Engineering Lipid Metabolism for Production of Oleochemicals in Saccharomyces cerevisiae

Doctoral thesis, 2018

Oleochemicals are chemicals usually derived from plant oils or animal fat. Large use of plant oil derivatives as replacements for petroleum-derived chemicals brings sustainability issues from extensive cultivation of oil plants in restricted regions. This project studied and developed the baker’s yeast Saccharomyces cerevisiae as a platform for sustainable production of oleochemical precursors. The first part of this work studied the dynamics of free fatty acids (FFAs) production. First, an alternative fatty acid synthesis system based on the reverse β-oxidation pathway was evaluated for its in vivo function but concluding that it was not an efficient route for fatty acid synthesis. The subsequent studies were based on high level production of FFA and secretion to the extracellular medium through removal of acyl-CoA synthase activity by deleting the FAA1-4 genes. This phenotype was coupled to a pathway that converts FFA to fatty alcohols, which allowed the observation that while FFA are more efficiently converted to fatty alcohols during growth on glucose, the production of FFA is highly increased during growth on ethanol. Fine-tuning of FAA1 expression resulted in improved production of fatty alcohols without FFA secretion in this strain. Following up, the pathways leading to FFA formation in a Δfaa1 Δfaa4 background were studied through construction of a strain with a constrained lipid metabolism network. It was observed that upon removing storage lipid formation, phospholipid synthesis had a strong correlation with FFA production and FFA formation was mostly derived from phospholipid hydrolysis. On the second part of this work, S. cerevisiae was engineered for the highest TAG production levels reported so far. This relied on overexpressing genes involved in malonyl-CoA supply and TAG synthesis from acyl-CoA, and removing genes involved in TAG hydrolysis, β-oxidation and glycerol-3-phosphate usage. On a second approach, TAG accumulation properties were further improved in these strains through enhancing lipid droplet assembly processes. This was achieved through expression of perilipins and FIT proteins and through stimulation of ER stress mechanisms. In conclusion, lipid metabolism is an important part of cell homeostasis and engineering this system requires overcoming its tight regulation networks and mastering the processes involved in the physical structural organization of the system. Here this was highlighted using both knowledge-driven studies and engineering approaches, leading to important advancements in the field.