numerical labels 34,35, 03-05, and 36-43 in FIG. 3-isopropylmalate? (R)-2-methylmalate dehydratase large 4) in a host oxyphotobacterium such as Thermosynechococ subunit (YP 002930810), a 231-bp nirA promoter from cus elongatus BP1. Briefly, SEQID NO:58 presents example 35 Thermosynechococcus elongatus BP1 (2013-2243), a 3-iso 58 of a designer nirA-promoter-controlled NADPH-depen propylmalate/(R)-2-methylmalate dehydratase small sub dent Glyceraldehyde-3-Phosphate Dehydrogenase (34) DNA unit-encoding sequence (2244-2738) selected/modified from construct (1417 bp) that comprises: a PCR FD primer (se the sequences of an Eubacterium eligens ATCC 277503-iso quence 1-20), a 231-bp nirA promoter from Thermosynecho propylmalate/(R)-2-methylmalate dehydratase small subunit coccus elongatus BP1 (21-251), an enzyme-encoding 40 (YP 00293.0809), a 120-bp rbcS terminator from BP1 sequence (252-1277) selected/modified from the sequences (2739-2858), and a PCRRE primer (2859-2878) at the 3' end. of a Staphylococcus aureus 04-02981 NADPH-dependent SEQ ID NO: 65 presents example 65 of a designer nirA glyceraldehyde-3-phosphate dehydrogenase (GenBank: promoter-controlled 3-Isopropylmalate Dehydratase (38) ADC37857), a 120-bp rbcS terminator from BP1 (1278 DNA construct (2380 bp) comprises: a PCRFD primer (se 1397), and a PCR RE primer (1398-1417) at the 3' end. 45 quence 1-20), a 231-bp nirA promoter from Thermosynecho SEQID NO. 59 presents example 59 of a designer nirA coccus elongatus BP1 (21-251), a 3-isopropylmalate dehy promoter-controlled NAD-dependent glyceraldehyde-3- dratase large Subunit-encoding sequence (252-1508) phosphate dehydrogenase (35) DNA construct (1387 bp) that selected/modified from the sequences of a Thermotoga petro comprises: a PCRFD primer (sequence 1-20), a 231-bp nirA phila RKU-1 3-isopropylmalate dehydratase large subunit promoter from Thermosynechococcus elongatus BP1 (21 50 (ABQ46641), a 231-bp nirA promoter from Thermosynecho 251), an enzyme-encoding sequence (252-1247) selected/ coccus elongatus BP1 (1509-1739), a 3-isopropylmalate modified from the sequences of an Edwardsiellatarda FL6-60 dehydratase small subunit-encoding sequence (1740-2240) NAD-dependent glyceraldehyde-3-phosphate dehydroge selected/modified from the sequences of a Thermotoga petro nase (GenBank: ADM41489), a 120-bp rbcS terminator from phila RKU-1 3-isopropylmalate dehydratase small subunit BP1 (1248-1367), and a PCR RE primer (1368-1387) at the 3' 55 (ABQ46640), a 120-bp rbcS terminator from BP1 (2241 end. 2360), and a PCR RE primer (2361-2380) at the 3' end. SEQID NO: 60 presents example 60 of a designer nirA SEQ ID NO: 66 presents example 66 of a designer nirA promoter-controlled Phosphoglycerate Mutase (03) DNA promoter-controlled 3-Isopropylmalate Dehydrogenase (39) construct (1627 bp) that includes a PCRFD primer (sequence DNA construct (1456 bp) consisting of a PCR FD primer 1-20), a 231-bp nirA promoter from Thermosynechococcus 60 (sequence 1-20), a 231-bp nirA promoter from Thermosyn elongatus BP1 (21-251), an enzyme-encoding sequence echococcus elongatus BP1 (21-251), a 3-isopropylmalate (252-1487) selected/modified from the sequences of a Oce dehydrogenase-encoding sequence (252-1316) selected/ anithermus profundus DSM 14977 phosphoglycerate mutase modified from the sequences of a Thermotoga petrophila (GenBank: ADR35708), a 120-bp rbcS terminator from BP1 RKU-1 3-isopropylmalate dehydrogenase (GenBank: (1488-1607), and a PCR RE primer (1608-1627) at the 3' end. 65 CP000702 Region 349983.351047), a 120-bp rbcS termina SEQID NO: 61 presents example 61 of a designer nirA tor from BP1 (1317-1436), and a PCR RE primer (1437 promoter-controlled Enolase (04) DNA construct (1678 bp) 1456) at the 3' end. US 8,986,963 B2 79 80 SEQID NO: 67 presents example 67 of a designer nirA ID NO: 69 and SEQID NO: 71 (and/or SEQID NO:70) can promoter-controlled 2-Isopropylmalate Synthase (40, EC result in the production of alcohol mixtures rather than single 4.1.3.12) DNA construct (1933 bp) consisting of: a PCR FD alcohols since all 2-keto acids can be converted to alcohols by primer (sequence 1-20), a 231-bp nirA promoter from Ther the two broad substrate specificity enzymes. Therefore, to mosynechococcus elongatus BP1 (21-251), an enzyme-en improve the specificity for 1-butanol production, it is a pre coding sequence (252-1793) selected/modified from the ferred practice to use a more Substrate-specific butanol dehy sequences of a Thermotoga petrophila RKU-1 3-isopropyl drogenase 12. SEQ ID NO: 72 presents example 72 of a malate dehydrogenase (CP000702 Region: 352811.354352), designer nir A-promoter-controlled NADH-dependent a 120-bp rbcS terminator from BP1 (1794-1913), and a PCR Butanol Dehydrogenase (12a) DNA construct (1555 bp) that RE primer (1914-1933) at the 3' end. 10 includes a PCR FD primer (sequence 1-20), a 231-bp nirA SEQID NO: 68 presents example 68 of a designer nirA promoter from Thermosynechococcus elongatus BP1 (21 promoter-controlled Isopropylmalate Isomerase (41) DNA 251), an enzyme-encoding sequence (252-1415) selected/ construct (2632 bp) comprises: a PCRFD primer (sequence modified from the sequences of a Geobacillus kaustophilus 1-20), a 231-bp nirA promoter from Thermosynechococcus HTA426 NADH-dependent butanol dehydrogenase (YP elongatus BP1 (21-251), a isopropylmalate isomerase large 15 148778), a 120-bp rbcS terminator from BP1 (1416-1535), subunit-encoding sequence (252-1667) selected/modified and a PCR RE primer (1536-1555) at the 3' end. from the sequences of a Geobacillus kaustophilus HTA426 SEQ ID NO: 73 presents example 73 of a designer nirA 3-isopropylmalate isomerase large subunit (YP 148509), a promoter-controlled NADPH-dependent Butanol Dehydro 231-bp nirA promoter from Thermosynechococcus elongatus genase (12b) DNA construct (1558 bp) consisting of a PCR BP1 (1668-1898), a isopropylmalate isomerase small sub FD primer (sequence 1-20), a 231-bp nirA promoter from unit-encoding sequence (1899-2492) selected/modified from Thermosynechococcus elongatus BP1 (21-251), a NADPH the sequences of a Geobacillus kaustophilus HTA426 isopro dependent butanol dehydrogenase-encoding sequence (252 pylmalate isomerase small subunit (YP 148508), a 120-bp 1418) selected/modified from the sequences of a Clostridium rbcSterminator from BP1 (2493-2612), and a PCRRE primer perfingens str. 13 NADPH-dependent butanol dehydroge (2613-2632) at the 3' end. 25 nase (NP 562172), a 120-bp rbcS terminator from BP1 SEQID NO: 69 presents example 69 of a designer nirA (1419-1528), and a PCRRE primer (1529-1558) at the 3' end. promoter-controlled 2-Keto Acid Decarboxylase (42) DNA Use of SEQID NOS: 72 and/or 73 (12a and/or 12b) along construct (2035 bp) consisting of a PCR FD primer (se with SEQID NOS:58-69 represents a specific Calvin-cycle quence 1-20), a 231-bp nirA promoter from Thermosynecho channeled 1-butanol production pathway numerically labeled coccus elongatus BP1 (21-251), a 2-keto acid decarboxylase 30 as 34, 35, 03-05, 36-42 and 12 in FIG. 4. encoding sequence (252-1895) selected/modified from the SEQID NOS: 74-81 represent an alternative (amino acids sequences of a Lactococcus lactis branched-chain alpha-ke metabolism-related) pathway (45-52 in FIG. 4) that branches toacid decarboxylase (AAS49166), a 120-bp rbcS terminator from the point of phosphoenolpyruvate and merges at the from BP1 (1896-2015), and a PCR RE primer (2016-2035) at point of 2-ketobutyrate in the Calvin-cycle-channeled 1-bu the 3' end. 35 tanol production pathway. Briefly, SEQID NO: 74 presents SEQID NO: 70 presents example 70 of a designer nirA example 74 of a designer nirA-promoter-controlled Phospho promoter-controlled NAD-dependent Alcohol Dehydroge enolpyruvate Carboxylase (45) DNA construct (3646 bp) nase (43) DNA construct (1426 bp) consisting of: a PCRFD consisting of: a PCR FD primer (sequence 1-20), a 231-bp primer (sequence 1-20), a 231-bp nirA promoter from Ther nirA promoter from Thermosynechococcus elongatus BP1 mosynechococcus elongatus BP1 (21-251), an enzyme-en 40 (21-251), an enzyme-encoding sequence (252-3506) coding sequence (252-1286) selected/modified from the selected/modified from the sequences of a Thermaerobacter sequences of an Aeropyrum pernix K, NAD-dependent alco subterraneus DSM 13965 Phosphoenolpyruvate carboxylase holdehydrogenase (NP 148480), a 120-bp rbcS terminator (EFR61439), a 120-bp rbcS terminator from BP1 (3507 from BP1 (1287-1406), and a PCR RE primer (1407-1426) at 3626), and a PCR RE primer (3627-3646) at the 3' end. the 3' end. 45 SEQ ID NO: 75 presents example 75 of a designer nirA As mentioned before, use of an NADPH-dependent alco promoter-controlled Aspartate Aminotransferase (46) DNA holdehydrogenase 44 that can use NADPH as the source of construct (1591 bp) that includes a PCRFD primer (sequence reductant can help alleviate the requirement of NADH supply 1-20), a 231-bp nirA promoter from Thermosynechococcus for enhanced photobiological production ofbutanol and other elongatus BP1 (21-251), an enzyme-encoding sequence alcohols. SEQID NO: 71 presents example 71 of a designer 50 (252-1451) selected/modified from the sequences of a Ther nirA-promoter-controlled NADPH-dependent Alcohol motoga lettingae TMO aspartate aminotransferase (YP Dehydrogenase (44) DNA construct (1468 bp) that com 001470 126), a 120-bp rbcS terminator from BP1 (1452 prises: a PCR FD primer (sequence 1-20), a 231-bp nirA 1471), and a PCR RE primer (1472-1591) at the 3' end. promoter from Thermosynechococcus elongatus BP1 (21 SEQ ID NO: 76 presents example 76 of a designer nirA 251), an enzyme-encoding sequence (252-1328) selected/ 55 promoter-controlled Aspartate Kinase (47) DNA construct modified from the sequences of a Pichia pastoris GS115 (1588 bp) that includes a PCRFD primer (sequence 1-20), a NADPH-dependent medium chain alcohol dehydrogenase 231-bp nirA promoter from Thermosynechococcus elongatus with broad substrate specificity (XP 002494.014), a 120-bp BP1 (21-251), an enzyme-encoding sequence (252-1448) rbcSterminator from BP1 (1329-1458), and a PCRRE primer selected/modified from the sequences of a Thermotoga let (1459–1468) at the 3' end. In one of the examples, this type of 60 tingae TMO aspartate kinase (YP 001470361), a 120-bp NADPH-dependent alcohol dehydrogenase gene (SEQ ID rbcSterminator from BP1 (1449-1568), and a PCRRE primer NO: 71) is also used in construction of Calvin-cycle-chan (1569-1588) at the 3' end. neled butanol production pathway. SEQ ID NO: 77 presents example 77 of a designer nirA However, because of the broad substrate specificity of the promoter-controlled Aspartate-Semialdehyde Dehydroge 2-keto acid decarboxylase (42, SEQ ID NO: 69) and the 65 nase (48) DNA construct (1411 bp) that includes a PCR FD alcohol dehydrogenase (43, SEQID NO: 70; or 44, SEQID primer (sequence 1-20), a 231-bp nirA promoter from Ther NO: 71), the pathway expressed with designer genes of SEQ mosynechococcus elongatus BP1 (21-251), an enzyme-en US 8,986,963 B2 81 82 coding sequence (252-1271) selected/modified from the of (as shown with the numerical labels 34,35, 03-05, 36-39, sequences of a Thermotoga lettingae TMO aspartate-semial 53-55, 42, 43 or 44/56 in FIG. 5): NADPH-dependent glyc dehyde dehydrogenase (YP 001470981), a 120-bp rbcSter eraldehyde-3-phosphate dehydrogenase 34, NAD-dependent minator from BP1 (1272-1391), and a PCR RE primer (1392 glyceraldehyde-3-phosphate dehydrogenase 35, phospho 1411) at the 3' end. glycerate mutase 03, enolase 04, pyruvate kinase 05, citra SEQID NO: 78 presents example 78 of a designer nirA malate synthase 36, 2-methylmalate dehydratase 37, 3-iso promoter-controlled Homoserine Dehydrogenase (49) DNA propylmalate dehydratase 38, 3-isopropylmalate construct (1684 bp) that includes a PCRFD primer (sequence dehydrogenase 39, acetolactate synthase 53, ketol-acid 1-20), a 231-bp nirA promoter from Thermosynechococcus reductoisomerase 54, dihydroxy-acid dehydratase 55, 2-keto elongatus BP1 (21-251), an enzyme-encoding sequence 10 acid decarboxylase 42, and NAD-dependent alcohol dehy (252-1544) selected/modified from the sequences of a Syn drogenase 43 (or NADPH-dependent alcohol dehydrogenase trophothermus lipocalidus DSM 12680 homoserine dehydro 44; more preferably, 2-methylbutyraldehyde reductase 56). genase (ADIO2231), a 120-bp rbcS terminator from BP1 In another embodiment, a designer Calvin-cycle-chan (1545-1664), and a PCR RE primer (1665-1684) at the 3' end. neled 2-methyl-1-butanol production pathway is created that SEQID NO: 79 presents example 79 of a designer nirA 15 takes the intermediate product, 3-phosphoglycerate, and con promoter-controlled Homoserine Kinase (50) DNA construct verts it into 2-methyl-1-butanol by using, for example, a set of (1237 bp) that includes a PCRFD primer (sequence 1-20), a enzymes consisting of (as shown with the numerical labels 231-bp nirA promoter from Thermosynechococcus elongatus 34, 35, 03, 04, 45-55, 42, 43 or 44/56 in FIG. 5): NADPH BP1 (21-251), an enzyme-encoding sequence (252-1097) dependent glyceraldehyde-3-phosphate dehydrogenase 34. selected/modified from the sequences of a Thermotoga petro NAD-dependent glyceraldehyde-3-phosphate dehydroge phila RKU-1 Homoserine Kinase (YP 001243979), a 120 nase 35, phosphoglycerate mutase 03, enolase 04, phospho bp rbcS terminator from BP1 (1098-1217), and a PCR RE enolpyruvate carboxylase 45, aspartate aminotransferase 46, primer (1218-1237) at the 3' end. aspartokinase 47, aspartate-semialdehyde dehydrogenase 48, SEQID NO: 80 presents example 80 of a designer nirA homoserine dehydrogenase 49, homoserine kinase 50, threo promoter-controlled Threonine Synthase (51) DNA construct 25 nine synthase 51, threonine ammonia-lyase 52, acetolactate (1438 bp) that includes a PCRFD primer (sequence 1-20), a synthase 53, ketol-acid reductoisomerase 54, dihydroxy-acid 231-bp nirA promoter from Thermosynechococcus elongatus dehydratase 55, 2-keto acid decarboxylase 42, and NAD BP1 (21-251), an enzyme-encoding sequence (252-1298) dependent alcohol dehydrogenase 43 (or NADPH dependent selected/modified from the sequences of a Thermotoga petro alcohol dehydrogenase 44; more preferably, 2-methylbu phila RKU-1 Threonine Synthase (YP 001243978), a 120 30 tyraldehyde reductase 56). bp rbcS terminator from BP1 (1299-1418), and a PCR RE These pathways (FIG. 5) are quite similar to those of FIG. primer (1419-1438) at the 3' end. 4, except that acetolactate synthase 53, ketol-acid reductoi SEQID NO: 81 presents example 81 of a designer nirA somerase 54, dihydroxy-acid dehydratase 55, and 2-methyl promoter-controlled Threonine Ammonia-Lyase (52) DNA butyraldehyde reductase 56 are used to produce 2-Methyl-1- construct (1600 bp) consisting of a PCRFD primer (sequence 35 Butanol. 1-20), a 231-bp nirA promoter from Thermosynechococcus SEQ ID NO: 82 presents example 82 of a designer nirA elongatus BP1 (21-251), an enzyme-encoding sequence promoter-controlled Acetolactate Synthase (53) DNA con (252-1460) selected/modified from the sequences of a Geo struct (2107 bp) that includes a PCR FD primer (sequence bacillus kaustophilus HTA426 threonine ammonia-lyase 1-20), a 231-bp nirA promoter from Thermosynechococcus (BAD75876), a 120-bp rbcS terminator from BP1 (1461 40 elongatus BP1 (21-251), an acetolactate synthase-encoding 1580), and a PCR RE primer (1581-1600) at the 3' end. sequence (252-1967) selected/modified from the sequences Note, SEQID NOS: 58-61, 74-81, 66-69, and 72 (and/or of a Bacillus subtilis subsp. subtilis str. 168 acetolactate syn 73) represent a set of sample designer genes that can express thase (CAB07802), a 120-bp rbcS terminator from BP1 a Calvin-cycle 3-phophoglycerate-branched photosynthetic (1968-2087), and a PCRRE primer (2088-2107) at the 3' end. NADPH-enhanced 1-butanol production pathway of 34, 35, 45 SEQ ID NO: 83 presents example 83 of a designer nirA 03, 04, 45-52 40, 41, 39, 42, and 12 while SEQ ID NOS: promoter-controlled Ketol-Acid Reductoisomerase (54) 58-69 and 72 (and/or 73) represent another set of sample DNA construct (1405 bp) that includes a PCR FD primer designer genes that can express another Calvin-cycle 3-pho (sequence 1-20), a 231-bp nirA promoter from Thermosyn phoglycerate-branched photosynthetic NADPH-enhanced echococcus elongatus BP1 (21-251), a ketol-acid reductoi 1-butanol production pathway as numerically labeled as 34. 50 Somerase-encoding sequence (252-1265) selected/modified 35, 03-05, 36-42, and 12 in FIG. 4. The net results of the from the sequences of a Syntrophothermus lipocalidus DSM designer photosynthetic NADPH-enhanced pathways in 12680 ketol-acid reductoisomerase (ADIO2902), a 120-bp working with the Calvin cycle are photobiological production rbcSterminator from BP1 (1266-1385), and a PCRRE primer of 1-butanol (CHCHCHCH-OH) from carbon dioxide (1386-1405) at the 3' end. (CO) and water (H2O) using photosynthetically generated 55 SEQ ID NO: 84 presents example 84 of a designer nirA ATP (Adenosine triphosphate) and NADPH (reduced nicoti promoter-controlled Dihydroxy-Acid Dehydratase (55) namide adenine dinucleotide phosphate) according to the DNA construct (2056 bp) that includes a PCR FD primer following process reaction: (sequence 1-20), a 231-bp nirA promoter from Thermosyn echococcus elongatus BP1 (21-251), an enzyme-encoding 60 sequence (252-1916) selected/modified from the sequences Designer Calvin-Cycle-Channeled 2-Methyl-1-Butanol Pro of a Thermotoga petrophila RKU-1 dihydroxy-acid dehy ducing Pathways dratase (YP 001243973), a 120-bp rbcS terminator from According to one of the various embodiments, a designer BP1 (1917-2036), and a PCR RE primer (2037-2056) at the 3' Calvin-cycle-channeled 2-Methyl-1-Butanol production end. pathway is created that takes the Calvin-cycle intermediate 65 SEQ ID NO: 85 presents example 85 of a designer nirA product, 3-phosphoglycerate, and converts it into 2-methyl promoter-controlled 2-Methylbutyraldehyde Reductase (56) 1-butanol by using, for example, a set of enzymes consisting DNA construct (1360 bp) that includes a PCR FD primer US 8,986,963 B2 83 84 (sequence 1-20), a 231-bp nirA promoter from Thermosyn erably, 3-methylbutanal reductase 57). The net result of this echococcus elongatus BP1 (21-251), an enzyme-encoding pathway in working with the Calvin cycle is photobiological sequence (252-1220) selected/modified from the sequences production of 3-methyl-1-butanol (CHCH(CH) of a Schizosaccharomyces japonicus yFS275 2-methylbu CHCH-OH) from carbon dioxide (CO) and water (H2O) tyraldehyde reductase (XP 002173231), a 120-bp rbcS ter using photosynthetically generated ATP and NADPH accord minator from BP1 (1221-1340), and a PCR RE primer (1341 ing to the following process reaction: 1360) at the 3' end. Note, SEQ ID NOS: 58-66, 82-84, 69 and 85 represent another set of sample designer genes that can express a These designer pathways (FIG. 6) share a number of Calvin-cycle 3-phophoglycerate-branched photosynthetic 10 designer pathway enzymes with those of FIGS. 4 and 5. NADPH-enhanced 2-methyl-1-butanol production pathway except that a 3-methylbutanal reductase 57 is preferably used numerically labeled as 34,35,03-05,36-39,53-55, 42 and 56; for production of 3-methyl-1-butanol; they all have a com while SEQID NOS: 58-61, 74-84, 69 and 85 represent a set mon feature of using an NADPH-dependent glyceraldehyde of sample designer genes that can express another Calvin 3-phosphate dehydrogenase 34 and an NAD-dependent glyc cycle 3-phophoglycerate-branched photosynthetic NADPH 15 eraldehyde-3-phosphate dehydrogenase 35 as an NADPH/ enhanced 2-methyl-1-butanol production pathway of 34, 35, NADH conversion mechanism to covert certain amount of 03. 