While the use of CO2 remains limited in biotechnology, it is possible to convert CO2 into methanol by electro-chemistry, which can later be used by methylotrophs – organisms capable to use reduced one carbon (C1) compounds for growth and energy. A particular feature of methylotrophy in yeast is that this metabolism is encapsulated into a compartment, the peroxisome. In FUNCEMM, a Franco-Austrian collaboration project led by Prof. Stéphanie Heux (TBI, Toulouse, France) and Univ.Prof. Diethard Mattanovich (BOKU, Vienna, Austria), we want to elucidate the meaning and importance of such a spatial organization to apply it in the establishment of synthetic methylotrophy.

More specifically, the aim of this PhD is to understand the role of the methylotrophic peroxisomal compartmentation in K. phaffii by relocating and optimizing the pathway into the cytosol by genome editing, using CRISPR-Cas9 technology. To confirm the pathway relocation, cellular organelles will be isolated by density gradient ultracentrifugation and their content further assessed by mass spectrometry (MS). Then, in collaboration with the partners from TBI, the engineered pathways will be analyzed in vivo and in vitro by dynamic metabolomics analysis (e.g. 13C-labelling MS and nuclear magnetic resonance (NMR)) to generate their kinetic models.

Subsequently, we want to harness this knowledge to build an artificial bacterial organelle mimicking the natural methylotrophic peroxisome. In collaboration with TBI, kinetic models of the synthetic bacterial strains will be developed to compare with the yeast strains, and to further improve their capacity to efficiently use methanol as a C1 carbon source via adaptive laboratory evolution; thus supporting the establishment of a C1-based bio economy.