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

生物燃料是可再生燃料，可以取代不可再生的化石燃料，从而减少美国对石油的依赖。酵母等微生物产生的脂质基生物燃料被认为是石油的可行替代品。 然而，微生物燃料的大规模生产一直滞后，因为产量低，使经济不利。该项目旨在研究在酵母细胞中将原材料转化为脂质生物燃料的催化剂。这一知识对于提高基于脂质的生物燃料的产量非常重要。下一步将是利用这些知识来设计更有效的生物燃料生产工艺。此外，这项研究将为非洲裔美国研究生和本科生提供培训机会。具体而言，第一代非裔美国本科生将成为该项目的重点，因为与白色同龄人相比，他们的毕业率和研究生入学率较低。PI将提供的实践培训和强化指导将提高这些学生的保留率和毕业率，并为他们的研究生课程做好准备。 磷脂酸磷酸酶（PAP）是真核细胞中脂质生物合成的主要调节因子。哺乳动物细胞表达三种PAP酶，称为脂蛋白，而酵母表达由PAH 1基因编码的单一脂蛋白同系物。 PAP酶催化磷脂酸的Mg+2依赖性去磷酸化，并且产物二酰基甘油用于合成储存脂质三酰基甘油。脂蛋白还充当转录激活剂。在产油酵母Yarrowia lipolytica中，PAP活性在脂肪生成培养基中生长的细胞中被诱导，并且这种诱导与细胞中增加的脂质量相关。在这项研究中，PAH 1在脂肪生成中的作用将使用联合收割机生物化学和分子遗传学与组学研究相结合的方法进行更详细的研究。 缺乏Pah 1的突变细胞（即，pah 1 Δ）或表达无催化活性形式的蛋白质将用于确定Pah 1在脂肪生成中的作用。 Pah 1是一种胞质蛋白，其易位至底物磷脂酸所在的膜部分以催化其反应。 脂肪生成过程中的蛋白质亚细胞定位将通过分级分离结合免疫印迹分析和显微镜进行研究。pah 1 Δ突变对细胞生理学的整体影响将通过转录组学和脂质组学分析进行研究。该项目由历史上黑人学院和大学-卓越研究计划（EiR），刺激竞争研究的既定计划（EPSCoR），历史黑人学院和大学-本科课程（HBCU-UP）该奖项反映了NSF的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

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.