04, 45-55, 42 and 56 in FIG. 5. These designer genes can photosynthetically generated NADPH to NADH which can be used in combination with other pathway gene(s) to express be used by NADH-requiring pathway enzymes such as an certain other pathways such as a Calvin-cycle Fructose-6- NADH-requiring alcohol dehydrogenase 43. phosphate branched 2-methyl-1-butanol production pathway SEQ ID NO: 86 presents example 86 of a designer nirA numerically labeled as 13-26, 36-39, 53-55, 42 and 56 (and/ promoter-controlled 3-Methylbutanal Reductase (57) DNA or, as 13-25, 45-55, 42 and 56) in FIG. 5 as well. The net construct (1420 bp) that includes a PCRFD primer (sequence results of the designer photosynthetic NADPH-enhanced 1-20), a 231-bp nirA promoter from Thermosynechococcus pathways in working with the Calvin cycle are production of elongatus BP1 (21-251), an enzyme-encoding sequence 2-methyl-1-butanol CHCH-CH(CH)CH-OH from car 25 (252-1280) selected/modified from the sequences of a Sac bon dioxide (CO) and water (HO) using photosynthetically charomyces cerevisiae S288c 3-Methylbutanal reductase generated ATP and NADPH according to the following pro (DAA10635), a 120-bp rbcS terminator from BP1 (1281 cess reaction: 1400), and a PCR RE primer (1401-1420) at the 3' end. SEQ ID NOS: 58-62, 82-84, 69, 70 (or 71) represent a set 30 of sample designer genes that can express a Calvin-cycle Designer Calvin-Cycle-Channeled Pathways for Production 3-phosphoglycerate-branched photosynthetic NADPH-en of Isobutanol and 3-Methyl-1-Butanol hanced isobutanol production pathway (34,35, 03-05,53-55, According to one of the various embodiments, a designer 42, 43 or 44); while SEQ ID NOS: 58-62, 82-84, 65-67, 69 Calvin-cycle-channeled pathway is created that takes the and 86 represent another set of sample designer genes that can Calvin-cycle intermediate product, 3-phosphoglycerate, and 35 express a Calvin-cycle 3-phosphoglycerate-branched photo converts it into isobutanol by using, for example, a set of synthetic NADPH-enhanced 3-methyl-1-butanol production enzymes consisting of (as shown with numerical labels 34. pathway (numerical labels 34, 35, 03-05, 53-55, 40, 38, 39, 35, 03-05, 53-55, 42, 43 (or 44) in FIG. 6): NADPH-depen 42, and 57 in FIG. 6). dent glyceraldehyde-3-phosphate dehydrogenase 34, NAD These designer genes can be used with certain other dependent glyceraldehyde-3-phosphate dehydrogenase 35, 40 designer genes to express certain other pathways such as a phosphoglycerate mutase 03, enolase 04, pyruvate kinase 05, Calvin-cycle Fructose-6-phosphate-branched 3-methyl-1- acetolactate synthase 53, ketol-acid reductoisomerase 54. butanol production pathway shown as 13-26, 53-54, 39-40, dihydroxy-acid dehydratase 55, 2-keto acid decarboxylase 42 and 57 (or 43/44) in FIG. 6 as well. The net results of the 42, and NAD-dependent alcohol dehydrogenase 43 (or designer photosynthetic NADPH-enhanced pathways in NADPH-dependent alcohol dehydrogenase 44). The net 45 working with the Calvin cycle are also production of isobu result of this pathway in working with the Calvin cycle is tanol ((CH) CHCH-OH) and/or 3-methyl-1-butanol photobiological production of isobutanol ((CH) (CHCH(CH)CHCH-OH) from carbon dioxide (CO) and CHCH-OH) from carbon dioxide (CO) and water (H2O) water (HO) using photosynthetically generated ATP and using photosynthetically generated ATP and NADPH accord NADPH. ing to the following process reaction: 50 Designer Calvin-Cycle-Channeled Pathways for Production of 1-Hexanol and 1-Octanol According to one of the various embodiments, a designer According to another embodiment, a designer Calvin Calvin-cycle-channeled pathway is created that takes the cycle-channeled pathway is created that takes the intermedi Calvin-cycle intermediate product, 3-phosphoglycerate, and ate product, 3-phosphoglycerate, and converts it into 3-me 55 converts it into 1-hexanol by using, for example, a set of thyl-1-butanol by using, for example, a set of enzymes enzymes consisting of (as shown with the numerical labels consisting of (as shown with the numerical labels 34, 35, 34, 35, 03-10. 07-12' in FIG. 7): NADPH-dependent glycer 03-05, 53-55, 40, 38, 39, 42, 43 (or 44/57) in FIG. 6): aldehyde-3-phosphate dehydrogenase 34, NAD-dependent NADPH-dependent glyceraldehyde-3-phosphate dehydro glyceraldehyde-3-phosphate dehydrogenase 35, phospho genase 34, NAD-dependent glyceraldehyde-3-phosphate 60 glycerate mutase 03, enolase 04, pyruvate kinase 05, pyru dehydrogenase 35, phosphoglycerate mutase 03, enolase 04, vate-ferredoxin oxidoreductase 06, thiolase 07, 3-hydroxy pyruvate kinase 05, acetolactate synthase 53, ketol-acid butyryl-CoA dehydrogenase 08, crotonase 09, butyryl-CoA reductoisomerase 54, dihydroxy-acid dehydratase 55, 2-iso dehydrogenase 10, designer 3-ketothiolase 07", designer propylmalate synthase 40,3-isopropylmalate dehydratase 38, 3-hydroxyacyl-CoA dehydrogenase 08", designer enoyl-CoA 3-isopropylmalate dehydrogenase 39, 2-keto acid decarboxy 65 dehydratase 09", designer 2-enoyl-CoA reductase 10'. lase 42, and NAD-dependent alcohol dehydrogenase 43 (or designer acyl-CoA reductase 11", and hexanol dehydrogenase NADPH-dependentalcoholdehydrogenase 44; or more pref 12. The net result of this designer pathway in working with US 8,986,963 B2 85 86 the Calvin cycle is photobiological production of 1-hexanol 1-20), a 231-bp nirA promoter from Thermosynechococcus (CHCHCHCHCHCH-OH) from carbon dioxide (CO) elongatus BP1 (21-251), an enzyme-encoding sequence and water (H2O) using photosynthetically generated ATP and (252-1421) selected/modified from the sequences of a Xan NADPH according to the following process reaction: thomonas campestris pv. Campestris 2-Enoyl-CoA Reduc tase (CAP53709), a 120-bp rbcS terminator from BP1 (1422 1541), and a PCR RE primer (1542-1561) at the 3' end. According to another embodiment, a designer Calvin SEQ ID NO: 91 presents example 91 of a designer nirA cycle-channeled pathway is created that takes the intermedi promoter-controlled Acyl-CoA Reductase (11') DNA con ate product, 3-phosphoglycerate, and converts it into 1-oc struct (1747 bp) that includes a PCR FD primer (sequence tanol by using, for example, a set of enzymes consisting of (as 10 1-20), a 231-bp nirA promoter from Thermosynechococcus shown with the numerical labels 34, 35, 03-10, 07-10, and elongatus BP1 (21-251), an enzyme-encoding sequence 07"-12" in FIG. 7): NADPH-dependent glyceraldehyde-3- (252-1607) selected/modified from the sequences of a phosphate dehydrogenase 34, NAD-dependent glyceralde Clostridium cellulovorans 743B Acyl-CoA reductase (YP hyde-3-phosphate dehydrogenase 35, phosphoglycerate 0.03845606), a 120-bp rbcS terminator from BP1 (1608 mutase 03, enolase 04, pyruvate kinase 05, pyruvate-ferre 15 1727), and a PCR RE primer (1728-1747) at the 3' end. doxin oxidoreductase 06, thiolase 07, 3-hydroxybutyryl-CoA SEQ ID NO: 92 presents example 92 of a designer nirA dehydrogenase 08, crotonase 09, butyryl-CoA dehydroge promoter-controlled Hexanol Dehydrogenase (12") DNA nase 10, designer 3-ketothiolase 07", designer 3-hydroxyacyl construct (1450 bp) that includes a PCRFD primer (sequence CoA dehydrogenase 08", designer enoyl-CoA dehydratase 1-20), a 231-bp nirA promoter from Thermosynechococcus 09', designer 2-enoyl-CoA reductase 10", designer 3-ke elongatus BP1 (21-251), an enzyme-encoding sequence tothiolase 07", designer 3-hydroxyacyl-CoA dehydrogenase (252-1310) selected/modified from the sequences of a Myco 08", designer enoyl-CoA dehydratase 09", designer 2-enoyl bacterium chubuense NBB4 hexanol dehydrogenase CoA reductase 10", designer acyl-CoA reductase 11", and (ACZ56328), a 120-bp rbcS terminator from BP1 (1311 octanol dehydrogenase 12". 1430), and a PCR RE primer (1431-1450) at the 3' end. These pathways represent a significant upgrade in the path 25 SEQ ID NO: 93 presents example 93 of a designer nirA way designs with part of a previously disclosed 1-butanol promoter-controlled Octanol Dehydrogenase (12") DNA production pathway (03-10). The key feature is the utilization construct (1074 bp) that includes a PCRFD primer (sequence of an NADPH-dependent glyceraldehyde-3-phosphate dehy 1-20), a 231-bp nirA promoter from Thermosynechococcus drogenase 34 and an NAD-dependent glyceraldehyde-3- elongatus BP1 (21-251), an enzyme-encoding sequence phosphate dehydrogenase 35 as a mechanism for NADPH/ 30 (252-934) selected/modified from the sequences of a Droso NADH conversion to drive an NADH-requiring designer phila subobscura octanol dehydrogenase (AB065263), a hydrocarbon chain elongation pathway (07-10) for 1-hex 120-bp rbcS terminator from BP1 (935-1054), and a PCRRE anol production (07-12 as shown in FIG. 7). primer (1055-1074) at the 3' end. SEQID NOS: 87-92 represent a set of designer genes that Note, the designer enzymes of SEQID NOS: 87-91 have can express the designer hydrocarbon chain elongation path 35 certain broad Substrate specificity. Consequently, they can way for 1-hexanol production (07-12 as shown in FIG. 7). also be used as designer 3-ketothiolase 07", designer 3-hy Briefly, SEQ ID NO: 87 presents example 87 of a designer droxyacyl-CoA dehydrogenase 08", designer enoyl-CoA nirA-promoter-controlled 3-Ketothiolase (07) DNA con dehydratase 09", designer 2-enoyl-CoA reductase 10", and struct (1540 bp) that includes a PCR FD primer (sequence designer acyl-CoA reductase 11". Therefore, SEQID NOS: 1-20), a 231-bp nirA promoter from Thermosynechococcus 40 87-91 and 93 represent a set of designer genes that can elongatus BP1 (21-251), an enzyme-encoding sequence express another designer hydrocarbon chain elongation path (252-1400) selected/modified from the sequences of a Geo way for 1-octanol production (07-10' and 07"-12" as shown bacillus kaustophilus HTA426 3-Ketothiolase (YP in FIG. 7). SEQID NO: 93 (encoding for octanol dehydro 147173), a 120-bp rbcS terminator from BP1 (1401-1520), genase 12") is one of the key designer genes that enable and a PCR RE primer (1521-1540) at the 3' end. 45 production of 1-octanol production in this pathway. The net SEQID NO: 88 presents example 88 of a designer nirA result of this pathway in working with the Calvin cycle are promoter-controlled 3-Hydroxyacyl-CoA Dehydrogenase photobiological production of 1-octanol (08) DNA construct (1231 bp) that includes a PCRFD primer (CHCHCHCHCHCHCHCH-OH) from carbon diox (sequence 1-20), a 231-bp nirA promoter from Thermosyn ide (CO) and water (HO) using photosynthetically gener echococcus elongatus BP1 (21-251), an enzyme-encoding 50 ated ATP and NADPH according to the following process sequence (252-1091) selected/modified from the sequences reaction: of a Syntrophothermus lipocalidus DSM 126803-Hydroxya cyl-CoA dehydrogenase (YP 003702743), a 120-bp rbcS terminator from BP1 (1092-1211), and a PCR RE primer (1212-1231) at the 3' end. 55 12O2 10 SEQID NO: 89 presents example 89 of a designer nirA Designer Calvin-Cycle-Channeled Pathways for Production promoter-controlled Enoyl-CoA Dehydratase (09) DNA of 1-Pentanol. 1-Hexanol and 1-Heptanol construct (1162 bp) that includes a PCRFD primer (sequence According to one of the various embodiments, a designer 1-20), a 231-bp nirA promoter from Thermosynechococcus Calvin-cycle-channeled pathway is created that takes the elongatus BP1 (21-251), an enzyme-encoding sequence 60 Calvin-cycle intermediate product, 3-phosphoglycerate, and (252-1022) selected/modified from the sequences of a Bor converts it into 1-pentanol, 1-hexanol, and/or 1-heptanol by detella petrii Enoyl-CoA dehydratase (CAP41574), a 120-bp using, for example, a set of enzymes consisting of (as shown rbcSterminator from BP1 (1023-1442), and a PCRRE primer with the numerical labels 34, 35, 03-05, 36-41, 39, 39-43', (1443-1162) at the 3' end. 39-43', 12", and 39"-43" in FIG. 8):NADPH-dependent glyc SEQID NO: 90 presents example 90 of a designer nirA 65 eraldehyde-3-phosphate dehydrogenase 34, NAD-dependent promoter-controlled 2-Enoyl-CoA Reductase (10) DNA glyceraldehyde-3-phosphate dehydrogenase 35, phospho construct (1561 bp) that includes a PCRFD primer (sequence glycerate mutase 03, enolase 04, pyruvate kinase 05, citra US 8,986,963 B2 87 88 malate synthase 36, 2-methylmalate dehydratase 37, 3-iso propylmalate dehydrogenase 39", designer2-keto acid decar propylmalate dehydratase 38, 3-isopropylmalate boxylase 42", and designer short-chain alcohol dehydroge dehydrogenase 39, 2-isopropylmalate synthase 40, isopropy nase 43". lmalate isomerase 41, 3-isopropylmalate dehydrogenase 39, In this case, proper selection of a short-chain alcoholdehy designer isopropylmalate synthase 40', designer isopropyl drogenase with certain promiscuity is also essential. SEQID malate isomerase 41', designer 3-isopropylmalate dehydro NO: 94 presents example 94 of a designer nirA-promoter genase 39", designer 2-keto acid decarboxylase 42", short controlled Short Chain Alcohol Dehydrogenase DNA con chain alcohol dehydrogenase 43', hexanol dehydrogenase struct (1096 bp) that includes a PCR FD primer (sequence 12, designer isopropylmalate synthase 40", designer isopro 1-20), a 231-bp nirA promoter from Thermosynechococcus pylmalate isomerase 41", designer 3-isopropylmalate dehy 10 drogenase 39", designer 2-keto acid decarboxylase 42", and elongatus BP1 (21-251), an enzyme-encoding sequence designer short-chain alcohol dehydrogenase 43". This (252-956) selected/modified from the sequences of a Pyro designer pathway works with the Calvin cycle using photo coccus furiosus DSM 3638 Short chain alcohol dehydroge synthetically generated ATP and NADPH for photobiological nase (AAC25556), a 120-bp rbcS terminator from BP1 (957 production of 1-pentanol (CHCH2CH2CHCH-OH), 1-hex 15 1076), and a PCR RE primer (1077-1096) at the 3' end. anol (CHCHCHCHCHCH-OH), and/or 1-heptanol Therefore, SEQ ID NOS: 58-69 and 94 represent a set of (CHCHCHCHCHCHCH-OH) from carbon dioxide designer genes that can express a designer Calvin-cycle (CO) and water (H2O) according to the following process 3-phosphoglycerate-braned photosynthetic NADPH-en reactions: hanced pathway for production of 1-pentanol, 1-hexanol, and/or 1-heptanol as shown with numerical labels 34, 35, 03-05, 36-41, 39, 39'-43', 39-43', 39"-43" in FIG. 8. Simi larly, SEQ ID NOS: 58-61, 74-81, 66-69, and 94 represent another set of sample designer genes that can express another Calvin-cycle 3-phophoglycerate-branched NADPH-en 25 hanced pathway for production of 1-pentanol, 1-hexanol, and/or 1-heptanol as numerically labeled as 34, 35, 03, 04, According to another embodiment, a designer Calvin 45-52, 40, 41, 39, 39-43', 39-43', 39"-43" in FIG. 8. Note, cycle-channeled pathway is created that takes the intermedi both of these two pathways produce alcohol mixtures with ate product, 3-phosphoglycerate, and converts it into 1-pen different chain lengths rather than single alcohols since all tanol, 1-hexanol, and/or 1-heptanol by using, for example, a 30 2-keto acids (such as 2-ketohexanoate, 2-ketaheptanoate, and set of enzymes consisting of (as shown with the numerical 2-ketooctanoate) can be converted to alcohol because of the labels 34, 35, 03, 04, 45-52, 40, 41, 39, 39-43', 39-43', 12", use of the promiscuity of designer 2-keto acid decarboxylase and 39"-43" in FIG. 8): NADPH-dependent glyceraldehyde 42 and designer short-chain alcohol dehydrogenase 43'. 