Master of Science / Biochemistry and Molecular Biophysics Interdepartmental Program / Timothy P. Durrett / Using vegetable oils directly as an alternative biofuel presents several problems as such oils typically possess poor fuel qualities including high viscosity, low volatility, and poor cold temperature properties. The ornamental shrub Euonymus alatus produces unusual acetyl-1,2-diacyl-sn-glycerols (acetyl-TAGs) that have an acetyl group in the sn-3 position instead of a long chain fatty acid. The presence of this sn-3 acetyl-group give acetyl-TAGs properties desirable for biofuels, such as reduced viscosity, comparted to the normal long chain triacyglycerols found in most vegetable oils. Acetyl-TAGs are synthesized by the Euonymus alatus diacylglycerol acetyltransferase (EaDAcT) and Euonymus fortunei diacylglycerol acetyltransferase (EfDAcT) enzymes. Both enzymes catalyze the transfer of an acetyl group from acetyl-CoA to diaclglycerol (DAG) to produce acetyl-TAGs. Previous work demonstrated that expression of EaDAcT combined with the suppression of a diacylglycerol aceyltransferase (DGAT1) in Camelina sativa led to seeds with 85 mol % acetyl-TAGs. Increasing acetyl-TAG levels further was explored using two strategies. Over expression of citrate lyase to increase the pool of acetyl-CoA to be used as a substrate for the acetyltransferase enzymes failed to increased levels of acetyl-TAGs. A second approach involved expressing EfDAcT in Camelina sativa. EfDAcT has demonstrated higher activity in vitro and in vivo and its expression in yeast leads to approximately 50 % higher levels of acetyl-TAGs compared to EaDAcT. The expression of EfDAcT coupled with the suppression of DGAT1 in Camelina sativa resulted in 90 mol % acetyl-TAGs in the transgenic seeds. Levels of EfDAcT protein analyzed in developing transgenic Camelina sativa seeds across a 40 day time period were highest at 15 and 20 days after flowering. Following these time points acetyl-TAG accumulation increased rapidly, coinciding with the higher enzyme expression levels. The optimization of additional promoters to ensure expression of EfDAcT in the last half of seed development could represent another way to further increase acetyl-TAGs in the future.

Doctor of Philosophy / Biochemistry and Molecular Biophysics Interdepartmental Program / Timothy P. Durrett / The direct use of vegetable oils as a biofuel suffers from problems such as high viscosity, low volatility and poor cold temperature properties. 3-acetyl-1,2-diacyl-sn-glycerols (acetyl-TAGs) have lower viscosity and freezing temperature than regular vegetable oils. However, by modifying their fatty acid composition, further improvement in their fuel properties is possible. Our goal was to develop plants that synthesize seed oils with further improved fuel properties. Euonymus alatus diacylglycerol acetyltransferase (EaDAcT) synthesizes acetyl-TAGs by the acetyl-CoA dependent acylation of diacylglycerol (DAG). Knowledge of the substrate specificity of EaDAcT for its acetyl-CoA donor and DAG acceptor substrates is important to generate the required acetyl-TAG composition in seed oil. A rapid method to quantify acetyl-TAGs was developed based on electrospray ionization mass spectrometry to gain information about the substrate specificity of EaDAcT. This method is as accurate and more rapid than the traditional radiolabeled substrate based assay and additionally provides information on acetyl-TAG molecular species present. Using this assay, EaDAcT specificity for different chain length acyl-CoA and DAGs was tested. It was found that although EaDAcT can use other short chain length acyl-CoAs as acyl donors, it has high preference for acetyl-CoA. Further, EaDAcT can acetylate a variety of DAGs with short, medium and long chain length fatty acids with high preference for DAGs containing unsaturated fatty acids. To generate acetyl-TAGs with lower molecular mass, EaDAcT was transformed into transgenic Camelina sativa lines producing high amounts of medium chain fatty acids (MCFAs). EaDAcT expression was also combined with the knockdown of DGAT1 and PDAT enzymes, which compete with EaDAcT for their common DAG substrate. High acetyl-TAG yielding homozygous T3 transgenic lines were generated but the incorporation of MCFAs into acetyl-TAGs was inefficient. A small increase in the viscosity of acetyl-TAGs from these lines was observed compared to acetyl-TAGs produced in wild type Camelina plant. The combined effect of insufficient lowering of molecular mass and increased fatty acid saturation levels of acetyl-TAGs might be responsible for this increased viscosity. Overall, it was concluded that the molecular mass and the saturation levels of fatty acids of acetyl-TAGs need to be considered at the same time in future attempts to further decrease their viscosity.