Comparative Multi-omics Reveals Conserved Membrane Architecture and Selective Metabolic Cargo Enrichment Across Yeast-Derived Extracellular Vesicles

Extracellular vesicles (EVs) are fundamental mediators of intercellular communication across all kingdoms of life, yet our understanding of EV biology has been derived predominantly from mammalian systems. In contrast, yeast-derived extracellular vesicles (YDEVs) remain poorly characterized despite their potential as scalable, non-pathogenic platforms for biotechnology and metabolic engineering. Here, we combined a high-yield isolation workflow with comparative multi-omics to define the metabolically active cargo and membrane composition of EVs derived from three yeast species and four strains: the conventional model yeast Saccharomyces cerevisiae (SC), the non-model oleaginous yeast Rhodotorula toruloides (RT-88), and two Yarrowia lipolytica strains, wild-type W29 and its engineered derivative Po1f. Using tangential flow filtration coupled with ultracentrifugation, we reproducibly isolated intact, bilayer-enclosed vesicles with conserved nanoscale morphology across all strains despite marked differences in EV yield. Comparative metabolomics revealed selective enrichment of distinct metabolically active cargo, including nucleotide metabolites in SC, fatty acids and acyl-carnitines in RT-88, and strain-specific metabolites within the Yarrowia isolates. Notably, nicotinamide adenine dinucleotide (NAD⁺) was enriched in SC and RT-88 EVs but was nearly absent from Yarrowia-derived vesicles, raising the possibility that EV encapsulation may facilitate protected transport of this otherwise membrane-impermeable cofactor. In contrast to these differences in cargo composition, comparative lipidomics demonstrated a broadly conserved membrane architecture across all strains, with only quantitative remodeling of specific lipid classes and membrane unsaturation profiles. Together, these findings support an organizational principle in which yeast-derived extracellular vesicles preserve a conserved membrane architecture while selectively enriching metabolically active cargo, establishing a comparative framework for understanding EV biology across evolutionarily diverse yeast species.