3-phosphate dehydrogenase 34, NAD-dependent glyceralde To improve product specificity, it is a preferred practice to hyde-3-phosphate dehydrogenase 35, phosphoglycerate 35 use Substrate specific designer enzymes. For example, use of mutase 03, enolase 04, phosphoenolpyruvate carboxylase 45. Substrate specific designer 1-hexanol dehydrogenase 12 aspartate aminotransferase 46, aspartokinase 47, aspartate (SEQ ID NO: 92) instead of short-chain alcohol dehydroge semialdehyde dehydrogenase 48, homoserine dehydrogenase nase with promiscuity (43") can improve product specificity 49, homoserine kinase 50, threonine synthase 51, threonine more toward 1-hexanol. Consequently, SEQID NOS: 58–69 ammonia-lyase 52.2-isopropylmalate synthase 40, isopropy 40 and 92 represent a set of designer genes that can express a lmalate isomerase 41, 3-isopropylmalate dehydrogenase 39, designer Calvin-cycle 3-phosphoglycerate-braned photosyn designer isopropylmalate synthase 40', designer isopropyl thetic NADPH-enhanced pathway for production of 1-hex malate isomerase 41', designer 3-isopropylmalate dehydro anol as shown with numerical labels 34,35, 03-05, 36-41, 39, genase 39", designer 2-keto acid decarboxylase 42", short 39-40', 39-42" and 12' in FIG. 8. chain alcohol dehydrogenase 43', hexanol dehydrogenase 45 Designer Calvin-Cycle-Channeled Pathways for Production 12, designer isopropylmalate synthase 40", designer isopro of 3-Methyl-1-Pentanol, 4-Methyl-1-Hexanol, and 5-Me pylmalate isomerase 41", designer 3-isopropylmalate dehy thyl-1-Heptanol drogenase 39", designer 2-keto acid decarboxylase 42", and According to one of the various embodiments, a designer designer short-chain alcohol dehydrogenase 43". Calvin-cycle-channeled pathway is created that takes the These pathways (FIG. 8) share a common feature of using 50 Calvin-cycle intermediate product, 3-phosphoglycerate, and an NADPH-dependent glyceraldehyde-3-phosphate dehy converts it into 3-methyl-1-pentanol, 4-methyl-1-hexanol, drogenase 34 and an NAD-dependent glyceraldehyde-3- and/or 5-methyl-1-heptanol by using, for example, a set of phosphate dehydrogenase 35 as a mechanism for NADPH/ enzymes consisting of (as shown with the numerical labels NADH conversion to drive production of 1-pentanol, 34, 35, 03-05, 36-39, 53-55, 39-43', 39-43', and 39"-43" in 1-hexanol, and/or 1-heptanol through a designer Calvin 55 FIG. 9): NADPH-dependent glyceraldehyde-3-phosphate cycle-channeled pathway in combination with a designer dehydrogenase 34, NAD-dependent glyceraldehyde-3-phos hydrocarbon chain elongation pathway (40'. 41', 39"). This phate dehydrogenase 35, phosphoglycerate mutase 03, eno embodiment also takes the advantage of the broad substrate lase 04, pyruvate kinase 05, citramalate synthase 36, 2-meth specificity (promiscuity) of 2-isopropylmalate synthase 40, ylmalate dehydratase 37, 3-isopropylmalate dehydratase 38, isopropylmalate isomerase 41, 3-isopropylmalate dehydro 60 3-isopropylmalate dehydrogenase 39, acetolactate synthase genase 39, 2-keto acid decarboxylase 42, and short-chain 53, ketol-acid reductoisomerase 54, dihydroxy-acid dehy alcohol dehydrogenase 43 so that they can be used also as: dratase 55, designer isopropylmalate synthase 40', designer designer isopropylmalate synthase 40', designer isopropyl isopropylmalate isomerase 41', designer 3-isopropylmalate malate isomerase 41', designer 3-isopropylmalate dehydro dehydrogenase 39", designer 2-keto acid decarboxylase 42", genase 39", designer2-keto acid decarboxylase 42, and short 65 short-chain alcohol dehydrogenase 43', designer isopropyl chain alcohol dehydrogenase 43', isopropylmalate synthase malate synthase 40", designer isopropylmalate isomerase 40", designer isopropylmalate isomerase 41", designer 3-iso 41", designer 3-isopropylmalate dehydrogenase 39", US 8,986,963 B2 89 90 designer 2-keto acid decarboxylase 42", and designer short heptanol (CHCH-CH(CH)CHCHCHCH-OH) from chain alcohol dehydrogenase 43". carbon dioxide (CO) and water (HO) using photosyntheti According to another embodiment, a designer Calvin cally generated ATP and NADPH according to the following cycle-channeled pathway is created that takes the intermedi process reactions: ate product, 3-phosphoglycerate, and converts it into 3-me thyl-1-pentanol, 4-methyl-1-hexanol, and/or 5-methyl-1- heptanol by using, for example, a set of enzymes consisting of (as shown with the numerical labels 34, 35, 03, 04, 45-55, 39-43', 39-43', and 39"-43" in FIG.9): NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase 34, NAD-de 10 pendent glyceraldehyde-3-phosphate dehydrogenase 35, phosphoglycerate mutase 03, enolase 04, phosphoenolpyru Designer Calvin-Cycle-Channeled Pathways for Production vate carboxylase 45, aspartate aminotransferase 46, aspar of 4-Methyl-1-Pentanol. 5-Methyl-1-Hexanol, and 6-Me tokinase 47, aspartate-semialdehyde dehydrogenase 48, thyl-1-Heptanol homoserine dehydrogenase 49, homoserine kinase 50, threo 15 According to one of the various embodiments, a designer nine synthase 51, threonine ammonia-lyase 52, acetolactate Calvin-cycle-channeled pathway is created that takes the synthase 53, ketol-acid reductoisomerase 54, dihydroxy-acid Calvin-cycle intermediate product, 3-phosphoglycerate, and dehydratase 55, designer isopropylmalate synthase 40'. converts it into 4-methyl-1-pentanol, 5-methyl-1-hexanol, designer isopropylmalate isomerase 41', designer 3-isopro and 6-methyl-1-heptanol by using, for example, a set of pylmalate dehydrogenase 39", designer 2-keto acid decar enzymes consisting of (as shown with the numerical labels boxylase 42, short-chain alcohol dehydrogenase 43', 34, 35, 03-05, 53-55, 40, 38,39,39-43', 39-43', and 39"-43" designer isopropylmalate synthase 40", designer isopropyl in FIG. 10): NADPH-dependent glyceraldehyde-3-phos malate isomerase 41", designer 3-isopropylmalate dehydro phate dehydrogenase 34, NAD-dependent glyceraldehyde-3- genase 39", designer 2-keto acid decarboxylase 42", and phosphate dehydrogenase 35, phosphoglycerate mutase 03. designer short-chain alcohol dehydrogenase 43". 25 enolase 04, pyruvate kinase 05, acetolactate synthase 53, These pathways (FIG. 9) are similar to those of FIG. 8, ketol-acid reductoisomerase 54, dihydroxy-acid dehydratase except they use acetolactate synthase 53, ketol-acid reductoi 55, isopropylmalate synthase 40, dehydratase 38, 3-isopro somerase 54, dihydroxy-acid dehydratase 55 as part of the pylmalate dehydrogenase 39, designer isopropylmalate Syn pathways for production of 3-methyl-1-pentanol, 4-methyl thase 40', designer isopropylmalate isomerase 41', designer 1-hexanol, and/or 5-methyl-1-heptanol. They all share a com 30 3-isopropylmalate dehydrogenase 39", designer 2-keto acid mon feature of using an NADPH-dependent glyceraldehyde decarboxylase 42, short-chain alcohol dehydrogenase 43', 3-phosphate dehydrogenase 34 and an NAD-dependent designer isopropylmalate synthase 40", designer isopropyl glyceraldehyde-3-phosphate dehydrogenase 35 as a mecha malate isomerase 41", designer 3-isopropylmalate dehydro nism for NADPH/NADH conversion to drive production of genase 39", designer 2-keto acid decarboxylase 42", and 3-methyl-1-pentanol, 4-methyl-1-hexanol, and/or 5-methyl 35 designer short-chain alcohol dehydrogenase 43". 1-heptanol through a designer Calvin-cycle-channeled path This pathway (FIG. 10) is similar to those of FIG. 8, except way in combination with a hydrocarbon chain elongation that it does not use citramalate synthase 36 and 2-methyl pathway (40, 41', 39"). This embodiment also takes the malate dehydratase 37, but uses acetolactate synthase 53, advantage of the broad Substrate specificity (promiscuity) of ketol-acid reductoisomerase 54, dihydroxy-acid dehydratase 2-isopropylmalate synthase 40, isopropylmalate isomerase 40 55 as part of the pathways for production of 4-methyl-1- 41, 3-isopropylmalate dehydrogenase 39, 2-keto acid decar pentanol, 5-methyl-1-hexanol, and/or 6-methyl-1-heptanol. boxylase 42, and short-chain alcohol dehydrogenase 43 so They all share a common feature of using an NADPH-depen that they can also serve as: designer isopropylmalate synthase dent glyceraldehyde-3-phosphate dehydrogenase 34 and an 40', designer isopropylmalate isomerase 41', designer 3-iso NAD-dependent glyceraldehyde-3-phosphate dehydroge propylmalate dehydrogenase 39', designer 2-keto acid decar 45 nase 35 as a mechanism for NADPH/NADH conversion to boxylase 42, and short-chain alcohol dehydrogenase 43'; drive production of 3-methyl-1-butanol, 4-methyl-1-butanol, designer isopropylmalate synthase 40", designer isopropyl and 5-methyl-1-butanol through a Calvin-cycle-channeled malate isomerase 41", designer 3-isopropylmalate dehydro pathway in combination with a designer hydrocarbon chain genase 39", designer 2-keto acid decarboxylase 42", and elongation pathway (40'. 41', 39"). This embodiment also designer short-chain alcohol dehydrogenase 43". 50 takes the advantage of the broad Substrate specificity (pro Therefore, SEQID NOS: 58-69, 82-84, and 94 representa miscuity) of 2-isopropylmalate synthase 40, isopropylmalate set of designer genes that can express a designer Calvin-cycle isomerase 41, 3-isopropylmalate dehydrogenase 39, 2-keto 3-phosphoglycerate-braned photosynthetic NADPH-en acid decarboxylase 42, and short-chain alcohol dehydroge hanced pathway for production of 3-methyl-1-pentanol, nase 43 so that they may also serve as: designer isopropyl 4-methyl-1-hexanol, and 5-methyl-1-heptanol as shown with 55 malate synthase 40', designer isopropylmalate isomerase 41', numerical labels 34,35, 03-05, 36-39, 53-55, 39-43', 39-43', designer 3-isopropylmalate dehydrogenase 39", designer and 39"-43" in FIG.9. Similarly, SEQID NOS:58-61, 74-81, 2-keto acid decarboxylase 42", and short-chain alcohol dehy 82-84, 66-69, and 94 represent another set of sample designer drogenase 43', designer isopropylmalate synthase 40". genes that can express another Calvin-cycle 3-phophoglyc designer isopropylmalate isomerase 41", designer 3-isopro erate-branched NADPH-enhanced pathway for production of 60 pylmalate dehydrogenase 39", designer 2-keto acid decar 3-methyl-1-pentanol, 4-methyl-1-hexanol, and/or 5-methyl boxylase 42", and designer short-chain alcohol dehydroge 1-heptanol as numerically labeled as 34, 35, 03, 04, 45-55, nase 43". 39-43', 39-43', 39"-43" in FIG. 9. The net results of the Therefore, SEQID NOS:58-62, 82-84, 65-69 and 94 rep designer photosynthetic NADPH-enhanced pathways in resent a set of sample designer genes that can be used to working with the Calvin cycle are production of 3-methyl-1- 65 express a designer Calvin-cycle 3-phosphoglycerate-braned pentanol (CHCH-CH(CH)CHCH-OH), 4-methyl-1-hex photosynthetic NADPH-enhanced pathway for production of anol (CHCH-CH(CH)CHCHCH-OH), and 5-methyl-1- 4-methyl-1-pentanol, 5-methyl-1-hexanol, and/or 6-methyl US 8,986,963 B2 91 92 1-heptanol as shown with numerical labels 34, 35, 03-05, echocyitis PCC 6714 phycocyanin-deficient mutant PD-1, 53-55, 40, 38,39,39-43', 39-43', and 39"-43" in FIG.10. The Cyanothece strain 51142, Cyanothece sp. CCYO110, Oscil net results of the designer photosynthetic NADPH-enhanced latoria limosa, Lyngbya majuscula, Symploca muscorum, pathway in working with the Calvin cycle are production of Gloeobacter violaceus, Prochloron didemni, Prochlorothrix 4-methyl-1-pentanol (CHCH(CH)CHCHCH-OH), hollandica, Prochlorococcus marinus, Prochlorococcus 5-methyl-1-hexanol (CHCH(CH)CHCHCHCH-OH), SS120, Synechococcus WH8102, Lyngbya majuscula, Sym and 6-methyl-1-heptanol (CHCH(CH.) ploca muscorum, Synechococcus bigranulatus, cryophilic CHCHCHCHCH-OH) from carbon dioxide (CO) and Oscillatoria sp., Phormidium sp., Nostoc sp.-1, Calothrix water (HO) using photosynthetically generated ATP and parietina, thermophilic Synechococcus bigranulatus, Syn NADPH according to the following process reactions: 10 echococcus lividus, thermophilic Mastigocladus laminosus, Chlorogloeopsis fritschii PCC 6912, Synechococcus vulca nus, Synechococcus sp. strain MA4, Synechococcus sp. Strain MA19, and Thermosynechococcus elongatus. According to one of the examples, use of designer DNA 15 constructs such as SEQID NOS: 58-94 in genetic transform of certain oxyphotobacteria hosts such as Thermosynechoc occus elongatus BP1 can create a series of designer trans Designer Oxyphotobacteria with Calvin-Cycle-Channeled genic oxyphotobacteria with Calvin-cycle-channeled path Pathways for Production of Butanol and Related Higher ways for production of butanol and related higher alcohols. Alcohols Consequently, SEQ ID NOS: 58-61, 74-81, 66-69, and 72 According to one of the various embodiments, use of (and/or 73) represent a designer transgenic oxyphotobacte designer DNA constructs in genetic transform of certain oxy rium such as a designer transgenic Thermosynechococcus photobacteria hosts can create various designer transgenic that comprises the designer genes of a Calvin-cycle 3-pho oxyphotobacteria with Calvin-cycle-channeled pathways for phoglycerate-branched photosynthetic NADPH-enhanced photobiological production of butanol and related higher 25 pathway (numerically labeled as 34,35, 03.04, 45-52, 39-42, alcohols from carbon dioxide and water. To ensure biosafety and 12 in FIG. 4) for photobiological production of 1-butanol for use of the designer transgenic photosynthetic organism from carbon dioxide and water. SEQID NOS: 58–69 and 72 based biofuels production technology, it is a preferred prac (and/or 73) represent another designer transgenic oxyphoto tice to incorporate biosafety-guarded features into the bacterium Such as designer transgenic Thermosynechococcus designer transgenic photosynthetic organisms as well. There 30 that comprises the designer genes of a Calvin-cycle 3-pho fore, in accordance with the present invention, various phoglycerate-branched photosynthetic NADPH-enhanced designer photosynthetic organisms including designer trans pathway (numerically labeled as 34,35, 03-05, 36-42, and 12 genic oxyphotobacteria are created with a biosafety-guarded in FIG. 4) for photobiological production of 1-butanol from photobiological biofuel-production technology based on carbon dioxide and water as well. cell-division-controllable designer transgenic photosynthetic 35 Similarly, SEQID NOS:58-66,82-84, 69 and 85 represent organisms. The cell-division-controllable designer photosyn another designer transgenic oxyphotobacterium such as thetic organisms contain two key functions: a designer bio designer transgenic Thermosynechococcus with a Calvin safety mechanism(s) and a designer biofuel-production path cycle 3-phophoglycerate-branched photosynthetic NADPH way(s). The designer biosafety feature(s) is conferred by a enhanced pathway (numerically labeled as 34, 35, 03-05, number of mechanisms including: a) the inducible insertion 40 36-39, 53-55, 42 and 56 in FIG. 5) for photobiological pro of designer proton-channels into cytoplasm membrane to duction of 2-methyl-1-butanol production from carbon diox permanently disable any cell division and/or mating capabil ide and water; while SEQID NOS: 58-61, 74-84, 69 and 85 ity, b) the selective application of designer cell-division-cycle represent another designer transgenic Thermosynechococcus regulatory protein or interference RNA (iRNA) to perma with a Calvin-cycle 3-phophoglycerate-branched photosyn nently inhibit the cell division cycle and preferably keep the 45 thetic NADPH-enhanced 2-methyl-1-butanol production cell at the G phase or Go State, and c) the innovative use of a pathway (34, 35, 03, 04, 45-55, 42 and 56 in FIG. 5) for high-CO-requiring host photosynthetic organism for expres photobiological production of 2-methyl-1-butanol produc sion of the designer biofuel-production pathway(s). The tion from carbon dioxide and water. designer cell-division-control technology can help ensure SEQ ID NOS: 58–63, 82-84, 69, 70 (or 71) represent biosafety in using the designer organisms for photosynthetic 50 another designer transgenic oxyphotobacterium such as biofuel production. designer transgenic Thermosynechococcus with a Calvin Oxyphotobacteria (including cyanobacteria and oxychlo cycle 3-phosphoglycerate-branched photosynthetic robacteria) that can be selected for use as host organisms to NADPH-enhanced isobutanol production pathway (34, 35, create designer transgenic oxyphotobacteria for photobio 03-05, 53-5, 42,43 or 44); while SEQID NOS:58-62,81-83, logical production of butanol and related higher alcohols 55 65-67, 69 and 86 represent another designer transgenic Ther include (but not limited to): Thermosynechococcus elongatus mosynechococcus with a Calvin-cycle 3-phosphoglycerate BP-1, Nostoc sp. PCC 7120, Synechococcus elongatus PCC branched photosynthetic NADPH-enhanced 3-methyl-1-bu 6301, Syncechococcus sp. strain PCC 7942, Syncechococcus tanol production pathway (numerical labels 34, 35, 03-05, sp. strain PCC 7002, Syncechocystis sp. strain PCC 6803, 53-55, 40, 38, 39, 42, and 57 in FIG. 6). Prochlorococcus marinus MED4, Prochlorococcus marinus 60 SEQID NOS: 87-92 represent another designer transgenic MIT 9313, Prochlorococcus marinus NATL1A, Prochloro Thermosynechococcus with a designer hydrocarbon chain coccus SS120, Spirulina platensis (Arthrospira platensis), elongation pathway (07-12 as shown in FIG. 7) for photo Spirulina pacifica, Lyngbya majuscule, Anabaena sp., Syn biological production of 1-hexanol. SEQID NOS: 87-91 and echocystis sp., Synechococcus elongates, Synechococcus 93 represent another designer transgenic Thermosynechococ (MC-A), Trichodesmium sp., Richelia intracellularis, Syn 65 cus with a designer hydrocarbon chain elongation pathway echococcus WH7803, Synechococcus WH8102, Nostoc (07-10' and 07"-12" as shown in FIG. 7) for photobiological punctiforme, Syncechococcus sp. strain PCC 7943, Syn production of 1-octanol. US 8,986,963 B2 93 94 SEQ ID NOS: 58-69 and 92 represent another designer coccus sp. strain PCC 7942 nitrite-reductase-gene promoter transgenic Thermosynechococcus with a designer Calvin sequence, an enzyme-encoding sequence (109-1752) cycle 3-phosphoglycerate-braned photosynthetic NADPH selected and modified from a Lactococcus lactis branched enhanced pathway (34,35, 03-05, 36-41,39,39-40', 39-42 chain alpha-ketoacid decarboxylase (GenBank accession and 12" in FIG. 8) for photobiological production of 1-hex number: AAS49166), a 308-bp Synechococcus sp. strain PCC anol from carbon dioxide and water. 