Characterization of triacylglycerol (TAG) lipase candidates in Chlamydomonas reinhardtii
Xiaobo Li1,2, Eric R. Moellering,3, Barbara B. Sears2, Rachel E. Mille1,4, Min-Hao Kuo3, and Christoph Benning3
1) MSU-DOE Plant Research Laboratory, 2) Department of Plant Biology, 3) Department of Biochemistry and Molecular Biology, 4) Cell and Molecular Biology Program, Michigan State University, East Lansing, MI 48824
Oils from microalgae with triacylglycerols (TAGs) as the major component provide an excellent biodiesel feedstock. Oil production in microalgae is induced by nutrient deprivation such as nitrogen deprivation. TAG lipase is responsible for the first step of TAG degradation and thus, is a potential target for genetic engineering to improve the oil content. Among the many lipase domain-containing proteins predicted in the Chlamydomonas genome, one was found to be down-regulated under nitrogen deprivation conditions by a transcript profiling approach. Heterologous expression of this putative lipase complemented growth of a yeast mutant defective in lipases. Further analysis shows this protein lowers the cellular TAG content in yeast. In vitro assays with yeast protein extracts to confirm the TAG lipase activity are in progress. Overexpression and knock down strains are being constructed to study the function of this protein. Additional lipase candidates show transcription al regulation by nitrogen deprivation in our recent deep transcript profiling using Illumina technology. These genes will also be tested for lipase activity by yeast complementation. Besides the directed approach targeting TAG lipases, in order to find more central genes and regulators involved in TAG biosynthesis and degradation, a plasmid insertion mutant screen was conducted to identify mutants with higher or lower TAG content than wild-type. SiteFinding PCR enabled the identification of a disrupted putative TAG lipase, confirming the important role of lipases in TAG metabolism. Lipid analysis of meiotic progeny suggests co-segregation between the lipid phenotype and the insertion. Complementation tests are in progress to further confirm the relationship between the mutation and the TAG phenotype observed.
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