Multilevel Engineering of Saccharomyces cerevisiae for Scalable Production of Ergosterol as a Vitamin D2 Precursor

Abstract Ergosterol, a nutritionally relevant fungal sterol and vitamin D2 precursor, is valuable for food and feed applications, but its overproduction in Saccharomyces cerevisiae is constrained by stringent sterol homeostasis, limited organelle capacity, and the coordination of gene-dense pathways. A multilevel engineering framework was established by relieving inhibitory regulation, enhancing acetyl-CoA supply, strengthening rate-limiting steps, expanding endoplasmic reticulum capacity, and rewiring ethanol assimilation. A bidirectional-terminator-based integration toolkit enabled programmable chromosomal integration, modular pathway refactoring, and tunable expression partitioning. Stimulated Raman scattering (SRS) microscopy provided label-free visualization of intracellular ergosterol distribution and storage at single-cell resolution, supporting sterol homeostasis optimization. Robustness-oriented engineering of central carbon metabolism, mitochondrial function, and epigenetic regulation enhanced cellular fitness and fermentation performance. The resulting strain produced 0.983 g/L ergosterol in shake-flask cultures and 4.39 g/L in a 5-L bioreactor, corresponding to 51.7 mg/g dry cell weight (DCW), demonstrating the potential for scalable ergosterol biomanufacturing.