7942 rbcS terminator (1753-2060), and a PCR RE primer SEQ ID NOS: 58-69, 82-84, and 94 represent a designer (2061-2080) at the 3' end. transgenic Thermosynechococcus with a designer Calvin SEQ ID NO: 98 presents a detailed DNA construct (1603 cycle 3-phosphoglycerate-braned photosynthetic NADPH bp) of a designer NADH-dependent butanol dehydrogenase enhanced pathway (34,35, 03-05, 36-39, 53-55, 39-43', 39 10 (12a) gene that include a PCRFD primer (sequence by 1-20), 43', 39"-43" in FIG. 9) for production of 3-methyl-1- a 88-bp nirA promoter (21-108) selected from the Synecho pentanol, 4-methyl-1-hexanol, and 5-methyl-1-heptanol coccus sp. strain PCC 7942 nitrite-reductase-gene promoter from carbon dioxide and water. Similarly, SEQ ID NOS: sequence, an enzyme-encoding sequence (109-1275) 58-61, 74-81, 82-84, 66-69, and 94 represent another selected and modified from a Clostridium carboxidivorans designer transgenic Thermosynechococcus with a Calvin 15 P7 NADH-dependent butanol dehydrogenase (GenBank cycle 3-phophoglycerate-branched NADPH-enhanced path accession number: AD012118), a 308-bp Synechococcus sp. way (34,35,03,04,45-55,39-43', 39-43', 39"-43" in FIG.9) strain PCC 7942 rbcS terminator (1276-1583), and a PCRRE for photobiological production of 3-methyl-1-pentanol, primer (1584-1603) at the 3' end. 4-methyl-1-hexanol, and 5-methyl-1-heptanol from carbon SEQ ID NO: 99 presents example 99 of a detailed DNA dioxide and water as well. construct (1654 bp) of a designer NADPH-dependent SEQ ID NOS: 58-62, 82-84, 65-69 and 94 represent a Butanol Dehydrogenase (12b) gene including: a PCR FD designer transgenic Thermosynechococcus with a designer primer (sequence by 1-20), a 88-bp nirA promoter (21-108) Calvin-cycle 3-phosphoglycerate-braned photosynthetic selected from the Synechococcus sp. strain PCC 7942 nitrite NADPH-enhanced pathway labels (34,35, 03-05, 53-55, 40, reductase-gene promoter sequence, an enzyme-encoding 38,39,39-43', 39-43', and 39"-43" in FIG. 10) for photobio 25 sequence (109-1326) selected and modified from a Butyriv logical production of 4-methyl-1-pentanol, 5-methyl-1-hex ibrio crossotus DSM 2876 NADPH-dependent butanol dehy anol, and/or 6-methyl-1-heptanol from carbon dioxide and drogenase (GenBank accession number: EFF67629), a 308 Water. bp Synechococcus sp. strain PCC 7942 rbcS terminator Use of other host oxyphotobacteria Such as Synechococcus (1327-1634), and a PCRRE primer (1635-1654) at the 3' end. sp. strain PCC 7942, Synechocystis sp. strain PCC 6803, 30 Note, in the designer transgenic Synechococcus that is Prochlorococcus marinus, Cyanothece sp. ATCC 51142, for represented by SEQID NOS: 95-98 (and/or 99), Synechoc genetic transformation with proper designer DNA constructs occuss's native enzymes of 03-05, 36-41 and 45-52 are used (genes) can create other designer oxyphotobacteria for pho in combination with the designer nirA-promoter-controlled tobiological production of butanol and higher alcohols as enzymes of 34, 35, 42 and 12 encoded by SEQ ID NOS: well. For example, use of Synechococcus sp. strain PCC 7942 35 95-98 (and/or 99) to confer the Calvin-cycle 3-phophoglyc as a host organism in genetic transformation with SEQ ID erate-branched photosynthetic NADPH-enhanced pathways NOS: 95-98 (and/or 99) can create a designer transgenic for photobiological production of 1-butanol from carbon Synechococcus for photobiological production of 1-butanol. dioxide and water (FIG. 4). Briefly, SEQ ID NO: 95 presents example 95 of a detailed Similarly, use of Synechocystis sp. strain PCC 6803 as a DNA construct (1438 base pairs (bp)) of a designer NADPH 40 host organism in genetic transformation with SEQID NOS: dependent Glyceraldehyde-3-Phosphate-Dehydrogenase 100-102 (and/or 103) creates a designer transgenic Syn (34) gene that includes a PCRFD primer (sequence by 1-20), echocystis for photobiological production of 1-butanol. a 88-bp nirA promoter (21-108) selected from the Synecho Briefly, SEQID NO: 100 presents example 100 of a designer coccus sp. strain PCC 7942 (freshwater cyanobacterium) nirA-promoter-controlled NAD-dependent Glyceraldehyde nitrite-reductase-gene promoter sequence, an enzyme-encod 45 3-Phosphate Dehydrogenase (35) DNA construct (1440 bp) ing sequence (109-1110) selected and modified from a Sta that includes a PCR FD primer (sequence 1-20), a 89-bp phylococcus lugdunensis HKUO9-01 NADPH-dependent Synechocystis sp. strain PCC 6803 nitrite-reductase nirA pro glyceraldehyde-3-phosphate-dehydrogenase sequence (Gen moter (21-109), an enzyme-encoding sequence (110-1011) Bank accession number: YP 0.03471459), a 308-bp Syn selected from a Streptococcus pyogenes NZ131 NAD-depen echococcus sp. strain PCC 7942 rbcS terminator (1111 50 dent Glyceraldehyde-3-phosphate dehydrogenase (Gen 1418), and a PCR RE primer (1419-1438) at the 3' end. Bank: YP 002285269), a 409-bp Synechocystis sp. PCC SEQ ID NO: 96 presents example 96 of a detailed DNA 6803 rbcS terminator (1012-1420), and a PCR RE primer construct (1447 bp) of a designer NAD-dependent Glyceral (1421-1440). dehyde-3-Phosphate-Dehydrogenase (35) gene that includes SEQ ID NO: 101 presents example 101 of a designer a PCRFD primer (sequence by 1-20), a 88-bp nirA promoter 55 nirA-promoter-controlled 2-Keto Acid Decarboxylase (42) (21-108) selected from the Synechococcus sp. strain PCC DNA construct (2182 bp) that includes a PCR FD primer 7942 nitrite-reductase-gene promoter sequence, an enzyme (sequence 1-20), a 89-bp Synechocystis sp. strain PCC 6803 encoding sequence (109-1119) selected and modified from a nitrite-reductase nirA promoter (21-109), an enzyme-encod Staphylococcus aureus 04-02981 NAD-dependent glyceral ing sequence (110-1753) selected from a Lactococcus lactis dehyde-3-phosphate-dehydrogenase sequence (GenBank 60 branched-chain alpha-ketoacid decarboxylase (GenBank: accession number: ADC36961), a 308-bp Synechococcus sp. AAS49166), a 409-bp Synechocystis sp. PCC 6803 rbcS ter strain PCC 7942 rbcS terminator (1120-1427), and a PCRRE minator (1754-2162), and a PCR RE primer (2163-2182). primer (1428-1447) at the 3' end. SEQ ID NO: 102 presents example 102 of a designer SEQ ID NO: 97 presents example 97 of a detailed DNA nirA-promoter-controlled NADH-dependent Butanol Dehy construct (2080 bp) of a designer2-Keto Acid Decarboxylase 65 drogenase (12a) DNA construct (1705 bp) that includes a (42) gene that includes a PCRFD primer (sequence by 1-20), PCRFD primer (sequence 1-20), a 89-bp Synechocystis sp. a 88-bp nirA promoter (21-108) selected from the Synecho strain PCC 6803 nitrite-reductase nirA promoter (21-109), an US 8,986,963 B2 95 96 enzyme-encoding sequence (110-1276) selected from a toc sp. strain PCC 7120 gor terminator (1873-2304), and a Clostridium carboxidivorans P7 NADH-dependent butanol PCR RE primer (2305-2324) at the 3' end. dehydrogenase (GenBank: AD012118), a 409-bp Syn SEQ ID NO: 108 presents example 108 of a designer echocystis sp. PCC 6803 rbcS terminator (1277-1685), and a hoX-promoter-controlled branched-chain alpha-Ketoacid PCR RE primer (1686-1705). 5 Decarboxylase (42) DNA construct (2288 bp) that includes a SEQ ID NO: 103 presents example 103 of a designer PCRFD primer (sequence 1-20), a 172-bp Nostoc sp. strain nirA-promoter-controlled NADPH-dependent butanol dehy PCC 7120 (Anabaena PCC 7120) hox promoter (21-192), an drogenase (12b) DNA construct (1756 bp) that includes a enzyme-encoding sequence (193-1836) selected/modified PCR FD primer (sequence 1-20), a 89-bp Synechocystis sp. from the sequence of a Lactococcus lactis branched-chain strain PCC 6803 nitrite-reductase nirA promoter (21-109), an 10 alpha-ketoacid decarboxylase (GenBank: AAS49166), a enzyme-encoding sequence (110-1327) selected from a 432-bp Nostoc sp. strain PCC 7120 gor terminator (1837 Butyrivibrio crossotus DSM 2876 NADPH-dependent 2268), and a PCR RE primer (2269-2288) at the 3' end. butanol dehydrogenase (GenBank: EFF67629), a 409-bp SEQ ID NO: 109 presents example 109 of a designer Synechocystis sp. PCC 6803 rbcS terminator (1328-1736), hox-promoter-controlled 2-Methylbutyraldehyde Reductase and a PCR RE primer (1737-1756). 15 (56) DNA construct (1613 bp) that includes a PCRFD primer Note, in the designer transgenic Synechocystis that con (sequence 1-20), a 172-bp Nostoc sp. strain PCC 7120 (Ana tains the designer genes of SEQ ID NOS: 100-102 (and/or baena PCC 7120) hox promoter (21-192), an enzyme-encod 103), Synechocystis's native enzymes of 34,03-05, 36-41 and ing sequence (193-1461) selected/modified from the 45-52 are used in conjunction with the designer nir A-pro sequence of a Schizosaccharomyces japonicusyFS2752-me moter-controlled enzymes of 35, 42 and 12 encoded by SEQ thylbutyraldehyde reductase (GenBank: XP 002173231), a ID NOS: 100-102 (and/or 103) to confer the Calvin-cycle 432-bp Nostoc sp. strain PCC 7120 gor terminator (1462 3-phophoglycerate-branched photosynthetic NADPH-en 1893), and a PCR RE primer (1894-1613) at the 3' end. hanced pathways for photobiological production of 1-butanol Note, in the designer transgenic Nostoc that contains from carbon dioxide and water (FIG. 4). designer hox-promoter-controlled genes of SEQ ID NOS: Use of Nostoc sp. strain PCC 7120 as a host organism in 25 104-109, Nostoc's native enzymes (genes) of 34, 03-05, genetic transformation with SEQID NOS: 104-109 can cre 36-39 and 45-52 are used in combination with the designer ate a designer transgenic Nostoc for photobiological produc hox-promoter-controlled enzymes of 35, 53-55, 42 and 56 tion of 2-methyl-1-butanol (FIG.5). Briefly, SEQID NO: 104 (encoded by DNA constructs of SEQ ID NOS: 104-109) to presents example 104 of a designer hoX-promoter-controlled confer the Calvin-cycle 3-phophoglycerate-branched photo NAD-dependent Glyceraldehyde-3-Phosphate Dehydroge 30 synthetic NADPH-enhanced pathways for photobiological nase (35) DNA construct (1655 bp) that includes a PCR FD production of 2-methyl-1-butanol from carbon dioxide and primer (sequence 1-20), a 172-bp Nostoc sp. strain PCC 7120 water (FIG. 5). (Anabaena PCC 7120) hox promoter (21-192), an enzyme Use of Prochlorococcus marinus MIT 9313 as a host encoding sequence (193-1203) selected/modified from the organism in genetic transformation with SEQID NOS: 110 sequence of a Streptococcus pyogenes NZ131 NAD-depen 35 122 can create a designer transgenic Prochlorococcus mari dent glyceraldehyde-3-phosphate dehydrogenase (GenBank: nus for photobiological production of isobutanol and/or YP 002285269), a 432-bp Nostoc sp. strain PCC 7120 gor 3-methyl-1-butanol (FIG. 6). Briefly, SEQ ID NO:110 pre terminator (1204-1635), and a PCR RE primer (1636-1655) sents example 110 for a designer groE-promoter-controlled at the 3' end. NAD-dependent Glyceraldehyde-3-Phosphate Dehydroge SEQ ID NO: 105 presents example 105 of a designer 40 nase (35) DNA construct (1300 bp) that includes a PCR FD hox-promoter-controlled Acetolactate Synthase (53) DNA primer (sequence 1-20), a 137-bp Prochlorococcus marinus construct (2303 bp) that includes a PCRFD primer (sequence MIT 9313 heat- and light-responsive groE promoter (21 1-20), a 172-bp Nostoc sp. strain PCC 7120 (Anabaena PCC 157), an enzyme-encoding sequence (158-1159) selected 7120) hoX promoter (21-192), an enzyme-encoding sequence from a Vibrio cholerae MJ-1236 NAD-dependent Glyceral (193-1851) selected/modified from the sequence of a Ther 45 dehyde-3-phosphate dehydrogenase (GenBank: mosynechococcus elongatus BP-1 acetolactate synthase ACQ61431), a 121-bp Prochlorococcus marinus MIT9313 (GenBank: NP 682614), a 432-bp Nostoc sp. strain PCC rbcS terminator (1160-1280), and a PCR RE primer (1281 7120 gor terminator (1852-2283), and a PCR RE primer 1300). (2284-2303) at the 3' end. SEQID NO: 111 presents example 111 for a designer groE SEQ ID NO: 106 presents example 106 of a designer 50 promoter-controlled Phosphoglycerate Mutase (O3) DNA hox-promoter-controlled Ketol-Acid Reductoisomerase (54) construct (1498 bp) that includes a PCRFD primer (sequence DNA construct (1661 bp) that includes a PCR FD primer 1-20), a 137-bp Prochlorococcus marinus MIT9313 heat (sequence 1-20), a 172-bp Nostoc sp. strain PCC 7120 (Ana and light-responsive groE promoter (21-157), an enzyme baena PCC 7120) hox promoter (21-192), an enzyme-encod encoding sequence (158-1357) selected from a Pelotomacu ing sequence (193-1209) selected/modified from the 55 lum thermopropionicum SI phosphoglycerate mutase (Gen sequence of a Calditerrivibrio nitroreducens DSM 19672 Bank: YP 001212148), a 121-bp Prochlorococcus marinus ketol-acid reductoisomerase (GenBank: YP 004.050904), a MIT9313 rbcSterminator (1358-1478), and a PCRRE primer 432-bp Nostoc sp. strain PCC 7120 gor terminator (1210 (1479-1498). 1641), and a PCR RE primer (1642-1661) at the 3' end. SEQID NO:112 presents example 112 for a designer groE SEQ ID NO: 107 presents example 107 of a designer 60 promoter-controlled Enolase (04) DNA construct (1588 bp) hox-promoter-controlled Dihydroxy-Acid Dehydratase (55) that includes a PCR FD primer (sequence 1-20), a 137-bp DNA construct (2324 bp) that includes a PCR FD primer Prochlorococcus marinus MIT9313 heat- and light-respon (sequence 1-20), a 172-bp Nostoc sp. strain PCC 7120 (Ana sive groE promoter (21-157), an enzyme-encoding sequence baena PCC 7120) hox promoter (21-192), an enzyme-encod (158-1447) selected from a Thermotoga petrophila RKU-1 ing sequence (193-1872) selected/modified from the 65 enolase (GenBank: ABQ46079), a 121-bp Prochlorococcus sequence of a Marivirga tractuosa DSM 4126 dihydroxy marinus MIT9313 rbcS terminator (1448-1568), and a PCR aciddehydratase (GenBank:YP 004053736), a 432-bp Nos RE primer (1569-1588). US 8,986,963 B2 97 98 SEQID NO: 113 presents example 113 for a designer groE tion of the following four designer groE promoter-controlled promoter-controlled Pyruvate Kinase (05) DNA construct genes (SEQ ID NO:119-122) results in another designer (1717 bp) that includes a PCRFD primer (sequence 1-20), a transgenic Prochlorococcus that can produce both isobutanol 137-bp Prochlorococcus marinus MIT9313 heat- and light and 3-methyl-1-butanol from carbon dioxide and water (35. responsive groE promoter (21-157), an enzyme-encoding 03-05, 53-55, 42, 43/44, plus 38-40 and 57 as shown in FIG. sequence (158-1576) selected from a Thermotoga lettingae 6). TMO pyruvate kinase (GenBank:YP 001471580), a 121-bp Briefly, SEQ ID NO: 119 presents example 119 for a Prochlorococcus marinus MIT9313 rbcS terminator (1577 designer groE-promoter-controlled 2-Isopropylmalate Syn 1697), and a PCR RE primer (1698-1717). thase (40) DNA construct (1816 bp) that includes a PCR FD SEQID NO:114 presents example 114 for a designer groE 10 primer (sequence 1-20), a 137-bp Prochlorococcus marinus promoter-controlled Acetolactate Synthase (53) DNA con MIT9313 heat- and light-responsive groE promoter (21-157), struct (2017 bp) that includes a PCR FD primer (sequence an enzyme-encoding sequence (158-1675) selected from a 1-20), a 137-bp Prochlorococcus marinus MIT 9313 heat Pelotomaculum thermopropionicum S12-isopropylmalate and light-responsive groE promoter (21-157), an enzyme synthase (GenBank: YP 001211081), a 121-bp Prochloro encoding sequence (158-1876) selected from a Bacillus 15 coccus marinus MIT9313 rbcS terminator (1676-1796), and licheniformis ATCC 14580 acetolactate synthase (GenBank: a PCR RE primer (1797-1816). AAU42663), a 121-bp Prochlorococcus marinus MIT 9313 SEQID NO:120 presents example 120 for a designer groE rbcS terminator (1877-1997), and a PCR RE primer (1998 promoter-controlled 3-Isopropylmalate Dehydratase (38) 2017). DNA construct (2199 bp) that includes a PCR FD primer SEQID NO: 115 presents example 115 for a designer groE (sequence 1-20), a 137-bp Prochlorococcus marinus promoter-controlled Ketol-Acid Reductoisomerase (54) MIT9313 heat- and light-responsive groE promoter (21-157), DNA construct (1588 bp) that includes a PCR FD primer a 3-isopropylmalate dehydratase large subunit-encoding (sequence 1-20), a 137-bp Prochlorococcus marinus sequence (158-1420) selected from a Pelotomaculum ther MIT9313 heat- and light-responsive groE promoter (21-157), mopropionicum S13-isopropylmalate dehydratase large Sub an enzyme-encoding sequence (158-1168) selected from a 25 unit (GenBank:YP 001211082), a 137-bp Prochlorococcus Thermotoga petrophila RKU-1 ketol-acid reductoisomerase marinus MIT9313 heat- and light-responsive groE promoter (GenBank: ABQ46398), a 400-bp Prochlorococcus marinus (1421-1557), a 3-isopropylmalate dehydratase small subunit MIT9313 rbcSterminator (1169-1568), and a PCRRE primer encoding sequence (1558-2058) selected from a Pelotomacu (1569-1588). lum thermopropionicum S13-isopropylmalate dehydratase SEQID NO: 116 presents example 116 for a designer groE 30 small subunit (GenBank: YP 001211083), a 121-bp promoter-controlled Dihydroxy-Acid Dehydratase (55) Prochlorococcus marinus MIT9313 rbcS terminator (2059 DNA construct (1960 bp) that includes a PCR FD primer 2179), and a PCR RE primer (2180-2199). (sequence 1-20), a 137-bp Prochlorococcus marinus SEQID NO: 121 presents example 121 for a designer groE MIT9313 heat- and light-responsive groE promoter (21-157), promoter-controlled 3-Isopropylmalate Dehydrogenase (39) an enzyme-encoding sequence (158-1819) selected from a 35 DNA construct (1378 bp) that includes a PCR FD primer Syntrophothermus lipocalidus DSM 12680 dihydroxy-acid (sequence 1-20), a 137-bp Prochlorococcus marinus dehydratase (GenBank: ADIO2905), a 121-bp Prochlorococ MIT9313 heat- and light-responsive groE promoter (21-157), cus marinus MIT9313 rbcS terminator (1820-1940), and a an enzyme-encoding sequence (158-1237) selected from a PCR RE primer (1941-1960). Syntrophothermus lipocalidus DSM 12680 3-isopropyl SEQID NO: 117 presents example 117 for a designer groE 40 malate dehydrogenase (GenBank: ADIO2898), a 121-bp promoter-controlled 2-Keto Acid Decarboxylase (42) DNA Prochlorococcus marinus MIT9313 rbcS terminator (1238 construct (1945bp) that includes a PCRFD primer (sequence 1358), and a PCR RE primer (1359-1378). 1-20), a 137-bp Prochlorococcus marinus MIT9313 heat SEQID NO: 122 presents example 122 for a designer groE and light-responsive groE promoter (21-157), an enzyme promoter-controlled 3-Methylbutanal Reductase (57) DNA encoding sequence (158-1804) selected from a Lactococcus 45 construct (1327 bp) that includes a PCRFD primer (sequence lactis subsp. lactis KF 147 Alpha-ketoisovalerate decarboxy 1-20), a 137-bp Prochlorococcus marinus MIT9313 heat lase (GenBank: ADA65057), a 121-bp Prochlorococcus and light-responsive groE promoter (21-157), an enzyme marinus MIT9313 rbcS terminator (1805-1925), and a PCR encoding sequence (158-1186) selected from a Saccharomy RE primer (1926-1945). ces cerevisiae S288c 3-Methylbutanal reductase (GenBank: SEQID NO: 118 presents example 118 for a designer nirA 50 DAA 10635), a 121-bp Prochlorococcus marinus MIT9313 promoter-controlled Alcohol Dehydrogenase (43/44) DNA rbcS terminator (1187-1307), and a PCR RE primer (1308 construct (1138 bp) that includes a PCRFD primer (sequence 1327). 