Abstract

Extracellular vesicles (EVs) are emerging as versatile therapeutic platforms, yet the mechanisms governing their biogenesis in yeast remain incompletely understood. Saccharomyces cerevisiae , a well-characterised and safe microbial chassis, naturally secretes abundant EVs and provides an attractive system for mechanistic dissection and engineering. Here, we establish S. cerevisiae as a tractable model for elucidating EV cargo loading. By combining multicopy expression of chicken interferon-λ (ChiIFN-λ) with cell wall perturbation, we achieved a tenfold increase in EV yield and efficient incorporation of ChiIFN-λ into EVs. Quantitative proteomics identified 1555 EV-associated proteins, including 501 predicted transmembrane proteins derived from multiple organelles. ChiIFN-λ overexpression and cell wall stress selectively reduced the abundance of key vesicle trafficking regulators, including SNARE, ESCRT and Rab proteins, indicating reprogramming of intracellular membrane trafficking pathways. Functional analyses further demonstrated that the SNARE proteins Sso2 and Nyv1 are enriched in the EV membrane and modulate EV size distribution and subpopulation composition. Together, these results reveal conserved protein-sorting machinery underlying yeast-derived extracellular vesicles (YDEVs) biogenesis and establish S. cerevisiae as a powerful platform for engineered EV production. Graphical Engineered Saccharomyces cerevisiae reveals how cell wall remodelling and membrane trafficking coordinate extracellular vesicle biogenesis. Interferon overexpression and chs3 deletion enhance EV secretion and reshape SNARE-, Rab- and ESCRT-associated pathways, establishing yeast as a versatile platform for EV engineering and therapeutic cargo loading.