(582bo) Engineering Lipid Droplet Targeting Domains in S.Cerevisiae

In eukaryotes, lipid droplets are dynamic membrane-bound lipid stores that expand and regress to regulate neutral lipid storage and metabolism. As the active compartment involved in fatty acid storage and mobilization, the lipid droplet membrane presents a novel intracellular target for enzyme localization. We envision the targeting of enzymes and enzyme pathways from a variety of industrially interesting pathways including the biosynthesis and downstream modification of triacylgylcerols (TAGs) and sterol esters (SE). Here, we investigate and characterize a series of putative lipid droplet-targeting domains with the goal of engineering a set of genetic fusions that will target a protein or enzyme of interest to lipid droplets in Saccharomyces cerevisiae. Among the N-terminal lipid droplet tags identified in previous mammalian cell studies, murine Viperin (1-42), murine ALD1 (1-27), and human AAM-B (1-28) appear to retain lipid droplet targeting in S. cerevisiae. Fluorescence microscopy and western blot analysis of lipid droplet fractions are used to confirm targeting and quantify the targeting specificity of each domain. Additional variable repeats of basic amino acid residues are incorporated in the juxtamembrane and C-terminal regions, and may further facilitate sorting from the endoplasmic reticulum to lipid droplets. We anticipate that the targeting domains developed here will be useful in the metabolic engineering of S. cerevisiae, the engineering of reaction pathways in other yeast species such as Yarrowia lipolytica, and in the study of eukaryotic lipid droplet biology.