1-20), a 251-bp Prochlorococcus marinus MIT9313 nirA Note, the use of SEQ ID NOS: 110-117 and 119-122 in promoter (21-271), an enzyme-encoding sequence (272-997) genetic transformation of Prochlorococcus marinus MIT selected from a Geobacillus kaustophilus HTA426 short 55 9313 creates another designer transgenic Prochlorococcus chain alcohol dehydrogenase (GenBank: YP 146837), a marinus with a groE promoter-controlled designer Calvin 121-bp Prochlorococcus marinus MIT9313 rbcS terminator cycle-channeled pathway (identified as 34 (native), 35, (998-1118), and a PCR RE primer (1119-1138). 03-05, 53-55, 38-40, 42 and 57 in FIG. 6) for photobiological Note, in the designer transgenic Prochlorococcus that con production of 3-methyl-1-butanol from carbon dioxide and tains the designer genes of SEQID NOS: 110-118, Prochlo 60 Water. rococcus’s native gene (enzyme) of 34 is used in combination Use of Cyanothece sp. ATCC 51142 as a host organism in with the designer groE and nirA-promoters-controlled genes genetic transformation with SEQID NOS: 123-128 can cre (enzymes) of 35, 03-05, 53-55, 42 and 43/44 (encoded by ate a designer transgenic Cyanothece for photobiological pro DNA constructs of SEQ ID NOS: 110-118) to confer the duction of 1-pentanol, 1-hexanol, and/or 1-heptanol (FIG. 8). Calvin-cycle 3-phophoglycerate-branched photosynthetic 65 Briefly, SEQID NO:123 presents example 123 for a designer NADPH-enhanced pathways for photobiological production nirA-promoter-controlled 2-Isopropylmalate Synthase (40) of isobutanol from carbon dioxide and water (FIG. 6). Addi DNA construct (2004 bp) that includes a PCR FD primer US 8,986,963 B2 99 100 (sequence 1-20), a 203-bp Cyanothece sp. ATCC 51142 nirA and water (FIG. 8). Addition of a designer nirA-promoters promoter (21-223), an enzyme-encoding sequence (224 controlled gene (SEQID NO: 128) of a short chain alcohol 1783) selected from a Hydrogenobacter thermophilus TK-6 dehydrogenase 43’ (43") with promiscuity results in another 2-isopropylmalate synthase sequence (GenBank: designer transgenic Cyanothece containing a Calvin-cycle BAI69273), a 201-bp Cyanothece sp. ATCC 51142 rbcS ter channeled pathway (35, 39-41, 39-43', 39-43', and 39"-43" minator (1784-1984), and a PCR RE primer (1985-2004). as shown in FIG. 8) that can produce 1-pentanol, 1-hexanol, SEQID NO.124 presents example 124 for a designer nirA and 1-hexanol from carbon dioxide and water. promoter-controlled Isopropylmalate Isomerase (41) large? Designer Advanced Photosynthetic Organisms with Calvin small subunits DNA construct (2648 bp) that includes a PCR Cycle-Channeled Pathways for Production of Butanol and FD primer (sequence 1-20), a 203-bp Cyanothece sp. ATCC 10 Related Higher Alcohols 51142 nirA promoter (21-223), an enzyme-large-subunit-en According to one of the various embodiments, use of cer coding sequence (224-1639) selected from a Anoxybacillus tain designer DNA constructs in genetic transformation of flavithermus WK1 isopropylmalate isomerase large subunit eukaryotic photosynthetic organisms such as plant cells, sequence (GenBank:YP 002314962), a 203-bp Cyanothece eukaryotic aquatic plants (including, for example, eukaryotic sp. ATCC 51142 nirA promoter (1640-1842), an enzyme 15 algae, Submersed aquatic herbs, duckweeds, water cabbage, small-subunit-encoding sequence (1843-2427) selected from water lily, water hyacinth, Bolbitis heudelotii, Cabomba sp., a Anoxybacillus flavithermus WK1 isopropylmalate and seagrasses) can create designer transgenic eukaryotic isomerase Small subunit sequence (GenBank: photosynthetic organisms for production of butanol and YP 002314963), a 201-bp Cyanothece sp. ATCC 51142 related higher alcohols from carbon dioxide and water. rbcS terminator (2428-1628), and a PCR RE primer (2629 Eukaryotic algae that can be selected for use as host organ 2648). isms to create designer algae for photobiological production SEQ ID NO: 125 presents example 125 for a designerg ofbutanol and related higher alcohols include (but not limited nir A-promoter-controlled 3-Isopropylmalate Dehydroge to): Dunaliella salina, Dunaliella viridis, Dunaliella bar nase (39) DNA construct (1530 bp) that includes a PCR FD dowil, Crypthecodinium cohnii, Schizochytrium sp., Chlamy primer (sequence 1-20), a 203-bp Cyanothece sp. ATCC 25 domonas reinhardtii, Platymonas subcordiformis, Chlorella 51142 nirA promoter (21-223), an enzyme-encoding fisca, Chlorella Sorokiniana, Chlorella vulgaris, 'Chlorella sequence (224-1309) selected from a Thermosynechococcus ellipsoidea, Chlorella spp., Haematococcus pluvialis, elongatus BP-1 3-isopropylmalate dehydrogenase sequence Parachlorella kessleri, Betaphycus gelatinum, Chondrus (GenBank: BAC09152), a 201-bp Cyanothece sp. ATCC crispus, Cyanidioschyzon merolae, Cyanidium caldarium, 51142 rbcS terminator (1310-1310), and a PCR RE primer 30 Galdieria sulphuraria, Gelidiella acerosa, Graciliaria (1311-1530). changii, Kappaphycus alvarezii, Porphyra miniata, Ostreo SEQIDNO:126 presents example 126 for a designer nir A coccus tauri, Porphyra yezoensis, Porphyridium sp., Pal promoter-controlled 2-Keto Acid Decarboxylase (42") DNA maria palmata, Graciliaria spp., Isochrysis galbana, Kappa construct (2088 bp) that includes a PCRFD primer (sequence phycus spp., Laminaria japonica, Laminaria spp., 1-20), a 203-bp Cyanothece sp. ATCC 51142 nirA promoter 35 Monostroma spp., Nannochloropsis oculata, Porphyra spp., (21-223), an enzyme-encoding sequence (224-1867) selected Porphyridium spp., Undaria pinnatifida, Ulva lactuca, Ulva from a Lactococcus lactis 2-keto acid decarboxylase (Gen spp., Undaria spp., Phaeodactylum Tricornutum, Navicula Bank: AAS49166), a 201-bp Cyanothece sp. ATCC 51142 Saprophila, Cylindrotheca fisiiformis, Cyclotella cryptica, rbcS terminator (1868-2068), and a PCR RE primer (2069 Euglena gracilis, Amphidinium sp., Symbiodinium microad 2088). 40 riaticum, Macrocystis pyrifera, Ankistrodesmus braunii, SEQID NO:127 presents example 127 for a designer nirA Scenedesmus obliquus, Stichococcus sp., Platymonas sp., promoter-controlled Hexanol Dehydrogenase (12") DNA Dunalielki sauna, and Stephanoptera gracilis. construct (1503 bp) that includes a PCRFD primer (sequence According to another embodiment, the transgenic photo 1-20), a 203-bp Cyanothece sp. ATCC 51142 nirA promoter synthetic organism comprises a designer transgenic plant or (21-223), an enzyme-encoding sequence (224-1282) selected 45 plant cells selected from the group consisting of aquatic from a Mycobacterium chubuense NBB4 hexanol dehydro plants, plant cells, green algae, red algae, brown algae, blue genase (GenBank: ACZ56328), a 201-bp Cyanothece sp. green algae (oxyphotobacteria including cyanobacteria and ATCC 51142 rbcS terminator (1283-1483), and a PCR RE oxychlorobacteria), diatoms, marine algae, freshwater algae, primer (1484-1503). salt-tolerant algal strains, cold-tolerant algal strains, heat SEQID NO: 128 presents example 128 for a designer nirA 50 tolerant algal strains, antenna-pigment-deficient mutants, promoter-controlled short-chain Alcohol Dehydrogenase butanol-tolerant algal strains, higher-alcohols-tolerant algal (43', 43") DNA construct (1149 bp) that includes a PCR FD strains, butanol-tolerant oxyphotobacteria, higher-alcohols primer (sequence 1-20), a 203-bp Cyanothece sp. ATCC tolerant oxyphotobacteria, and combinations thereof. 51142 nirA promoter (21-223), an enzyme-encoding According to another embodiment, said transgenic photo sequence (224-928) selected from a Pyrococcus firiosus 55 synthetic organism comprises a biosafety-guarded feature DSM 3638 Short chain alcohol dehydrogenase (GenBank: selected from the group consisting of a designer proton AAC25556), a 201-bp Cyanothece sp. ATCC 51142 rbcS channel gene inducible under pre-determined inducing con terminator (929-1129), and a PCR RE primer (1130-1149). ditions, a designer cell-division-cycle iRNA gene inducible Note, in the designer transgenic Cyanothece that contains under pre-determined inducing conditions, a high-CO2-re designer nirA promoter-controlled genes of SEQ ID NOS: 60 quiring mutant as a host organism for transformation with 123-127, Cyanothece's native enzymes of 34,03-05, 36-38, designer biofuel-production-pathway genes in creating and 45-52 are used in combination with the designer nirA designer cell-division-controllable photosynthetic organ promoters-controlled enzymes of 35, 39-41 (39-41', 39-41'). isms, and combinations thereof. 42 and 12" (encoded by DNA constructs of SEQ ID NOS: The greater complexity and compartmentalization of 123-127) to confer the Calvin-cycle 3-phophoglycerate 65 eukaryotic plant cells allow for creation of a wider range of branched photosynthetic NADPH-enhanced pathways for photobiologically active designer organisms and novel meta photobiological production of 1-hexanol from carbon dioxide bolic pathways compartmentally segregated for production US 8,986,963 B2 101 102 of butanol and/or higher alcohols from water and carbon primer (sequence 1-20), a 2x84-bp Chlamydomonas rein dioxide. In a eukaryotic algal cell, for example, the translation hardtii Nial promoter (21-188), a 135-bp Chlamydomonas of designer nuclear genes occurs in cytosol whereas the pho reinhardtii RbcS2 transit peptide (189-323), an enzyme-en tosynthesis/Calvin cycle is located inside an algal chloro coding sequence (324-1742) selected/modified from Cyan plast. This clear separation of algal chloroplast photosynthe 5 othece sp. PCC 8802 pyruvate-kinase (YP 003138017), a sis from other subcellular functions such as the functions of 223-bp Chlamydomonas reinhardtii RbcS2 terminator cytoplasm membrane, cytosol and mitochondria can be used (1743-1965), and a PCR RE primer (1966-1985). as an advantage in creation of a biosafety-guarded designer SEQ ID NO. 132 presents example 132 for a designer algae through an inducible insertion of designer proton-chan Nial-promoter-controlled chloroplast-targeted NADPH-de nels into cytoplasm membrane to permanently disable any 10 pendent Glyceraldehyde-3-Phosphate Dehydrogenase (34) cell division and/ormating capability while keeping the algal DNA construct (1568 bp) that includes a PCR FD primer chloroplast functional work with the designer biofuel produc (sequence 1-20), a 2x84-bp Chlamydomonas reinhardtii tion, pathways to produce butanol and related higher alco Nial promoter (21-188), a 135-bp Chlamydomonas rein hols. However, it is essential to genetically deliver designer hardtii RbcS2 transit peptide (189-323), a NADPH-depen enzyme(s) into the chloroplast to tame the Calvin cycle and 15 dent Glyceraldehyde-3-phosphate dehydrogenase-encoding funnel metabolism toward butanol directly from CO, and sequence (324-1325) selected/modified from Staphylococcus H2O. This requires more complicated gene design to achieve lugdunensis HKUO9-01 NADPH-dependent glyceralde desirable results. hyde-3-phosphate dehydrogenase (ADC87332), a 223-bp According to one of various embodiments, designer Chlamydomonas reinhardtii RbcS2 terminator (1326-1548), Calvin-cycle-channeled pathway enzymes encoded with and a PCR RE primer (1549-1568). designer unclear genes are targetedly expressed into algal SEQ ID NO. 133 presents example 133 for a designer chloroplast through use of a transit signal peptide sequence. Nial-promoter-controlled chloroplast-targeted NAD-depen The said signal peptide is selected from the group consisting dent Glyceraldehyde-3-phosphate dehydrogenase (35) DNA of the hydrogenase transit-peptide sequences (HydA1 and construct (1571 bp) that includes a PCRFD primer (sequence HydA2), ferredoxin transit-peptide sequence (Frx1), thiore 25 1-20), a 2x84-bp Chlamydomonas reinhardtii Nia1 (nitrate doxin-m transit-peptide sequence (Trx2), glutamine synthase reductase) promoter (21-188), a 135-bp Chlamydomonas transit-peptide sequence (GS2), LhcII transit-peptide reinhardtii RbcS2 transit peptide (189-323), a NAD-depen sequences, PSII-T transit-peptide sequence (PsbT), PSII-S dent Glyceraldehyde-3-phosphate dehydrogenase-encoding transit-peptide sequence (PsbS), PSII-W transit-peptide sequence (324-1328) selected/modified from Flavobacteri sequence (PsbW), CFCF subunit-Y transit-peptide 30 aceae bacterium 3519-10 NAD-dependent Glyceraldehyde sequence (AtpC), CFCF subunit-6 transit-peptide sequence 3-phosphate dehydrogenase (YP 003095198), a 223-bp (AtpD), CFoCF subunit-II transit-peptide sequence (AtpG). Chlamydomonas reinhardtii RbcS2 terminator (1329-1551), photosystem I (PSI) transit-peptide sequences, Rubisco SSU and a PCR RE primer (1552-1571). transit-peptide sequences, and combinations thereof. Pre SEQ ID NO. 134 presents example 134 for a designer ferred transit peptide sequences include the Hyd1 transit pep 35 Nial-promoter-controlled chloroplast-targeted Citramalate tide, the FrX1 transit peptide, and the Rubisco SSU transit Synthase (36) DNA construct (2150 bp) that includes a PCR peptides (such as RbcS2). FD primer (sequence 1-20), a 2x84-bp Chlamydomonas rein SEQ ID NOS. 129-165 present examples for designer hardtii Nia1 (nitrate reductase) promoter (21-188), a 135-bp DNA constructs of designer chloroplast-targeted enzymes for Chlamydomonas reinhardtii RbcS2 transit peptide creation of designer eukaryotic photosynthetic organisms 40 (189-323), a Citramalate Synthase-encoding sequence (324 Such as designer algae with Calvin-cycle-channeled photo 1907) selected/modified from Hydrogenobacter thermophi synthetic NADPH-enhanced pathways for photobiological lus TK-6 Citramalate Synthase (AD045737), a 223-bp production of butanol and related higher alcohols. Briefly, Chlamydomonas reinhardtii RbcS2 terminator (1908-2130), SEQID NO. 129 presents example 129 for a designer Nial and a PCR RE primer (2131-2150). promoter-controlled chloroplast-targeted Phosphoglycerate 45 SEQ ID NO. 135 presents example 135 for a designer Mutase (03) DNA construct (1910 bp) that includes a PCR Nial-promoter-controlled chloroplast-targeted 3-Isopropyl FD primer (sequence 1-20), a 2x84-bp Chlamydomonas rein malate/(R)-2-Methylmalate Dehydratase (37) large/small hardtii Nia1 (nitrate reductase) promoter (21-188), a 135-bp subunits DNA construct (3125 bp) that includes a PCR FD Chlamydomonas reinhardtii RbcS2 transit peptide primer (sequence 1-20), a 2x84-bp Chlamydomonas rein (189-323), a Phosphoglycerate Mutase-encoding sequence 50 hardtii Nial promoter (21-188), a 135-bp Chlamydomonas (324-1667) selected/modified from Nostoc azolae' 0708 reinhardtii RbcS2 transit peptide (189-323), a 3-isopropyl Phosphoglycerate Mutase (ADI65627), a 223-bp Chlamy malate? (R)-2-methylmalate dehydratase large Subunit-en domonas reinhardtii RbcS2 terminator (1668-1890), and a coding sequence (324-2084) selected/modified from Eubac PCR RE primer (1891-1910). terium eligens ATCC 27750 3-isopropylmalate/(R)-2- SEQ ID NO. 130 presents example 130 for a designer 55 methylmalate dehydratase large subunit (YP 00293.0810), a Nial-promoter-controlled chloroplast-targeted Enolase (04) 2x84-bp Chlamydomonas reinhardtii Nial promoter (2085 DNA construct (1856 bp) that includes a PCR FD primer 2252), a 135-bp Chlamydomonas reinhardtii RbcS2 transit (sequence 1-20), a 2x84-bp Chlamydomonas reinhardtii peptide (2253-2387), a 3-isopropylmalate/(R)-2-methyl Nial promoter (21-188), a 135-bp Chlamydomonas rein malate dehydratase Small subunit-encoding sequence (2388 hardtii RbcS2 transit peptide (189-323), an Enolase-encod 60 2882) selected/modified from Eubacterium eligens ATCC ing sequence (324-1613) selected/modified from Nostoc 27750 3-isopropylmalate/(R)-2-methylmalate dehydratase azolae '0708 Enolase (ADI63801), a 223-bp Chlamydomo small subunit (YP 00293.0809), a 223-bp Chlamydomonas nas reinhardtii RbcS2 terminator (1614-1836), and a PCRRE reinhardtii RbcS2 terminator (2883-3105), and a PCR RE primer (18837-1856). primer (3106-3125). SEQ ID NO. 131 presents example 131 for a designer 65 SEQ ID NO. 136 presents example 136 for a designer Nial-promoter-controlled chloroplast-targeted Pyruvate-Ki Nial-promoter-controlled chloroplast-targeted 3-Isopropyl nase (05) DNA construct (1985 bp) that includes a PCR FD malate Dehydratase (38) large/small subunits DNA construct US 8,986,963 B2 103 104 (2879 bp) that includes a PCRFD primer (sequence 1-20), a SEQ ID NO. 141 presents example 141 for a designer 2x84-bp Chlamydomonas reinhardtii Nial promoter Nial-promoter-controlled chloroplast-targeted NADH-de (21-188), a 135-bp Chlamydomonas reinhardtii RbcS2 tran pendent Alcohol Dehydrogenase (43) DNA construct (1724 sit peptide (189-323), a 3-isopropylmalate dehydratase large bp) that includes a PCRFD primer (sequence 1-20), a 2x84 subunit-encoding sequence (324-1727) selected/modified bp Chlamydomonas reinhardtii Nial promoter (21-188), a from Cyanothece sp. PCC 7822 3-isopropylmalate dehy 135-bp Chlamydomonas reinhardtii RbcS2 transit peptide dratase large subunit (YP 003886427), a 2x84-bp Chlamy (189-323), a NADH-dependent alcohol dehydrogenase-en domonas reinhardtii Nial promoter (1727-1894), a 135-bp coding sequence (324-1481) selected/modified from Glucon Chlamydomonas reinhardtii RbcS2 transit peptide (1895 acetobacter hansenii ATCC 23769 NADH-dependent alco 10 hol dehydrogenase (ZP 06834544), a 223-bp 2029), a 3-isopropylmalate dehydratase small subunit-encod Chlamydomonas reinhardtii RbcS2 terminator (1482-1704), ing sequence (2030-2636) selected/modified from Cyanoth and a PCR RE primer (1705-1724). ece sp. PCC 7822 3-isopropylmalate dehydratase small SEQ ID NO. 142 presents example 142 for a designer subunit (YP 003889452), a 223-bp Chlamydomonas rein Nial-promoter-controlled chloroplast-targeted NADPH-de hardtii RbcS2 terminator (2637-2859), and a PCR RE primer 15 pendent Alcohol Dehydrogenase (44) DNA construct (1676 (2860-2879). bp) that includes a PCRFD primer (sequence 1-20), a 2x84 SEQ ID NO. 137 presents example 137 for a designer bp Chlamydomonas reinhardtii Nial promoter (21-188), a Nial-promoter-controlled chloroplast-targeted 3-Isopropyl 135-bp Chlamydomonas reinhardtii RbcS2 transit peptide malate Dehydrogenase (39) DNA construct (1661 bp) that (189-323), a NADPH-dependent alcohol dehydrogenase-en includes a PCR FD primer (sequence 1-20), a 2x84-bp coding sequence (324-1433) selected/modified from Fuso Chlamydomonas reinhardtii Nia1 (nitrate reductase) pro bacterium sp. 7 1 NADPH-dependent alcohol dehydroge moter (21-188), a 135-bp Chlamydomonas reinhardtii RbcS2 nase (ZP 04573952), a 223-bp Chlamydomonas reinhardtii transit peptide (189-323), a 3-isopropylmalate dehydroge RbcS2 terminator (1434-1656), and a PCR RE primer (1657 nase-encoding sequence (324-1418) selected/modified from 1676). Cyanothece sp. PCC 78223-isopropylmalate dehydrogenase 25 Note, use of SEQID NOS. 129-141 (and/or 142) in genetic (YP 003888480), a 223-bp Chlamydomonas reinhardtii transformation of an eukaryotic photosynthetic organism RbcS2 terminator (1419-1641), and a PCR RE primer (1642 Such as Chlamydomonas can create a designer eukaryotic 1661). photosynthetic organism such as designer Chlamydomonas SEQ ID NO. 138 presents example 138 for a designer with a Calvin-cycle 3-phosphogylcerate-branched NADPH Nial-promoter-controlled chloroplast-targeted 2-Isopropyl 30 enhanced pathway (03-05, 34-43/44 in FIG. 4) for photobio malate Synthase (40) DNA construct (2174 bp) that includes logical production of 1-butanol from carbon dioxide and a PCRFD primer (sequence 1-20), a 2x84-bp Chlamydomo Water. nas reinhardtii Nial promoter (21-188), a 135-bp Chlamy SEQ ID NO. 143 presents example 143 for a designer domonas reinhardtii RbcS2 transit peptide (189-323), a Nial-promoter-controlled chloroplast-targeted Phospho 2-isopropylmalate synthase-encoding sequence (324-1931) 35 enolpyruvate Carboxylase (45) DNA construct (3629 bp) that selected/modified from Cyanothece sp. PCC 7822 2-isopro includes a PCR FD primer (sequence 1-20), a 2x84-bp pylmalate synthase (YP 003890 122), a 223-bp Chlamy Chlamydomonas reinhardtii Nial promoter (21-188), a 135 domonas reinhardtii RbcS2 terminator (1932-2154), and a bp Chlamydomonas reinhardtii RbcS2 transit peptide (189 PCR RE primer (2155-2174). 