Metabolic engineering approaches reveal widespread physiological functions of membrane lipids for Saccharomyces cerevisiae

摘要

The lipid composition of biological membranes can differ significantly between organisms and even between organelles of the same cell in terms of lipid compounds and specific ratios of lipid classes. Referring to this, every membrane features a characteristic lipid composition that is thought to regulate its physicochemical properties and cellular function by providing lipid environments supporting the integrity of membrane-localized protein machinery and membrane-associated processes. Chapter I gives a brief overview of the interlinkage between the chemical nature of membrane lipids, the structural and functional organization as well as the physicochemical properties of lipid bilayers and their influence on membrane-embedded proteins.udStudies to gain detailed knowledge on how membrane lipid composition influences the physiology of cells and regulates cellular processes require tools to manipulate lipid composition in vivo. By employing metabolic engineering approaches based on titratable gene expression tools, sets of Saccharomyces cerevisiae strains in which membrane lipid composition is under experimental control were engineered. The study described in Chapter II addresses OLE1, encoding for the sole fatty acid desaturase of budding yeast, to control the extent of acyl unsaturation of fatty acids incorporated in phospholipids. This approach revealed cellular roles for the physical state of cell membranes, so called membrane fluidity, on yeast flocculation and hypoxic growth. It is shown, how the endogenous lipid homeostasis machinery of budding yeast is adapted to carry out a broad response to oxygen limitation (hypoxia) and how it activates a non-canonical yeast flocculation pathway involving FLO1, which encodes for cell wall glycoproteins that mediate cell-cell-interactions by binding cell wall mannose residues of adjacent cells.udIn Chapter III, the previously generated strain in which expression of OLE1 is under experimental control was used as a cellular platform to assay the activity of heterologously expressed stearoyl-CoA desaturases (SCDs). Putative SCDs from human pathogens T. brucei and T. cruzi were functionally expressed in S. cerevisiae, thereby additionally confirming their SCD activity in vivo. The presented assay might also provide a tool to screen for inhibitors of SCDs, which are interesting drug targets in the treatment of bacterial and parasitic infections in humans.udThe study presented in Chapter IV addresses ERG9, an essential gene involved in the ergosterol biosynthetic pathway and used a metabolic engineering approach to achieve control over the total sterol biosynthetic activity of the cell. Cells that allowed for manipulating the native sterol homeostasis were employed to unveil physiological effects of ergosterol and total sterol depletion on the cell’s general viability as well as on fundamental membrane associated processes such as protein sorting and endo- and exocytosis. By combining this metabolic engineering approach and the powerful method of marker-free CRISPR/Cas9-mediated gene tagging, it was possible to establish a cellular system for investigating the impact of sterol depletion on the lateral distribution pattern of lipid-raft associated GFP-tagged membrane proteins within the plasma membrane of yeast.udChapter V introduces a novel set of all-in-one constitutive and inducible CRISPR/Cas9 vectors that allow for a very easy and highly convenient application of the technology in S. cerevisiae. The simplicity of the inducible system is based on the possibility of introducing a desired gRNA targeting sequence with homologous recombination-mediated assembly of overlapping single-stranded oligonucleotides. The inducible Cas9 expression approach also introduces the novel concept of chronologically separating the cloning procedure from the actual genome editing step by preloading cells with an all-in-one CRISPR/Cas9 plasmid. This way, CRISPR/Cas9-supported genome editing can be obtained with high efficiency and effectivity by just transforming a desired preloaded target strain with donor DNA to be genomically integrated without the need of co-introducing any of the CRISPR system components. These novel CRISPR/Cas9 systems will help to overcome limitations often observed for challenging metabolic and genetic engineering approaches that can be e.g. used for following studies to reveal physiological roles of membrane lipids for budding yeast.