Unlabelled: Phytosphingosine (PHS), a sphingolipid-derived bioactive compound, exhibits multifunctional properties including antimicrobial activity, skin moisturization, and hydration, rendering it highly valuable for cosmetic and pharmaceutical applications. Through systematic metabolic engineering of Saccharomyces cerevisiae, we achieved 82.62 mg/g DCW phytosphingosine (PHS) production via integrated pathway optimization and stress mitigation. Key strategies involved: (1) Knockout of LCB4 (sphingoid long-chain base kinase), SHM2 (serine hydroxymethyltransferase), and CHA1 (l-serine deaminase) to block competitive pathways; (2) Overexpression of TSC10 (3-ketosphingosine reductase), SYR2 (sphingosine hydroxylase), and LCB1/LCB2 (serine palmitoyltransferase) to amplify the PHS synthesis flux. Initial shake flask fermentation (96 h) yielded 15.31 mg/g DCW of PHS, with ORM2 knockout providing a 73.6% productivity increase (26.54 mg/g DCW) despite inducing growth defects from sphingosine accumulation. We hypothesized that disrupted ORM2-mediated control of serine palmitoyltransferase activity might compromise ER homeostasis through sphingolipid imbalance, which was alleviated through HAC1 overexpression to enhance unfolded protein response (UPR) capacity. Fed-batch fermentation under optimized conditions (40 mM serine, 0.5 mM palmitic acid, pH 5) demonstrated scalable production, delivering a 5.4-fold improvement over baseline. This work establishes UPR engineering as a critical strategy for resolving lipid toxicity constraints in yeast sphingolipid biosynthesis, while highlighting S. cerevisiae's potential as an industrial PHS production platform through coordinated pathway and stress response manipulation.
Supplementary information: The online version contains supplementary material available at 10.1007/s13205-025-04417-z.
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