What´s in there? - Developing rapid and high-purity organelle isolation methods for advanced subcellular metabolomics in yeast

Background.

In nature, dedicated biochemical conversions are carried out in specific compartments or organelles, which contain a specialized subset of enzymes, confine potentially toxic pathway intermediates and provide a defined chemical environment (e.g. redox potential, cofactors, pH). Cellular metabolomics is at the core of metabolic engineering, driving microbial cell factories towards sustainability. Despite its importance, most metabolomics measurements are conducted in whole cells, primarily due to technical limitations. Thus, there is an urgent demand for subcellularly resolved metabolomics data. The miniaturization of the complete analytical process - to be developed by the research team of Gunda Koellensperger at the University of Vienna - relies on a key prerequisite: the rapid isolation of highly pure organelles.

Aims

The primary goal of this PhD project that is embedded within the Cluster of Excellence “Circular Bioengineering” is to develop rapid, high-purity isolation methods of native and synthetic organelles in yeast to enable unbiased subcellular metabolomics. The focus will be on isolating key organelles, including mitochondria, microsomes, vacuoles/lysosomes, peroxisomes, secretory organelles, and newly developed synthetic compartments. The project aims to overcome the limitations of traditional centrifugation-based techniques by introducing novel approaches that ensure high efficiency and precision in organelle isolation.

Methods.

• Engineer yeast cells with suitable organelle-specific tags.
• Validate the tagged organelles using fluorescent microscopy and proteomics to ensure accuracy and purity.
• Test novel and faster experimental procedures for subcellular fractionation, such as solid-phase separations and differential filtration.
• Develop and apply strains with tagged organelles for affinity purification methods to ensure rapid and precise organelle isolation.

Collaborations.

The work will be carried out at the BOKU - Institute of Microbiology and Microbial Biotechnology. Close collaboration within the COE “Circular Bioengineering”, esp. with analytical chemistry groups of Gunda Koellensperger (University of Vienna) for lipidomics and metabolomics. Protocols for organelle isolation will be shared within the consortium. They are also the basis of subcellular flux analysis that will be developed by the groups of Stephan Hann and Diethard Mattanovich (BOKU).