Understanding and Engineering Algal Lipid Metabolism in Chlamydomonas reinhardtii

Recent research has focused on understanding and using microalgal metabolic pathways to produce triacylglycerol (TAG), the biodiesel precursor. However, our current knowledge on microalgal lipid metabolism is primarily limited in predictive models without experimental verification. Here I describe three projects that broaden our understanding of microalgal lipid biology. First, we investigated a mechanism of TAG biosynthesis in microalgae. TAGs in Chlamydomonas reinhardtii, a model green microalga, predominantly have 16 carbon fatty acids (C16) at the sn-2 position. According to plant lipid biochemistry, lysophosphatidic acid acyltransferases (LPAATs) in chloroplasts prefer C16 to acylate the sn-2 position of glycerolipids, and LPAATs in the endoplasmic reticulum (ER) prefer C18. This information led to a postulation that TAGs in C. reinhardtii came from the chloroplast. However, researchers have not reported the delivery of TAG from plastids to the ER. We found an enzyme called CrLPAAT2 in the C. reinhardtii proteome database. This enzyme exists only in green microalgal species, localized in the ER, preferentially used C16 fatty, and played an important role in TAG accumulation. Second, we aimed to engineer microalgal metabolism to accumulate lipids (biodiesel precursors) in fast-growing conditions without growth retardation. Using C. reinhardtii, we simultaneously overexpressed genes encoding a pyruvate transporter (BASS1), a fatty acid thioesterase (FAT1), a glycerol-3- phosphate dehydrogenase (GPD2), and a pyruvate kinase (PYK5), which presumably rerouted pyruvate and glycerol-3-phosphate to lipid metabolism. The overexpressing strains achieved less protein content (0.8-fold) and higher quantities of total biomass (1.3- fold) and molecules including starch (2.2-fold), various small carbon molecules, and plastid lipids such as monogalactosyldiacylglycerol (MGDG, 1.4-fold), free fatty acids (FFAs, fold-change various to each FFA) and phytol (1.9-fold). However, total lipid content did not change. Third, we investigated the function of the phosphatase-fused glycerol-3-phosphate dehydrogenase (GPD2) in C. reinhardtii. I predicted two catalytically essential aspartates in the phosphatase domain. I also found that GPD2 participates in the biosynthesis of most glycerolipids. In addition, our phylogenetic analysis suggests that GPD2 have derived from gene duplication and fusion at the early evolution of eukaryotes. In conclusion, we provided the experimental evidence on the microalgal TAG biosynthetic pathway and lipid metabolic engineering