Microbial lipids offer a range of applications. Unlike their counterparts - animal fats and vegetable oils, they present a more sustainable production model that uses less land and water. Crucially, their production doesn't compete with food land-use, freeing up cropland for vital food crops rather than biofuel cultivation. Oleaginous yeasts have the remarkable capability to produce a diverse range of lipids. These lipids are not only prevalent in common food and cosmetic products like palm oil, but also find substantial use in various industries. They contribute significantly to the production of biofuels, lubricants, and inks. Moreover, they also have a use-case for more specialised applications such as the production of nutraceuticals, THC-precursors, and vital omega-3 fatty acids. Yarrowia lipolytica, an oleaginous yeast organism, has received extensive study over the last 15 years due to its lipid-producing capacity. Researchers such as Dr. Ledesma-Amaro's group have significantly advanced synthetic biology tools for this host, simplifying the engineering process. However, the economic feasibility of microbial lipid production encounters an obstacle in the downstream process, specifically lipid extraction. Microbial lipids are stored within cells, necessitating the breaking open of cells for extraction, a process that is energy-intensive and requires solvents or enzymes, driving up production costs. Furthermore, lipid production is limited by the number of cells and available intracellular space. A secretion system can make the production of fatty acids significantly cheaper by simplifying the extraction process. This can save up to 40-80% (1)(2) of the total production costs which is highly desirable from an industrial point of view and can make the difference between a non-profitable and a commercially successful product. To circumvent these limitations and reduce costs, an innovative approach has been adopted to allow Yarrowia lipolytica to secrete lipids into the culture broth. This makes extraction and purification significantly easier (3). While the proof-of-concept study establishes the feasibility of the approach, there is potential for further enhancement. This ability to secrete lipids also opens the door for continuous fermentation processes, allowing more efficient use of carbon resources by negating the need for constant cell regeneration. The project's objective is to develop innovative bioprocesses for microbial lipid production, integrating genetic and metabolic engineering with bioreactor and downstream optimization. To accomplish these goals, we propose two primary strategies: 1. Developing non-Yarrowia oleaginous yeast strains with enhanced capacity to produce and secrete microbial lipids. 2. Investigating varied bioprocess types to enable continuous production of microbial oils. Sources (1) Single-Cell Oils as a Source of Omega-3 Fatty Acids: An Overview of Recent Advances. doi.org/10.1007/s11746-012-2154-3 (2) Fatty acid production in genetically modified cyanobacteria. doi.org/10.1073/pnas.1103014108 (3) Combining metabolic engineering and process optimization to improve production and secretion of fatty acids. doi.org/10.1016/j.ymben.2016.06.004