Many microalgae, including Chlamydomonas, accumulate triacylglycerols when cultures encounter certain environmental stresses such as nutrient limitation. However, the regulatory factors and enzymes that govern triacylglycerol biosynthesis in microalgae have not been studied at the molecular level. Chlamydomonas is used as a microalgal model to identify genes and regulatory mechanisms required for triacylglycerol biosynthesis following nutrient deprivation. Multiple global and focused approaches are pursued towards this goal: 1. A gene disruption mutant screen of 32,000 lines yielded 80 putative mutants, some of which are disrupted in genes central to the regulation of triacylglycerol biosynthesis. 2. Global expression analysis identified putative transcription factor genes induced under oil accumulation conditions. New expression vectors were produced for their analysis in Chlamydomonas. 3. Following proteomics analysis of isolated lipid droplets, the expression of a major lipid droplet associated protein was repressed by RNAi in Chlamydomonas resulting in increased oil body size. Betaine lipid biosynthetic enzymes were found associated with lipid droplets and are presumed to be critical to oil biosynthesis. 4. Of five putative genes present in the genome encoding diacylglycerol acyltransferases that catalyze the last reaction of triacylglycerol biosynthesis, two were confirmed to encode enzymes with oil biosynthetic activity following expression in a respective yeast mutant.  These newly identified genes will provide novel targets for future engineering approaches towards optimizing microalgae oil production strains.
Funding for this work is provided by the US Air Force Office of Scientific Research and the Michigan Agriculture Experiment Station.