Engineering Lipid Metabolism for Production of Oleochemicals in Saccharomyces cerevisiae

Oleochemicals are chemicals usually derived from plant oils or animal fat. Large use of plant oil derivatives as replacements for petroleum-derived chemicals or for large scale production of specialty chemicals brings sustainability issues from extensive cultivation of these specific oil plants in very restricted regions. This project studied and developed the baker’s yeast Saccharomyces cerevisiae as a platform for sustainable production of oleochemicals. The work aimed at production of two main chemical precursor species that allow for easy chemical conversion into all other basic oleochemical species: free fatty acids and triacylglycerols. The first part of this work related to studying the dynamics of FFA production. The first approach here was the development of an alternative fatty acid synthesis system based on the reverse β-oxidation pathway. The efficiency of this pathway was evaluated for its in vivo function but concluding that it was an efficient route for fatty acid synthesis. The subsequent studies were based on removal of acyl-CoA synthase activity through removal of FAA1-4 genes, which causes high level production of these and secretion to the extracellular medium. The work here studied this phenotype by coupling it to a pathway that converts FFA to the basic oleochemical fatty alcohols. The FFA production profile of a strain with this phenotype showed 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. This was further explored through fine-tuning of FAA1 expression which resulted in improved production of fatty alcohols without FFA secretion. Following up, the pathways leading to FFA formation in a Δfaa1 Δfaa4 background were studied through construction of a strain with an increasingly constrained lipid metabolism network. Under standard conditions, free fatty acids are a product of storage lipid and phospholipid remodeling, which are interconnected processes. Here, upon removing storage lipid formation, FFA formation fluxes were observed mostly through phospholipase-mediated phospholipid hydrolysis and phospholipid synthesis had a strong correlation with FFA production. 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 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 through the use of both knowledge-driven studies and engineering approaches, leading to important advancements in the field.