Molecular Characterization of Dunaliella spp. Growth and Primary Metabolism in Response to Environmental Changes

There is a renewed interest in the use of microalgae as alternative, carbon neutral and sustainable energy resources due to the increasing energy demand, depleting supply of fossil fuels and the environmental concerns associated with global warming. Marine microalgae are a promising feedstock for biofuels, since they are devoid of major drawbacks associated with terrestrial crop plants such as requirement of land or fresh water resources for cultivation. The use of marine microalgae like Dunaliella spp. is advantageous, because they grow fast, accumulate high levels of triacylgycerides (TAGs; oil) with little need for fresh water and lack a rigid cell wall, which makes the TAG extraction process less expensive. For maximal productivity, fast growth and accumulation of high quantities of oil are the most desirable traits, because they provide in a short amount of time large quantities of biomass with high levels of oils. Cell division rates and TAG accumulation exhibit an inverse relationship in algae, because cell division rates are maximal under optimal growth conditions while TAG as carbon and energy storage components accumulate during growth limited conditions. TAGs are direct precursors for biofuel production, however, very little is known about the molecular mechanism behind TAG accumulation in microalgae.;The research reported here focused on understanding the time-resolved physiological, metabolic and transcriptomic response of Dunaliella viridis dumsii to photoperiod, temperature and the integration of both environmental changes. We found that the rate of cell division in D. viridis increased under continuous light compared to light:dark cycles, while an increase in temperature from 25ºC to 35ºC did not significantly affect the cell division rate, but increased the TAG per cell several-fold under continuous light. The amount of saturated fatty acids in the TAG fraction was more responsive to an increase in temperature than to a change in the light regime. Transcriptome analysis showed that genes coding for fatty acid biosynthesis enzymes in response to elevated temperature are not transcriptionally regulated, while TAG biosynthesis under continuous light at elevated temperature is driven by transcriptional up-regulation of lipases involved in the recycling of fatty acids from membrane lipids. Starch metabolism was controlled via transcriptional regulation of its degradation enzymes and does not respond to temperature changes under light:dark cycles, but was sensitive to temperature under continuous light.;Dunaliella possess useful traits for biofuel production, although, the high cost of controlled growth and harvesting have prevented the development of this technology at commercial level. Expression of high-value co-products like industrial enzymes could offset the high costs of algae cultivation associated with photobioreactors and harvesting, and make algae-derived biofuels commercially viable. To produce recombinant enzymes in Dunaliella, methods for stable transformation are required. This study contributed to the development of molecular tools for nuclear transformation of D. viridis dumsii for genetic engineering. We isolated and used the endogenous promoters and terminators of the ribulose bisphosphate carboxylase/oxygenase small subunit (rbcS) of D. viridis to drive the expression of the reporter gene encoding the "Enhanced Green Fluorescent Protein" (EGFP) and Bleomycin (ble) conferring resistance to antibiotic zeocin. Transformation was attempted by mechanical stress using glass beads and by electroporation. A novel method of embedding D. viridis cells in solid growth media while selecting with zeocin was developed to improve the efficiency of colony forming units after transformation. Our attempt to transform D. viridis with a heterologous Thioesterase A from the halophilic bacterium Chromohalobacter salexigens resulted in transgenic lines, but did not provide stable integration of the transgenes into the nuclear genome.