This multidisciplinary R&D area involves synthetic biology and genetic transformation of photosynthetic organisms to create designer transgenic organisms that can photo­biologically produce biofuels such as hydrogen, lipids/biodiesel, ethanol, butanol, and/or other related higher alcohols (e. g., pentanol and hexanol), or hydrocarbons directly from water and carbon dioxide. Chapter 20 reports inventions on creating designer algae for photobiological production of hydrogen from water. In wild-type algae, there are four physiological problems associated with the proton gradient across the algal thylakoid membrane, which severely limit algal hydrogen production. These technical issues are: (1) accumulation of a proton gradient across the algal thylakoid membrane, (2) competition from carbon dioxide fixation, (3) requirement for bicarbonate binding at photosystem II (PSII) for efficient photosynthetic activity, and (4) competitive drainage of electrons by molecular oxygen. As reported in Chap. 20 one of the key inventions here is the genetic insertion of a proton channel into the algal thylakoid membrane to simultaneously eliminate all of the four tran — sthylakoid proton gradient-associated technical problems for enhanced photoauto­trophic hydrogen production.

In addition to the designer proton-channel algae, Chap. 20 describes a further invention on creating designer switchable PSII algae for robust photobiological production of hydrogen from water splitting, which can eliminate all the following three molecular oxygen (O2)-associated technical problems: (4) competitive drainage of electrons generated from photosynthetic water splitting by molecular oxygen, (5) oxygen sensitivity of algal hydrogenase, and (6) the H2-O2 gas separation and safety issue. Use of the two inventions (two US patents): (I) designer proton-channel algae [14] and (II) designer switchable PSII algae [15], may enable efficient and robust photobiological production of hydrogen with an enhanced yield likely more than ten times better than that of the wild-type.

This designer-algae synthetic biology approach can be applied not only for hydrogen production, but also for the production of other advanced biofuels of choice, such as ethanol and/or butanol, depending on specific metabolic pathway designs [16, 17]. Chapter 21 reports inventions on application of synthetic biology for photobiologically production of ethanol directly from carbon dioxide and water while Chap. 22 describes the methods of creating designer transgenic organisms for photobiological production of butanol and/or related higher alcohols from carbon dioxide and water. One of the key ideas here is to genetically introduce a set of specific enzymes to interface with the Calvin-cycle activity so that certain interme­diate product such as 3-phosphoglycerate (3-PGA) of the Calvin cycle could be con­verted immediately to biofuels such as butanol. The net result of the envisioned total process, including photosynthetic water splitting and proton-coupled electron trans­port for generation of NADPH and ATP that supports the Calvin cycle and the butanol production pathway is the conversion of CO2 and H2O to butanol (CH3CH2CH2CH2OH) and O2 as shown in (1). Therefore, theoretically, this could be a new mechanism to synthesize biofuels (e. g., butanol) directly from CO2 and H2O with the following photosynthetic process reaction:

4CO2 + 5H2O ^ CH3CH2CH2CH2OH + 6O2 (1)

This photobiological biofuel production process completely eliminates the problem of recalcitrant lignocellulosics by bypassing the bottleneck problem of the biomass technology. Since this approach could theoretically produce biofuels (such as hydrogen, ethanol, butanol, related higher alcohols, and/or hydrocarbons/ biodiesel) directly from water and carbon dioxide with high solar-to-biofuel energy efficiency, it may provide the ultimate green/clean renewable energy technology for the world as a long-term goal. According to a recent study [18] for this type of direct photosynthesis-to-biofuel process, the practical maximum solar-to-biofuel energy conversion efficiency could be about 7.2% while the theoretical maximum solar-to — biofuel energy conversion efficiency is calculated to be 12%.

The designer algae approach may also enable the use of seawater and/or ground­water for photobiological production of biofuels without requiring freshwater or agricultural soil, since the biofuel-producing function can be placed through molecular genetics into certain marine algae and/or cyanobacteria that can use seawater and/or certain groundwater. They may be used also in a sealed photobioreactor that could be operated on a desert for the production of biofuels with highly efficient use of water since there will be little or no water loss by evaporation and/or transpiration that a common crop system would suffer. That is, this designer algae approach could provide a new generation of renewable energy (e. g., butanol) production technology without requiring arable land or freshwater resources, which may be strategically important to many parts of the world for long-term sustainable development. Recently, certain independent studies [19, 20] have also applied synthetic biology in certain model cyanobacteria, such as Synecoccus elongatus PCC7942, for photobi­ological production of isobutanol and 1-butanol.

Furthermore, the designer algae approach may be applied for enhanced photo­biological production of other bioproducts, including (but not limited to) lipids, hydrocarbons, intermediate metabolites, and possibly high-value bioproducts such as docosahexaenoic acid (DHA) omega-3 fatty acid, eicosapentaenoic acid (EPA) omega-3 fatty acid, arachidonic acid (ARA) omega-6 fatty acid, chlorophylls, car­otenoids, phycocyanins, allophycocyanin, phycoerythrin, and their derivatives/ related product species.