Elucidation and Engineering of Sphingolipid Biosynthesis Pathway in Yarrowia lipolytica for Enhanced Production of Human-type Sphingoid Bases and Glucosylceramides

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Sphingolipids are vital membrane components in in mammalian cells, plants, and various microbes. We aimed to explore and exploit the sphingolipid biosynthesis pathways in an oleaginous and dimorphic yeast Yarrowia lipolytica by constructing and characterizing mutant strains with specific gene deletions and integrating exogenous genes to enhance the production of long-chain bases (LCBs) and glucosylceramides (GlcCers). To block the fungal/plant-specific phytosphingosine (PHS) pathway, we deleted the SUR2 gene encoding a sphinganine C4-hydroxylase, resulting in a remarkably elevated secretory production of dihydrosphingosine (DHS) and sphingosine (So) without acetylation. The Y. lipolytica SUR2 deletion (Ylsur2Δ) strain displayed retarded growth, increased pseudohyphal formation and stress sensitivity, along with the altered profiles of inositolphosphate-containing ceramides, GlcCers, and sterols. The subsequent disruption of the SLD1 gene, encoding a fungal/plant-specific Δ8 sphingolipid desaturase, restored filamentous growth in the Ylsur2Δ strain to a yeast-type form and further increased the production of human-type GlcCers. Additional introduction of mouse alkaline ceramidase 1 (maCER1) into the Ylsur2Δsld1Δ double mutants considerably increased DHS and So production while decreasing GlcCers. The production yields of LCBs from the Ylsur2Δsld1Δ/maCER1 strain increased in proportion to the C/N ratio in the N-source optimized medium, leading to production of 1.4 g/L non-acetylated DHS at the 5 L fed-batch fermentation with glucose feeding. This study highlights the feasibility of using the engineered Y. lipolytica strains as a cell factory for valuable sphingolipid derivatives for pharmaceuticals, cosmeceuticals, and nutraceuticals.