Functionalizing Yeast Lipid Droplets as Versatile Biomaterials

Lipid droplets (LD) are dynamic cellular organelles of ≈1 µm diameter in yeast where a neutral lipid core is surrounded by a phospholipid monolayer and attendant proteins. Beyond the storage of lipids, opportunities for LD engineering remain underdeveloped but they show excellent potential as new biomaterials. In this research, LD from yeast Saccharomyces cerevisiae is engineered to display mCherry fluorescent protein, Halotag ligand binding protein, plasma membrane binding v‐SNARE protein, and carbonic anhydrase enzyme via linkage to oleosin, an LD anchoring protein. Each protein‐oleosin fusion is coded via a single gene construct. The expressed fusion proteins are specifically displayed on LD and their functions can be assessed within cells by fluorescence confocal microscopy, TEM, and as isolated materials via AFM, flow cytometry, spectrophotometry, and by enzyme activity assay. LD isolated from the cell are shown to be robust and stabilize proteins anchored into them. These engineered LD function as reporters, bind specific ligands, guide LD and their attendant proteins into union with the plasma membrane, and catalyze reactions. Here, engineered LD functions are extended well beyond traditional lipid storage toward new material applications aided by a versatile oleosin platform anchored into LD and displaying linked proteins. This study diversified the functions of lipid droplets from Baker's yeast (Saccharomyces cerevisiae) for innovative in vivo and in vitro applications via protein engineering. By fusing diverse proteins with plant protein oleosin and anchoring these into lipid droplets, they gained new functions as biosensors, for transport of cargo to the plasma membrane, and as biocatalysts. This research highlights their potential as advanced biomaterials, opening new avenues beyond conventional lipid storage.