Redirection of lipid flux toward phospholipids in yeast increases fatty acid turnover and secretion

Significance Replacement of nonrenewable petrochemicals and liquid fuels requires sustainable production of oleochemicals. Free fatty acids (FFAs) are versatile molecules that can be produced by microbial fermentation and are used as precursors for production of these oleochemicals. In the past few years, we have seen major advancements in improving the yeast Saccharomyces cerevisiae for FFA production. Despite these successes, lipid metabolism is highly complex, and the pathways and metabolites involved in the formation of FFAs in yeast remain incompletely understood. In this work, we make important advancements in understanding the dynamics of FFA formation in the cell and explore the role of phospholipids in this process. Bio-based production of fatty acids and fatty acid-derived products can enable sustainable substitution of petroleum-derived fuels and chemicals. However, developing new microbial cell factories for producing high levels of fatty acids requires extensive engineering of lipid metabolism, a complex and tightly regulated metabolic network. Here we generated a Saccharomyces cerevisiae platform strain with a simplified lipid metabolism network with high-level production of free fatty acids (FFAs) due to redirected fatty acid metabolism and reduced feedback regulation. Deletion of the main fatty acid activation genes (the first step in β-oxidation), main storage lipid formation genes, and phosphatidate phosphatase genes resulted in a constrained lipid metabolic network in which fatty acid flux was directed to a large extent toward phospholipids. This resulted in simultaneous increases of phospholipids by up to 2.8-fold and of FFAs by up to 40-fold compared with wild-type levels. Further deletion of phospholipase genes PLB1 and PLB2 resulted in a 46% decrease in FFA levels and 105% increase in phospholipid levels, suggesting that phospholipid hydrolysis plays an important role in FFA production when phospholipid levels are increased. The multiple deletion mutant generated allowed for a study of fatty acid dynamics in lipid metabolism and represents a platform strain with interesting properties that provide insight into the future development of lipid-related cell factories.

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