Advanced gene manipulation tools are essential for an efficient application of genetic engineering technology, but they are still in the development stage. These gene manipulation tools include:

• Homologous gene replacement or nuclear gene targeting [346, 347]

• Inducible nuclear promoters [348]

• Gene silencing approaches [349-351]

• Gene expression regulation by riboswitches [352, 353]

• New protein tagging approaches [354]

Chloroplast Transformation Technologies

Commonly, wild-type green algae have heavily stacked thylacoids and large light­harvesting antenna complexes (LHC) acclimated for low light conditions that cause photoinhibition under high light conditions [355, 356]. To eliminate the formation of reactive oxygen species, the absorbed photons need to be released as fluorescence and waste heat. This release of energy reduces conversion efficiency of light energy to biomass [357]. One solution to increase the conversion efficiency is to reduce the LHC size and enhance light penetration to the growth media [357-360]. For exam­ple, RNA interference technology was applied to downregulate the entire LHC gene family in C. reinhardtii [361].

Metabolic Network Reconstruction and Simulation

A metabolic network model is a prospectively powerful tool for selection of the most suitable wild-type organism, as well as to provide direction for the genetic engineering of a more efficient mutant [362]. Combinations of knockout and added genes can be optimized for target product and/or biomass yield [363, 364]. A genome scale reconstructed metabolic network improved bioethanol production for Saccharomyces cerevisiae though genetic engineering and evolutionary adapta­tion [365, 366]. The optimal parameters of the designed bioprocess are the growth media composition, the level of irradiation, the temperature, the product yield, the physical dimensions, and the cost efficiency of the overall process [367] .