323), a Phosphoenolpyruvate Carboxylase-encoding SEQ ID NO. 139 presents example 139 for a designer 40 sequence (324-3386) selected/modified from Cyanothece sp. Nial-promoter-controlled chloroplast-targeted Isopropyl PCC 7822 Phosphoenolpyruvate Carboxylase (YP malate Isomerase (41) large/small subunit DNA construct 003887888), a 223-bp Chlamydomonas reinhardtii RbcS2 (2882 bp) that includes a PCRFD primer (sequence 1-20), a terminator (3387-3609), and a PCR RE primer (3610-3629). 2x84-bp Chlamydomonas reinhardtii Nial promoter SEQ ID NO. 144 presents example 144 for a designer (21-188), a 135-bp Chlamydomonas reinhardtii RbcS2 tran 45 Nial-promoter-controlled chloroplast-targeted Aspartate sit peptide (189-323), an isopropylmalate isomerase large Aminotransferase (46) DNA construct (1745 bp) that subunit-encoding sequence (324-1727) selected/modified includes a PCR FD primer (sequence 1-20), a 2x84-bp from Anabaena variabilis ATCC 29413 isopropylmalate Chlamydomonas reinhardtii Nial promoter (21-188), a 135 isomerase large subunit (YP 324467), a 2x84-bp Chlamy bp Chlamydomonas reinhardtii RbcS2 transit peptide (189 domonas reinhardtii Nial promoter (1728-1895), a 135-bp 50 323), a Aspartate Aminotransferase-encoding sequence (324 Chlamydomonas reinhardtii RbcS2 transit peptide (1896 1502) selected/modified from Synechococcus elongatus PCC 2030), an isopropylmalate isomerase Small subunit-encoding 6301 Aspartate Aminotransferase (YP 172275), a 223-bp sequence (2031-2639) selected/modified from Anabaena Chlamydomonas reinhardtii RbcS2 terminator (1503-1525), variabilis ATCC 29413 isopropylmalate isomerase small and a PCR RE primer (1526-1745). subunit (YP 324.466), a 223-bp Chlamydomonas reinhardtii 55 SEQ ID NO. 145 presents example 145 for a designer RbcS2 terminator (2640-2862), and a PCR RE primer (2863 Nial-promoter-controlled chloroplast-targeted Aspartoki 2882). nase (47) DNA construct (2366 bp) that includes a PCR FD SEQ ID NO. 140 presents example 140 for a designer primer (sequence 1-20), a 2x84-bp Chlamydomonas rein Nial-promoter-controlled chloroplast-targeted 2-Keto Acid hardtii Nial promoter (21-188), a 135-bp Chlamydomonas Decarboxylase (42) DNA construct (2210 bp) that includes a 60 reinhardtii RbcS2 transit peptide (189-323), an Aspartoki PCRFD primer (sequence 1-20), a 2x84-bp Chlamydomonas nase-encoding sequence (324-2123) selected/modified from reinhardtii Nial promoter (21-188), a 135-bp Chlamydomo Cyanothece sp. PCC 8802 Aspartokinase (YP 003136939), nas reinhardtii RbcS2 transit peptide (189-323), a 2-keto acid a 223-bp Chlamydomonas reinhardtii RbcS2 terminator decarboxylase-encoding sequence (324-1967) selected/ (2124-2346), and a PCR RE primer (2347-2366). modified from Lactococcus lactis 2-keto acid decarboxylase 65 SEQ ID NO. 146 presents example 146 for a designer (AAS49166), a 223-bp Chlamydomonas reinhardtii RbcS2 Nial-promoter-controlled chloroplast-targeted Aspartate terminator (1968-2190), and a PCR RE primer (2191-2210). Semialdehyde Dehydrogenase (48) DNA construct (1604 bp) US 8,986,963 B2 105 106 that includes a PCRFD primer (sequence 1-20), a 2x84-bp synthase (CAB07802), a 223-bp Chlamydomonas reinhardtii Chlamydomonas reinhardtii Nial promoter (21-188), a 135 RbcS2 terminator (2040-2262), and a PCR RE primer (2263 bp Chlamydomonas reinhardtii RbcS2 transit peptide (189 2282). 323), an Aspartate-semialdehyde dehydrogenase-encoding SEQ ID NO. 152 presents example 152 for a designer sequence (324-1361) selected/modified from Trichodesmium Nial-promoter-controlled chloroplast-targeted Ketol-Acid erythraeum IMS101 Aspartate-semialdehyde dehydrogenase Reductoisomerase (54) DNA construct (1562 bp) that (ABG50031), a 223-bp Chlamydomonas reinhardtii RbcS2 includes a PCR FD primer (sequence 1-20), a 2x84-bp terminator (1362-1584), and a PCR RE primer (1585-1604). Chlamydomonas reinhardtii Nial promoter (21-188), a 135 SEQ ID NO. 147 presents example 147 for a designer bp Chlamydomonas reinhardtii RbcS2 transit peptide (189 Nial-promoter-controlled chloroplast-targeted Homoserine 10 323), an enzyme-encoding sequence (324-1319) selected/ modified from Cyanothece sp. PCC 7822 ketol-acid Dehydrogenase (49) DNA construct (1868 bp) that includes a reductoisomerase (YP 003885458), a 223-bp Chlamy PCRFD primer (sequence 1-20), a 2x84-bp Chlamydomonas domonas reinhardtii RbcS2 terminator (1320-1542), and a reinhardtii Nial promoter (21-188), a 135-bp Chlamydomo PCR RE primer (1543-1562). nas reinhardtii RbcS2 transit peptide (189-323), a 15 SEQ ID NO. 153 presents example 153 for a designer homoserine dehydrogenase-encoding sequence (324-1625) Nial-promoter-controlled chloroplast-targeted Dihydroxy selected/modified from Cyanothece sp. PCC 7822 Acid Dehydratase (55) DNA construct (2252 bp) that homoserine dehydrogenase (YP 003887242), a 223-bp includes a PCR FD primer (sequence 1-20), a 2x84-bp Chlamydomonas reinhardtii RbcS2 terminator (1626-1848), Chlamydomonas reinhardtii Nial promoter (21-188), a 135 and a PCR RE primer (1849-1868). bp Chlamydomonas reinhardtii RbcS2 transit peptide (189 SEQ ID NO. 148 presents example 148 for a designer 323), a dihydroxy-acid dehydratase-encoding sequence (324 Nial-promoter-controlled chloroplast-targeted Homoserine 2009) selected/modified from Cyanothece sp. PCC 7822 Kinase (50) DNA construct (1472 bp) that includes a PCRFD dihydroxy-acid dehydratase (YP 003887466), a 223-bp primer (sequence 1-20), a 2x84-bp Chlamydomonas rein Chlamydomonas reinhardtii RbcS2 terminator (2010-2232), hardtii Nial promoter (21-188), a 135-bp Chlamydomonas 25 and a PCR RE primer (2233-2252). reinhardtii RbcS2 transit peptide (189-323), a Homoserine SEQ ID NO. 154 presents example 154 for a designer kinase-encoding sequence (324-1229) selected/modified Nial-promoter-controlled chloroplast-targeted 2-Methylbu from Cyanothece sp. PCC 7822 Homoserine kinase (YP tyraldehyde Reductase (56) DNA construct (1496 bp) that 003886645), a 223-bp Chlamydomonas reinhardtii RbcS2 includes a PCR FD primer (sequence 1-20), a 2x84-bp terminator (1230-1452), and a PCR RE primer (1453-1472). 30 Chlamydomonas reinhardtii Nial promoter (21-188), a 135 SEQ ID NO. 149 presents example 149 for a designer bp Chlamydomonas reinhardtii RbcS2 transit peptide (189 Nial-promoter-controlled chloroplast-targeted Threonine 323), an enzyme-encoding sequence (324-1253) selected/ Synthase (51) DNA construct (1655 bp) that includes a PCR modified from Pichia pastoris GS115 FD primer (sequence 1-20), a 2x84-bp Chlamydomonas rein 2-methylbutyraldehyde reductase (XP 002490018), a 223 hardtii Nial promoter (21-188), a 135-bp Chlamydomonas 35 bp Chlamydomonas reinhardtii RbcS2 terminator (1254 reinhardtii RbcS2 transit peptide (189-323), a Threonine syn 1476), and a PCR RE primer (1477-1496). thase-encoding sequence (324-1412) selected/modified from Note, use of SEQ ID NOS. 129-137,140, and 151-154 in Cyanothece sp. PCC 7425 Threonine synthase (YP genetic transformation of an eukaryotic photosynthetic 002485009), a 223-bp Chlamydomonas reinhardtii RbcS2 organism Such as Chlamydomonas can create a designer terminator (1413-1635), and a PCR RE primer (1636-1655). 40 eukaryotic photosynthetic organism Such as designer SEQ ID NO. 150 presents example 150 for a designer Chlamydomonas with a Calvin-cycle 3-phosphogylcerate Nial-promoter-controlled chloroplast-targeted Threonine branched NADPH-enhanced pathway (03-05, 34-39, 53-55, Ammonia-Lyase (52) DNA construct (2078 bp) that includes 42, and 56 in FIG.5) for photobiological production of 2-me a PCRFD primer (sequence 1-20), a 2x84-bp Chlamydomo thyl-1-butanol from carbon dioxide and water. nas reinhardtii Nial promoter (21-188), a 135-bp Chlamy 45 SEQ ID NO. 155 presents example 155 for a designer domonas reinhardtii RbcS2 transit peptide (189-323), a Nial-promoter-controlled chloroplast-targeted 3-Methylbu threonine ammonia-lyase-encoding sequence (324-1835) tanal Reductase (57) DNA construct (1595 bp) that includes selected/modified from Synechococcus sp. PCC 7335 threo a PCRFD primer (sequence 1-20), a 2x84-bp Chlamydomo nine ammonia-lyase (ZP 05035047), a 223-bp Chlamy nas reinhardtii Nial promoter (21-188), a 135-bp Chlamy domonas reinhardtii RbcS2 terminator (1836-2058), and a 50 domonas reinhardtii RbcS2 transit peptide (189-323), a PCR RE primer (2059-2078). 3-methylbutanal reductase-encoding sequence (324-1352) Note, use of SEQ ID NOS. 129,130,132,133, 143-150, selected/modified from Saccharomyces cerevisiae S288c 137-141 (and/or 141) through genetic transformation of an 3-methylbutanal reductase (DAA10635), a 223-bp Chlamy eukaryotic photosynthetic organism Such as Chlamydomonas domonas reinhardtii RbcS2 terminator (1353-1575), and a can create a designer eukaryotic photosynthetic organism 55 PCR RE primer (1576-1595). Such as designer Chlamydomonas with a Calvin-cycle Note, use of SEQID NOS. 129-133, 151-153,140 and 141 3-phosphogylcerate-branched NADPH-enhanced pathway (or 142) in genetic transformation of an eukaryotic photosyn (03,04,34,35,45-52,39-43/44 in FIG. 4) for photobiological thetic organism such as Chlamydomonas can create a production of 1-butanol from carbon dioxide and water. designer eukaryotic photosynthetic organism such as SEQ ID NO. 151 presents example 151 for a designer 60 designer Chlamydomonas with a Calvin-cycle 3-phospho Nial-promoter-controlled chloroplast-targeted Acetolactate gylcerate-branched NADPH-enhanced pathway (03-05, 34, Synthase (53) DNA construct (2282 bp) that includes a PCR 35, 53-55, 42, and 43 (44) in FIG. 6) for photobiological FD primer (sequence 1-20), a 2x84-bp Chlamydomonas rein production of isobutanol from carbon dioxide and water. hardtii Nial promoter (21-188), a 135-bp Chlamydomonas Whereas, SEQID NOS. 129-133, 151-153, 136-138,140 and reinhardtii RbcS2 transit peptide (189-323), an acetolactate 65 155 represent a designer eukaryotic photosynthetic organism synthase-encoding sequence (324-2039) selected/modified Such as designer Chlamydomonas with a Calvin-cycle from Bacillus subtilis subsp. subtilis str. 168 acetolactate 3-phosphogylcerate-branched NADPH-enhanced pathway US 8,986,963 B2 107 108 (03-05, 34,35,53-55, 40,38,39, 42, and 57 in FIG. 6) that can nas reinhardtii RbcS2 transit peptide (189-323), an enzyme photobiologically produce 3-methyl-1-butanol from carbon encoding sequence (324-1094) selected/modified from Bor dioxide and water. detella petrii DSM 12804 Enoyl-CoA dehydratase (YP SEQ ID NO. 156 presents example 156 for a designer 001629844), a 223-bp Chlamydomonas reinhardtii RbcS2 Nial-promoter-controlled chloroplast-targeted NADH-de terminator (1095-1317), and a PCR RE primer (1318-1337). pendent Butanol Dehydrogenase (12a) DNA construct (1739 SEQ ID NO. 161 presents example 161 for a designer bp) that includes a PCRFD primer (sequence 1-20), a 2x84 Nial-promoter-controlled 2-Enoyl-CoA reductase (10") bp Chlamydomonas reinhardtii Nia1 (nitrate reductase) pro DNA construct (1736 bp) that includes a PCR FD primer moter (21-188), a 135-bp Chlamydomonas reinhardtii RbcS2 (sequence 1-20), a 2x84-bp Chlamydomonas reinhardtii transit peptide (189-323), an enzyme-encoding sequence 10 Nial promoter (21-188), a 135-bp Chlamydomonas rein (324-1496) selected/modified from Clostridium perfingens hardtii RbcS2 transit peptide (189-323), an enzyme-encoding str. 13 NADH-dependent butanol dehydrogenase (NP sequence (324-1493) selected/modified from Xanthomonas 561774), a 223-bp Chlamydomonas reinhardtii RbcS2 termi campestris pv. Campestris str. B100 2-Enoyl-CoA reductase nator (1497-1719), and a PCR RE primer (1720-1739). (YP 001905744), a 223-bp Chlamydomonas reinhardtii SEQ ID NO. 157 presents example 157 for a designer 15 RbcS2 terminator (1494-1716), and a PCR RE primer (1717 Nial-promoter-controlled chloroplast-targeted NADPH-de 1736). pendent Butanol Dehydrogenase (12b) DNA construct (1733 SEQ ID NO. 162 presents example 162 for a designer bp) that includes a PCRFD primer (sequence 1-20), a 2x84 Nial-promoter-controlled chloroplast-targeted Acyl-CoA bp Chlamydomonas reinhardtii Nial promoter (21-188), a reductase (11') DNA construct (2036 bp) that includes a PCR 135-bp Chlamydomonas reinhardtii RbcS2 transit peptide FD primer (sequence 1-20), a 2x84-bp Chlamydomonas rein (189-323), an enzyme-encoding sequence (324-1490) hardtii Nial promoter (21-188), a 135-bp Chlamydomonas selected/modified from Clostridium saccharobutyllicum reinhardtii RbcS2 transit peptide (189-323), an enzyme-en NADPH-dependent butanol dehydrogenase (AAA83520), a coding sequence (324-1793) selected/modified from Ther 223-bp Chlamydomonas reinhardtii RbcS2 terminator mosphaera aggregains DSM 11486 Acyl-CoA reductase (1491-1713), and a PCR RE primer (1714-1733). 25 (YP 003649571), a 223-bp Chlamydomonas reinhardtii Note, use of SEQID NOS. 129-140 and 156 (and/or 157) RbcS2 terminator (1794-2016), and a PCR RE primer (2017 in genetic transformation of an eukaryotic photosynthetic 2036). organism Such as Chlamydomonas can create a designer SEQ ID NO. 163 presents example 163 for a designer eukaryotic photosynthetic organism Such as designer Nial-promoter-controlled chloroplast-targeted Hexanol Chlamydomonas with a Calvin-cycle 3-phosphogylcerate 30 Dehydrogenase (12") DNA construct (1625 bp) that includes branched NADPH-enhanced butanol production pathway a PCRFD primer (sequence 1-20), a 2x84-bp Chlamydomo (03-05, 34-42 and 12 in FIG. 4) for more specific photobio nas reinhardtii Nial promoter (21-188), a 135-bp Chlamy logical production of 1-butanol from carbon dioxide and domonas reinhardtii RbcS2 transit peptide (189-323), an water. Similarly, SEQID NOS. 129, 130,132,133, 143-150, enzyme-encoding sequence (324-1382) selected/modified 137-140, and 156 (and/or 157) represent another designer 35 from Mycobacterium chubuense NBB4 hexanol dehydroge eukaryotic photosynthetic organism Such as designer nase (ACZ56328), a 223-bp Chlamydomonas reinhardtii Chlamydomonas with a Calvin-cycle 3-phosphogylcerate RbcS2 terminator (1383-1605), and a PCR RE primer (1606 branched NADPH-enhanced butanol-production pathway 1625). (03, 04, 34,35, 45-52, 39-42 and 12 in FIG. 4) for photobio Note, use of SEQID NOS. 158-163 with other properDNA logical production of 1-butanol from carbon dioxide and 40 constructs such as SEQ ID NOS. 132 and 133 in genetic Water. transformation of an eukaryotic photosynthetic organism SEQ ID NO. 158 presents example 158 for a designer Such as Chlamydomonas can create a designer eukaryotic Nial-promoter-controlled chloroplast-targeted 3-Ketothio photosynthetic organism such as designer Chlamydomonas lase (07) DNA construct (1745 bp) that includes a PCR FD with a Calvin-cycle 3-phosphogylcerate-branched NADPH primer (sequence 1-20), a 2x84-bp Chlamydomonas rein 45 enhanced hexanol production pathway (34, 35, 03-10, and hardtii Nia1 (nitrate reductase) promoter (21-188), a 135-bp 07-12 in FIG.7) for photobiological production of 1-hexanol Chlamydomonas reinhardtii RbcS2 transit peptide from carbon dioxide and water. (189-323), a 3-Ketothiolase-encoding sequence (324-1502) SEQ ID NO. 164 presents example 164 for a designer selected/modified from Azohydromonas lata 3-Ketothiolase Nial-promoter-controlled chloroplast-targeted Octanol (AAD 10275), a 223-bp Chlamydomonas reinhardtii RbcS2 50 Dehydrogenase (12") DNA construct (1249 bp) that includes terminator (1503-1725), and a PCR RE primer (1726-1745). a PCRFD primer (sequence 1-20), a 2x84-bp Chlamydomo SEQID NO. 159 presents a designer Nial-promoter-con nas reinhardtii Nial promoter (21-188), a 135-bp Chlamy trolled chloroplast-targeted 3-Hydroxyacyl-CoA dehydroge domonas reinhardtii RbcS2 transit peptide (189-323), an nase (08) DNA construct (1439 bp) that includes a PCR FD enzyme-encoding sequence (324-1006) selected/modified primer (sequence 1-20), a 2x84-bp Chlamydomonas rein 55 from Drosophila subobscura Octanol dehydrogenase hardtii Nial promoter (21-188), a 135-bp Chlamydomonas (AB065263), a 223-bp Chlamydomonas reinhardtii RbcS2 reinhardtii RbcS2 transit peptide (189-323), an enzyme-en terminator (1007-1229), and a PCR RE primer (1230-1249). coding sequence (324-1196) selected/modified from Ocean Note, SEQ ID NOS. 132,133, and 158-163 represent a ithermus profundus DSM 149773-Hydroxyacyl-CoA dehy designer eukaryotic photosynthetic organism Such as a drogenase (ADR36325), a 223-bp Chlamydomonas 60 designer Chlamydomonas with a designer hydrocarbon chain reinhardtii RbcS2 terminator (1197–1419), and a PCR RE elongation pathway (34, 35,07-12 as shown in FIG. 7) for primer (1420-1439). photobiological production of 1-hexanol. SEQID NOS: 132, SEQ ID NO. 160 presents example 160 for a designer 133, 158-162 and 164 represent another designer eukaryotic Nial-promoter-controlled chloroplast-targeted Enoyl-CoA photosynthetic organism Such as a designer Chlamydomonas dehydratase (09') DNA construct (1337 bp) that includes a 65 with a designer hydrocarbon chain elongation pathway (34. PCRFD primer (sequence 1-20), a 2x84-bp Chlamydomonas 35,07-10' and 07"-12" as shown in FIG.7) for photobiologi reinhardtii Nial promoter (21-188), a 135-bp Chlamydomo cal production of 1-octanol. US 8,986,963 B2 109 110 SEQ ID NO. 165: a designer Nial-promoter-controlled NADPH-enhanced pathway gene(s) and biosafety-guarding chloroplast-targeted Short Chain Alcohol Dehydrogenase technology for enhanced photobiological production of (43') DNA construct (1769 bp) that includes a PCRFD primer butanol and related higher alcohols from carbon dioxide and (sequence 1-20), a 2x84-bp Chlamydomonas reinhardtii water. According to one of the various embodiments, it is a Nial promoter (21-188), a 135-bp Chlamydomonas rein preferred practice to grow designer photosynthetic organisms hardtii RbcS2 transit peptide (189-323), an enzyme-encoding photoautotrophically using carbon dioxide (CO) and water sequence (324-1526) selected/modified from Burkholderia (HO) as the sources of carbon and electrons with a culture vietnamiensis G4 Short chain alcohol dehydrogenase medium containing inorganic nutrients. The nutrient ele (AB056626), a 223-bp Chlamydomonas reinhardtii RbcS2 ments that are commonly required for oxygenic photosyn terminator (1527-1749), and a PCR RE primer (1750-1769). 