Complementation Studies of the Pah1‐encoded Phosphatidate Phosphatase in Yarrowia lipolytica

Abstract

The PAH1 gene encodes for phosphatidate (PA) phosphatase which catalyzes the dephosphorylation of PA to produce diacylglycerol that can be further converted to triacylglycerol (TAG). Our recent studies in the oleaginous yeast Yarrowia lipolytica showed that the pah1Δ mutation caused a significant decrease in total lipid content and TAG levels during the lipogenic phase, where cell growth ceases and lipid biosynthesis predominates. Also, the expression of the Pah1 protein is regulated under these conditions. To gain a better understanding of this regulation, integrative vectors that drive the expression of PAH1 under the control of its native promoter and either the LIP2 or the PEX20 terminator were constructed and transformed into pah1Δ cells. The expression of Pah1 in the transformants was confirmed by immunoblot analysis using antibodies directed against the protein, and its biological function was examined by analyzing the PA phosphatase activity, lipid content, and TAG levels. The results showed that the levels of the Pah1 protein expressed under theLIP2 and the PEX20 terminators were similar to the wild type levels. Also, the vector expressed Pah1 was able to restore the TAG levels in pah1Δ cells. The constructed vectors will be further used to study the function of Pah1 by mutating its catalytic motif using site-directed mutagenesis. We hypothesize that the catalytically inactive Pah1 protein will not complement the phenotype of the pah1Δ cells, thereby confirming that the catalytic activity of Pah1 is responsible for the phenotypical changes observed in the pah1Δ cells.