Metabolic engineering of Saccharomyces cerevisiae sphingolipid pathways for enhanced phytoceramide production

Phytoceramides are essential sphingolipids that support skin barrier integrity and hydration, making them valuable for cosmetic and pharmaceutical applications. However, their intricate structures and low natural abundance pose significant challenges for scalable production. Here, we present an integrated metabolic engineering and lipidomics study aimed at enhancing phytoceramide production in Saccharomyces cerevisiae. We implemented three strategies: (i) overexpression of SUR2 (sphinganine C4-hydroxylase) to boost phytosphingolipid formation; (ii) deletion of SCS7 (ceramide α-hydroxylase) to redirect flux toward non-hydroxylated phytoceramides; and (iii) overexpression of ISC1 (inositol phosphosphingolipid phospholipase) to recycle complex sphingolipids into ceramide pools. SUR2 overexpression showed the highest transcript levels, whereas lipidomics revealed that scs7Δ produced the highest phytoceramide enrichment with a 15-fold increase in phytoceramide abundance relative to the wild type. In terms of relative abundance within the quantified ceramide pool, phytoceramides increased from 5% in wild type to 46% in the SUR2-OE strain and 75% in the scs7Δ strain. The combined scs7Δ SUR2-OE strain did not exhibit additive metabolic effects on the lipid profile. The presence of residual hydroxylated ceramides indicated intrinsic regulatory constraints, aligning with the bypass mechanism proposed here whereby ceramide synthases can use pre-hydroxylated acyl-CoA. Importantly, this work contributes a comprehensive lipidomic profiling of S. cerevisiae, enabling clear discrimination between engineered and wild type strains and identification of genotypes exerting the greatest impact on phytoceramide accumulation. This approach advances sphingolipid pathway modulation and positions S. cerevisiae as a valuable model for studying phytoceramide-focused remodeling.