10 thetic organism growth are: N. P. and Kat the concentrations Note, use of SEQ ID NOS. 129-140 and 165 in genetic of about 1-10 mM, and Mg, Ca, S, and C1 at the concentra transformation of an eukaryotic photosynthetic organism tions of about 0.5 to 1.0 mM, plus some trace elements Mn, Such as Chlamydomonas can create a designer eukaryotic Fe, Cu, Zn, B, Co, Mo among others at LM concentration photosynthetic organism such as designer Chlamydomonas levels. All of the mineral nutrients can be supplied in an with a Calvin-cycle 3-phosphogylcerate-branched NADPH 15 aqueous minimal medium that can be made with well-estab enhanced pathway (03-05,34-41,39-43', 39-43' and 39"-43" lished recipes of oxygenic photosynthetic organism (such as in FIG. 8) for photobiological production of 1-pentanol, algal) culture media using water (freshwater for the designer 1-hexanol, and 1-heptanol from carbon dioxide and water. freshwater algae; seawater for the salt-tolerant designer Similarly, SEQID NOS. 129-140 and 163 represent another marine algae) and relatively small of inexpensive fertilizers designer eukaryotic photosynthetic organism Such as and mineral salts such as ammonium bicarbonate designer Chlamydomonas with a Calvin-cycle 3-phospho (NHHCO) (or ammonium nitrate, urea, ammonium chlo gylcerate-branched NADPH-enhanced pathway (03-05, ride), potassium phosphates (KHPO and KH2PO4), magne 34-41, 39-41', 39-42 and 12' in FIG. 8) for photobiological sium sulfate heptahydrate (MgSO.7H2O), calcium chloride production of 1-hexanol from carbon dioxide and water. (CaCl), Zinc sulfate heptahydrate (ZnSO.7H2O), iron (II) Likewise, use of SEQID NOS. 129-137, 151-153, 138-140 25 sulfate heptahydrate (FeSO.7H2O), and boric acid (HBO), and 165 through genetic transformation of an eukaryotic pho among others. That is, large amounts of designer algae (or tosynthetic organism such as Chlamydomonas can create a oxyphotobacteria) cells can be inexpensively grown in a short designer eukaryotic photosynthetic organism Such as period of time because, under aerobic conditions such as in an designer Chlamydomonas with a Calvin-cycle 3-phospho open pond, the designer algae can photoautotrophically grow gylcerate-branched NADPH-enhanced pathway (03-05, 30 by themselves using air CO as rapidly as their wild-type 34-39, 53-55, 39-43', 39-43', and 39"-43" in FIG. 9) for parental strains. This is a significant feature (benefit) of the photobiological production of 3-methyl-1-pentanol, 4-me invention that could provide a cost-effective solution in gen thyl-1-hexanol, and 5-methyl-1-heptanol from carbon diox eration of photoactive biocatalysts (the designer photosyn ide and water: The expression of SEQID NOS. 129, 130, 132, thetic biofuel-producing organisms such as designer algae or 133, 143-150, 151-153, 137-140 and 165 in an eukaryotic 35 oxyphotobacteria) for renewable Solar energy production. photosynthetic organism Such as a host Chlamydomonas rep According to one of the various embodiments, when resent another designer eukaryotic photosynthetic organism designer photosynthetic organism culture is grown and ready with a Calvin-cycle 3-phosphogylcerate-branched NADPH for photobiological production of butanol and/or related enhanced pathway (03,05,34,35, 42–55,39-43', 39-43', and higher alcohols, the designer photosynthetic organism cells 39"-43" in FIG.9) for photobiological production of 3-me 40 are then induced to express the designer Calvin-cycle chan thyl-1-pentanol, 4-methyl-1-hexanol, and 5-methyl-1-hep neled photosynthetic NADPH-enhanced pathway(s) to pho tanol from carbon dioxide and water; The expression of SEQ tobiologically produce butanol and/or related higher alcohols ID NOS. 129-133, 151-153, 136-140 and 165 in a host from carbon dioxide and water. The method of induction is eukaryotic photosynthetic organism Such as Chlamydomonas designer pathway gene(s) specific. For example, if/when a represent yet another designer eukaryotic photosynthetic 45 nirA promoter is used to control the designer Calvin-cycle organism with a Calvin-cycle 3-phosphogylcerate-branched channeled pathway gene(s) such as those of SEQ ID NOS: NADPH-enhanced pathway (03-05, 34,35,53-55, 40,38,39, 58-69 and 72 (and/or 73) which represent a designer trans 39-43', 39-43', and 39"-43" in FIG. 10) for photobiological genic Thermosynechococcus that comprises the designer production of 4-methyl-1-pentanol. 5-methyl-1-hexanol, and genes of a Calvin-cycle 3-phophoglycerate-branched photo 6-methyl-1-heptanol from carbon dioxide and water. 50 synthetic NADPH-enhanced pathway (numerically labeled Use of Designer Photosynthetic Organisms with Photobiore as 34,35, 03-05, 36-42, and 12 in FIG. 4) for photobiological actor for Production and Harvesting of Butanol and Related production of 1-butanol from carbon dioxide and water, the Higher Alcohols designer transgenic Thermosynechococcus is grown in a The designer photosynthetic organisms with designer minimal liquid culture medium containing ammonium (but Calvin-cycle channeled photosynthetic NADPH-enhanced 55 no nitrate) and other inorganic nutrients. When the designer pathways (FIGS. 1, and 4-10) can be used with photobiore transgenic Thermosynechococcus culture is grown and ready actors for production and harvesting ofbutanol and/or related for photobiological production of biofuel 1-butanol, nitrate higher alcohols. The said butanol and/or related higher alco fertilizer will then be added into the culture medium to induce hols are selected from the group consisting of 1-butanol, the expression of the designer nirA-controlled Calvin-cycle 2-methyl-1-butanol, isobutanol, 3-methyl-1-butanol, 1-hex 60 channeled pathway to photobiologically produce 1-butanol anol, 1-octanol. 1-pentanol, 1-heptanol, 3-methyl-1-pen from carbon dioxide and water in this example. tanol, 4-methyl-1-hexanol, 5-methyl-1-heptanol, 4-methyl For the designer photosynthetic organism(s) with anaero 1-pentanol, 5-methyl-1-hexanol, 6-methyl-1-heptanol, and bic promoter-controlled pathway(s) such as the designer combinations thereof. transgenic Nostoc that contains designer hoX-promoter-con The said designer photosynthetic organisms such as 65 trolled Calvin-cycle 3-phophoglycerate-branched pathway designer transgenic oxyphotobacteria and algae comprise genes of SEQID NOS. 104-109, anaerobic conditions can be designer Calvin-cycle-channeled and photosynthetic used to induce the expression of the designer pathway gene(s) US 8,986,963 B2 111 112 for photobiological production of 2-methyl-1-butanol from water splitting and proton gradient coupled electron transport carbon dioxide and water (FIG. 5). That is, when the designer process in the photobioreactor to synthesize butanol and transgenic Nostoc culture is grown and ready for photobio related higher alcohols from carbon dioxide and water; and c) logical biofuel production, its cells will then be placed (or using a product separation process to harvest the synthesized sealed) into certain anaerobic conditions to induce the expres butanol and/or related higher alcohols from the photobiore sion of the designer hoX-controlled pathway gene(s) to pho actOr. tobiologically produce 2-methyl-1-butanol from carbon In Summary, there are a number of embodiments on how dioxide and water. the designer organisms may be used for photobiological For those designer photosynthetic organism(s) that con butanol (and/or related higher alcohols) production. One of tains a heat- and light-responsive promoter-controlled and 10 the preferred embodiments is to use the designer organisms nir A-promoter-controlled pathway(s) such as the designer for direct photosynthetic butanol production from CO and transgenic Prochlorococcus that contains a set of designer H2O with a photobiological reactor and butanol-harvesting groE-promoter-controlled and nirA-promoter-controlled (filtration and distillation/evaporation) system, which Calvin-cycle 3-phophoglycerate-branched pathway genes of includes a specific operational process described as a series of SEQID NOS. 110-118, light and heat are used in conjunction 15 the following steps: a) Growing a designer transgenic organ of nitrate addition to induce the expression of the designer ism photoautotrophically in minimal culture medium using pathway genes for photobiological production of isobutanol air CO as the carbon Source under aerobic (normal) condi from carbon dioxide and water (FIG. 6). tions before inducing the expression of the designer butanol According to another embodiment, use of designer marine production-pathway genes; b) When the designer organism algae or marine oxyphotobacteria enables the use of seawater culture is grown and ready for butanol production, sealing or and/or groundwater for photobiological production of biofu placing the culture into a specific condition to induce the els without requiring freshwater or agricultural soil. For expression of designer Calvin-cycle-channeled pathway example, designer Prochlorococcus marinus that contains the genes; c) When the designer pathway enzymes are expressed, designer genes of SEQ ID NOS: 110-117 and 119-122 can Supplying visible light energy Such as Sunlight for the use seawater and/or certain groundwater for photoau 25 designer-genes-expressed cells to work as the catalysts for totrophic growth and synthesis of 3-methyl-1-butanol from photosynthetic production of butanol and/or related higher carbon dioxide and water with its groE promoter-controlled alcohols from CO and HO; d) Harvesting the product designer Calvin-cycle-channeled pathway (identified as 34 butanol and/or related higher alcohols by any method known (native), 35, 03-05, 53-55, 38-40, 42 and 57 in FIG. 6). The to those skilled in the art. For example, harvesting the butanol designer photosynthetic organisms can be used also in a 30 and/or related higher alcohols from the photobiological reac sealed photobioreactor that is operated on a desert for pro tor can be achieved by a combination of membrane filtration duction of isobutanol with highly efficient use of water since and distillation/evaporation butanol-harvesting techniques. there will be little or no water loss by evaporation and/or The above process to use the designer organisms for pho transpiration that a common crop system would suffer. That tosynthetic production and harvesting ofbutanol and related is, this embodiment may represent a new generation of renew 35 higher alcohols can be repeated for a plurality of operational able energy (butanol and related higher alcohols) production cycles to achieve more desirable results. Any of the steps a) technology without requiring arable land or freshwater through d) of this process described above can also be SOUCS. adjusted in accordance of the invention to Suit for certain According to another embodiment, use of nitrogen-fixing specific conditions. In practice, any of the steps a) through d) designer oxyphotobacteria enables photobiological produc 40 of the process can be applied in full or in part, and/or in any tion of biofuels without requiring nitrogen fertilizer. For adjusted combination as well for enhanced photobiological example, the designer transgenic Nostoc that contains production ofbutanol and higher alcohol inaccordance of this designer hox-promoter-controlled genes of SEQ ID NOS. invention. 104-109 is capable of both fixing nitrogen (N) and photo In addition to butanol and/or related higher alcohols pro biologically producing 2-methyl-1-butanol from carbon 45 duction, it is also possible to use a designer organism or part dioxide and water (FIG. 6). Therefore, use of the designer of its designer butanol-production pathway(s) to produce cer transgenic Nostoc enables photoautotrophic growth and tain intermediate products of the designer Calvin-cycle-chan 2-methyl-1-butanol synthesis from carbon dioxide and water. neled pathways (FIGS. 1 and 4-10) including (but not limited Certain designer oxyphotobacteria are designed to perform to): butyraldehyde, butyryl-CoA, crotonyl-CoA, 3-hydroxy multiple functions. For example, the designer transgenic 50 butyryl-CoA, acetoacetyl-CoA, acetyl-CoA, pyruvate, phos Cyanothece that contains designer nirA promoter-controlled phoenolpyruvate, 2-phosphoglycerate, 1,3-diphosphoglycer genes of SEQ ID NOS. 123-127 is capable of (1) using ate, glyceraldehye-3-phosphate, dihydroxyacetone seawater, (2)N fixing nitrogen, and photobiological produc phosphate, fructose-1,6-diphosphate, fructose-6-phosphate, ing 1-hexanol from carbon dioxide and water (FIG. 8). Use of glucose-6-phosphate, glucose, glucose-1-phosphate, citra this type of designer oxyphotobacteria enables photobiologi 55 malate, citraconate, methyl-D-malate, 2-ketobutyrate, 2-ke cal production of advanced biofuels such as 1-hexanol using tovalerate, Oxaloacetate, aspartate, homoserine, threonine, seawater without requiring nitrogen fertilizer 2-keto-3-methylvalerate, 2-methylbutyraldehyde, 3-methyl According to one of various embodiments, a method for butyraldehyde, 4-methyl-2-oxopentanoate, 3-isopropyl photobiological production and harvesting of butanol and malate, 2-isopropylmalate, 2-oxoisovalerate, 2,3-dihydroxy related higher alcohols comprises: a) introducing a transgenic 60 isovalerate, 2-acetolactate, isobutyraldehyde, 3-keto-C6 photosynthetic organism into a photobiological reactor sys acyl-CoA, 3-hydroxy-C6-acyl-CoA, C6-enoyl-CoA, tem, the transgenic photosynthetic organism comprising C6-acyl-CoA, 3-keto-C8-acyl-CoA, 3-hydroxy-C8-acyl transgenes coding for a set of enzymes configured to act on an CoA, C8-enoyl-CoA, C8-acyl-CoA, octanal, 1-pentanol, intermediate product of a Calvin cycle and to convert the 1-hexanal, 1-heptanal, 2-ketohexanoate, 2-ketoheptanoate, intermediate product into butanol and related higher alcohols; 65 2-ketooctanoate, 2-ethylmalate, 3-ethylmalate, 3-methyl-1- b) using reducing power and energy associated with the trans pentanal, 4-methyl-1-hexanal, 5-methyl-1-heptanal, 2-hy genic photosynthetic organism acquired from photosynthetic droxy-2-ethyl-3-oxobutanoate, 2,3-dihydroxy-3-methyl US 8,986,963 B2 113 114 pentanoate, 2-keto-4-methyl-hexanoate, 2-keto-5-methyl dehydrogenase or butyraldehyde dehydrogenase omitted heptnoate, 2-keto-6-methyl-octanoate, 4-methyl-1-pentanal, from the designer pathway(s) of FIG. 1 may be used to pro 5-methyl-1-hexanal, 6-methyl-1-heptanal, 2-keto-7-methyl duce butyraldehyde or butyryl-CoA, respectively. octanoate, 2-keto-6-methyl-heptanoate, and 2-keto-5-me While the present invention has been illustrated by descrip thyl-hexanoate. According to one of various embodiments, tion of several embodiments and while the illustrative therefore, a further embodiment comprises an additional step embodiments have been described in considerable detail, it is of harvesting the intermediate products that can be produced not the intention of the applicant to restrict or in any way limit also from an induced transgenic designer organism. The pro the scope of the appended claims to Such detail. Additional duction of an intermediate product can be selectively advantages and modifications will readily appear to those enhanced by Switching off a designer-enzyme activity that 10 skilled in the art. The invention in its broader aspects is catalyzes its consumption in the designer pathways. The pro therefore not limited to the specific details, representative duction of a said intermediate product can be enhanced also apparatus and methods, and illustrative examples shown and by using a designer organism with one or some of designer described. Accordingly, departures may be made from Such enzymes omitted from the designer butanol-production path details without departing from the spirit or scope of appli ways. For example, a designer organism with the butanol cant’s general inventive concept.

