Excellence in Research: PAH1-mediated regulation of lipid synthesis in the model oleaginous yeast Yarrowia lipolytica

Biofuels are renewable fuels that can replace nonrenewable fossil fuels and thus decrease US dependency on oil. Microorganisms such as yeasts produce lipid-based biofuels that are considered a viable alternative to oil. However, the large-scale production of microbial biofuels has lagged because the low yields make the economics unfavorable.

The project aims to study the catalysts that convert raw materials to lipid biofuels in yeast cells. This knowledge is important for increasing the yields of lipid-based biofuels. The next step will be to use this knowledge to design more efficient production processes for biofuels.

In addition, this research will provide training opportunities for African-American graduate and undergraduate students. Specifically, first-generation African American undergraduate students will be the focus of the project because they have lower graduation rates and lower graduate enrollment rates compared to white peers. The hands-on training and intensive mentoring that the PIs will provide will increase these students' retention and graduation rates and prepare them for graduate programs.

Phosphatidate phosphatase (PAP) enzymes are major regulators of lipid biosynthesis in eukaryotic cells. Mammalian cells express three PAP enzymes, named lipins, while yeasts express a single lipin homolog encoded by the PAH1 gene. PAP enzymes catalyze the Mg+2-dependent dephosphorylation of phosphatidate, and the product diacylglycerol is used to synthesize the storage lipid triacylglycerol.

Lipins also act as transcriptional activators. In the oleaginous yeast Yarrowia lipolytica, the PAP activity is induced in cells growing in lipogenic media, and this induction correlates with increased lipid amounts in the cells. In this research, the role of PAH1 in lipogenesis will be studied in more detail using approaches that combine biochemistry and molecular genetics with -omics studies.

Mutant cells that lack Pah1 (i.e., pah1Δ) or express catalytically inactive forms of the protein will be used to establish the role of Pah1 in lipogenesis. Pah1 is a cytosolic protein that translocates to the membrane fraction where the substrate phosphatidate resides to catalyze its reaction. The protein subcellular location during lipogenesis will be studied via fractionation coupled with immunoblot analysis and by microscopy.

The global effect of the pah1Δ mutation on cell physiology will be studied by transcriptomic and lipidomics analyses.

This project is jointly funded by the Historically Black Colleges and Universities - Excellence in Research Program (EiR), the Established Program to Stimulate Competitive Research (EPSCoR), and the Historically Black Colleges and Universities - Undergraduate Program (HBCU-UP) of the Division of Human Resource Development.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.