Student projects - Toon de Kroon
Depleting yeast of the major membrane lipid phosphatidylcholine
Phosphatidylcholine (PC) is the most abundant membrane lipid in most eukaryotes and considered essential. The yeast double deletion mutant cho2opi3 lacks the methyltransferases which convert phosphatidylethanolamine (PE) to PC. Consequently, the cho2opi3 mutant is a choline auxotroph that relies on supplementation with choline for the synthesis of PC by the CDP-choline route. However, recently we isolated cho2opi3 suppressor (cho2opi3S) clones that suppress the auxotrophy for choline, and show robust growth in the absence of choline or choline substitute. Preliminary analysis of the lipidome of cho2opi3S clones showed that PC is below the detection limit after culture without choline; instead PE has become the most abundant phospholipid. The neutral lipid triacylglycerol strongly accumulates in the PC-free cho2opi3 suppressors. Importantly, the lipidome of the PC-free cho2opi3S reveals an overall shift to shorter average acyl chain length, which is thought to play a key role in maintaining membrane physical properties. Whole genome sequencing of a subset of suppressor mutants suggested 2N-1 aneuploidy as mechanism underlying the adaptation, which was proven by engineering a corresponding 2N-1 cho2opi3 mutant.
To solve the mechanism that enables yeast to live without PC, we want to address the following questions:
- How does cho2opi3S shorten the average acyl chain length?
- What are the roles of PE and TAG metabolism in PC-free cho2opi3S?
- What is the molecular genetic mechanism of PC-free cho2opi3S?
- How is aneuploidy induced?
Lipid analysis: Thin layer chromatography (TLC), Gas chromatography (GC), Mass spectrometry (MS), Isotope labeling
Yeast molecular biology: Gene deletion by homologous recombination, CRISPR-Cas9, DNA and RNA isolation
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