SEQUENCE LISTING

<16 Os NUMBER OF SEO ID NOS : 165

<21 Os SEQ ID NO 1 &211s LENGTH: 1809 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Construct - Sequence No. 1, Example 1 : designer Butanol-Dehydrogenase DNA construct (1809 bp)

<4 OOs SEQUENCE: 1 agaaaatctg gCaccacacic tatatggtag ggtgcgagtg accc.cgc.gcg acttggagct 60

cgatggc.ccc gggttgtttgggggg.tc.cgc ct ct cqc.gct attctgagct ggaga.ccgag 12O

gcgcatgaaa atgcatt.cgc titccatagga cqctgcattg tdgcttgaag gttcaaggga 18O

agggttcaaa caccc.cgcc gtacgaactt ttgtcggggg gcqctic ccgg ccc.cgggctic 24 O

ttgttgcgc.gc attagggctt cqggit cqcaa gCaagacgat acatggcc.gc cqt cattgcc 3 OO aagtic ct cog to tcc.gcggc cgtggct cqc ccggc.ccgct coagcgtgcg ccc.catggcc 360 gcgctgaagc cc.gc.cgt caa ggctgcc.ccc gtggctg.ccc cq9ctic aggc calaccagatg 42O gagaattitta gatttaatgc at atacagag atgctttittg gaaagggaca aatagagaag 48O

Ctt CC agagg ttittaaaaag at atggtaala aatat attac ttgcatatgg tdgtggaagt 54 O

ataaaaaaga atggacticta tdatactatic caaaagctat tdaaagattt taat attgtt 6 OO

gaattaagtg gt attgaac C aaatccalaga attgaaactg. talagacgtgg agttgaactt 660

tgcagaaaaa at aaagtaga tigittattitta gctgttggtg gagggagtac aatagactgc 72O

tdaaaggitta taggggcagg ttatt attat gctggagatg catgggacct totaaaaaat 78O

Ccagctaaaa taggtgaggt tttaccalata gtgacagttt taacaatggc agct actggit 84 O

totgaaatga at agaaatgc tigittatttcaaagatggata caaatgaaaa gottggaaca 9 OO

ggat caccita agatgat coc to aaactitct attittagat c cagaat attt gtatacattg 96.O

ccagdaattic aaa.ca.gctgc aggttgttgct gatattatgt cacacatatt tdaacaat at 102O

tittaataaaa ctacagatgc titttgtacaa gataaatttg cqgaaggttt gttgcaaact 108O

tgtataaaat attgc cctdt togctittaaag galaccaaaga attatgaagic tagagcaaat 114 O

ataatgtggg ct agttcaat ggctcittaac ggactitt tag galagtgggala agctggagct 12 OO

tgg acttgtc atccaataga acatgaatta agtgcattitt atgatataac to atggagta 1260

ggit cittgcaa ttittaactcc aagttggatg agatatat ct taagtgatgt aacagttgat 132O

aagtttgtta acg tatggca tttagaacaa aaagaagata aatttgctct togcaaatgaa 138O