Characterization of lipases involved in triacylglycerol metabolism in Chlamydomonas reinhardtii

Triacylglycerol (TAG) is the major storage lipid for eukaryotic organisms, including animals, fungi, land plants and algae. With a higher energy density than carbohydrates and proteins, TAG from plant sources serves as an ideal feedstock for biofuel production. By transesterification, TAGs can be converted to fatty acid methylesters (FAMEs), which are similar to components of diesel derived from petroleum in carbon chain length and viscosity. Microalgae grow faster and have a larger potential for industrial biofuel production than land plants. In microalgae TAG production can be induced by nutrient stresses such as nitrogen (N) deprivation. Insights into the mechanism of oil production are required for further optimization of the biofuel production process. As an experimental organism for studying lipid metabolism, Chlamydomonas reinhardtii represents the most studied alga with multiple genetic and molecular biological tools available. In a transcript-profiling study to compare transcript abundance of genes in Chlamydomonas under normal growth conditions and N deprived conditions, a large number of predicted lipase-encoding genes appeared to be differentially regulated under the two conditions. This observation suggests that lipases play an important role in TAG metabolism of Chlamydomonas.;Conceivably, TAG production can be enhanced by suppressing TAG degradation enzymes, such as TAG lipases. One avenue of research pursued a reverse genetic screen of several lipase candidates by heterologous expression in a yeast mutant deficient in TAG lipases. Over-expression of one of the candidate genes, named CrLIP1, rescued the yeast mutant. Recombinant CrLIP1 protein exhibited lipolytic activity on diacylglycerol (DAG) and polar lipids, but not TAG. RNA suppression of the CrLIP1 gene led to delayed TAG degradation in Chlamydomonas, possibly through reduced capacity to degrade DAG, which is an intermediate in TAG turnover. It is also possible that CrLIP1 acts on TAG but requires an unknown cofactor which was missing in the lipase assays.;To complement the transcript profiling and reverse genetic approaches, an unbiased forward genetic screen was performed to obtain mutants with abnormal TAG amounts. Focus was given to a low TAG mutant disrupted in a gene designated Plastid Galactolipid Degradation 1 (PGD1), which was identified as a lipase-encoding gene. In the above-mentioned transcriptomic study, PGD1 was up-regulated following N deprivation. The pgd1 mutant is decreased in acyl fluxes from galactolipids to TAG. Recombinant PGD1 protein exhibited lipase activity on the substrate monogalactosyldiacylglycerol (MGDG). Multiple lines of evidence suggest PGD1 takes part in an acylation/deacylation cycle to export de novo synthesized fatty acids from the plastid.;The low TAG mutant pgd1 provides a tool for understanding the consequences of reduced TAG production. The pgd1 mutant exhibited chlorosis, accumulation of oxidative damage and loss of viability, which was reversed by the exposure to 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), a specific inhibitor for photosynthetic electron transport. This indicates that cells synthesize fatty acids and TAG to relieve the strain of excess electron transport and prevent the accompanying oxidative stress. These studies demonstrate the physiological significance of the accumulation of TAG under stress conditions and will impact how scientists approach the improvement of the production of biofuels from algae.