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ABSTRACT Extracellular vesicles (EVs) hold great promise as therapeutic delivery vehicles, leveraging their natural role as mediators of intercellular communication in all organisms studied. However, many barriers must be overcome to realize their full potential. Saccharomyces cerevisiae is an attractive chassis organism to explore solutions: It is used for drug biomanufacturing, it is amenable to complex genetic engineering, and their EVs can drive responses in human cells. To further develop this prospect, we sought to genetically modify S. cerevisiae EVs by devising a research framework amenable to iterative design, build, test, learn cycles – a core principle of synthetic biology. Using this approach, we focused on identifying new scaffolds – proteins that load cargoes into EVs – from a small pool of candidates. We first optimized a modular cloning strategy, called “EVclo”, for plasmid and genome-integrated candidate gene expression. Candidate genes were fused to EGFP, and after confirming expression in cells, we showed that scaffold-EFGP proteins colocalized with mRuby2-tagged Nhx1, a biomarker of multivesicular bodies, presumed sites of EV biogenesis. We triggered release of EVs by heat stress, isolated these EVs by ultrafiltration and size exclusion chromatography, and confirmed the presence of exosome-sized EVs in all samples. We find that candidate scaffold proteins did not affect EV size, morphology or titers. Further analysis of these samples indicated that some EGFP-tagged scaffolds are present in EVs: Bro1, a yeast ortholog of ALIX, was most abundant and ExoSignal showed highest enrichment of the human candidates. In all, we conclude that Bro1 is a good scaffold for future engineering strategies, and that human proteins can be sorted into yeast EVs suggesting conservation of the sorting machinery and demonstrating that yeast EVs can be humanized. This synthetic biology-based, proof-of-concept study establishes S. cerevisiae as a platform to engineer and bioproduce designer EVs for many applications. GRAPHICAL ABSTRACT HIGHLIGHTS AND TOC BLURB synthetic biology-based system was optimized to engineer EVs in S. cerevisiae EV scaffolds can be sorted to yeast EVs is an efficient scaffold to sort proteins into yeast EVs S. cerevisiae can be used to engineer designer EVs for drug delivery Extracellular vesicles (EVs) are a promising new modality for drug delivery. However, designer EVs must be engineered to broaden applications and improve efficacy. Here, Bouffard et al. optimize methods rooted in synthetic biology to genetically engineer EVs in S. cerevisiae , a yeast commonly used to manufacture biological drugs. They find that ectopically expressed human EV scaffolds (CD63, ExoSignal, PDGFR) can be sorted to yeast EVs, but Bro1 – the yeast ortholog of ALIX – was most efficient at sorting GFP into EVs. This proof-of-concept study demonstrates a single DBTL (design-build-test-learn) cycle that can be used to develop designer EVs for therapeutic applications.