One of the main problems facing America today is the reliance on fossil fuels for power and transportation. The combination of CO2 emission and declining petroleum reserves means that an alternative needs to be found. Biofuels are one of the main alternatives being considered, as they can act as a carbon sink during plant growth, minimizing the net CO2 released when consumed, and they can be used for transportation fuel. Biodiesel has many good fuel properties, such as high energy density and ability to be used with existing technologies, but obtaining a high enough yield to meet current transportation needs is still an issue. One approach to address this is to use algae for biodiesel production, as they are capable of yielding much higher oil (in the form of triacylglycerol, or TAG) per land used, compared to traditional land-based oil crops. However, research is still needed to identify effective production strains, and modify them to improve overall yield.Chlamydomonas reinhardtii was used as a model microalga to study TAG synthesis, as it is already well studied and has various molecular tools available. Additionally, TAG synthesis can be induced by nitrogen deprivation, making it easy to study in laboratory conditions. Two approaches were used to study TAG synthesis, the first focusing on the enzymes directly responsible for synthesis and the second looking at the larger metabolic changes that contribute to TAG synthesis, along with potential regulators of the process.The first project discovered five putative type 2 diacylglycerol acyltransferases (DGATs). Testing of these DGATs, named DGTT1-5, in yeast confirmed DGTT2, DGTT3 and DGTT4 have DGAT activity, and DGTT5 is likely a pseudogene. Testing in yeast also revealed a potential difference in fatty acyl preference between DGTT2, DGTT3 and DGTT4. Expression of DGTT2 in Arabidopsis further confirmed its DGAT activity, demonstrated its ability to synthesize TAG in vegetative tissues, and also showed its ability to incorporate long-chain fatty acyls into TAG. Testing of DGTT2 and DGTT3 in Chlamydomonas by over-expression and amiRNA was attempted, but repeatable changes in TAG phenotype were not detected.In the second project, a transcript profiling experiment was performed using high-throughput sequencing, comparing populations grown under nitrogen replete and nitrogen deprived conditions. Many metabolic pathways showed consistent changes in transcript level, indicative of the changes occurring within the cell during nitrogen deprivation, which may contribute to TAG accumulation. In addition, various genes of interest for further study were discovered, including several candidate transcription factors. These candidate genes were cloned and tested via over-expression in Chlamydomonas. However, after testing, these candidates were found not to have a detectable role in TAG synthesis. The transcript profile dataset continues to be a valuable resource for future research in Chlamydomonas.
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