Genetic engineering of microalgae for improved biomass production at large scales: proof of concept studies for advanced harvesting and pest-management strategies.

摘要

By midcentury it is estimated that food production must nearly double in order to meet global requirements and it remains unclear if this level of production will be possible due to limiting land, fresh-water, energy and nutrient resources; all-the-while, the deleterious impacts of climate change and first generation biofuel production threaten to make the situation even more perilous. Many experts agree that meeting these looming agricultural crises will require a complete overhaul of the current agricultural system. Yields on existing lands must increase but novel saline and wastewater tolerant crops, such as microalgae, will also be needed to meet production demands. Microalgae offer a particularly tantalizing solution but major cultivation and processing problems must first be overcome before their potential can be realized.;Harvesting microalgae at large scales is a significant barrier to economically feasible production of biofuels and other low-cost commodities from microalgal biomass. In chapter 2 of this dissertation, I demonstrate a strategy for reducing the costs of harvesting microalgae by generating transgenic strains of Chlamydomonas reinhardtii that express a heterologous cellular adhesion molecule (Algal-CAM) from the multicellular green alga Volvox carteri. Constitutive expression of heterologous Algal-CAM causes Chlamydomonas unicells to adhere together such that they settle out of suspension much more rapidly than controls.;Immunoblotting shows the heterologous Algal-CAM to be present in the extracellular matrix of Chlamydomonas transformants and apparently cross-linked with native glycoproteins there. We define this form of cell adhesion as genetically engineered (GE) flocculation to distinguish it from other flocculation strategies. Future development of this trait will include making expression of Algal-CAM inducible for controlled timing of GE flocculation and exploring regulated expression of additional cell adhesion molecules from the many other multicellular relatives of Chlamydomonas. Advanced forms of this technology could lead to production of novel biomaterials from single-celled algae by controlled expression of diverse cell adhesion molecules with different cross-linking properties.;The only problem affecting large-scale algaculture that is potentially more significant than harvesting is contamination control. Just as weeds can drastically reduce yields in traditional agricultural systems, so too can aquatic pests, fungi, bacteria and competing algae act to limit production from commercial algaculture operations. Terrestrial agriculture has largely turned to new genetically modified crops that are resistant to common herbicides in order to manage weeds and boost yields while reducing management costs. This transgenic approach to weed control has proven successful and could potentially be applied to algaculture for the control of contamination in large algal ponds or photobioreactors. In chapter 3, I outline a specific strategy to implement contamination control in algal growth systems by generating transgenic algae that possess greater resistance to hydrogen peroxide, a powerful broad-spectrum biocide, by heterologous expression of a yeast cytosolic catalase enzyme (CTT1). I then describe and demonstrate the technical aspects of generating these transgenic algae. Algae transformed with a CTT1 expression vector show significantly greater capacity to break down hydrogen peroxide to oxygen and water than do controls. Moreover, in-vivo catalase enzyme kinetic studies suggest that an additional catalase enzyme is functional in positively transformed lines when compared to transgenic control cell lines that were not transformed with the CTT1 gene. Catalase enzyme kinetics also show that positive transformant lines can break down hydrogen peroxide with greater efficiency and capacity than a control line (engineered cells demonstrate greater Vmax and decreased Km values). The enhanced ability of engineered cells to break down hydrogen peroxide may result in greater resistance to exogenously applied peroxide as an aquatic biocide. Further studies must now be done to demonstrate physiological responses of control and positive transgenic cell lines and verify if cytosolic catalase expression can enhance cell viability after exposure to otherwise lethal doses of hydrogen peroxide.