Abstract

Extracellular vesicles (EVs) represent a promising new modality for drug delivery. We use Saccharomyces cerevisiae (baker’s yeast) – an organism used for drug biomanufacturing – as a platform to design, build, and test engineered EVs for therapeutic applications. This involves modifying their contents and surfaces by adding human or yeast proteins with diverse functionalities requiring testing of thousands of modifications (individually or in combination) to optimize EV cargo loading, cell targeting, and content delivery tailoring to specific outcomes in patients. To support our studies, I sought to establish high throughput cloning and phenotyping protocols to generate libraries of genetically modified S. cerevisiae strains. I employed Golden Gate and Gateway cloning strategies based on the modular Yeast Toolkit, enabling use of new constructs by the synthetic biology community. Candidate genes were introduced into donor plasmids and then integrated into expression vectors containing a strong promotor (TDH3) and the Nanoluciferase (NLuc) gene. PCR, genetic assemblies, bacterial transformations and colony selection were conducted in 96-well plate format by robotic equipment housed in Concordia University’s Genome Foundry. After assembled vectors were validated by pooled nanopore sequencing, robots were used to transform them into S. cerevisiae (e.g. wild type BY4741), to select clonal transformants, and to prepare furizamine-based assays for detection of candidate proteins tagged with the luminescent biomarker nanoluciferase (NLuc) within whole cell lysates or EV-containing samples, measured using a plate-reading luminometer. As proof-of-concept, I implemented an automated procedure to generate an initial set of 96 yeast strains each expressing a candidate protein fused to (NLuc). Despite using a single promotor, I observed variable expression levels of human and yeast candidate proteins within S. cerevisiae cells. Initial phenotyping of extracellular media containing EVs revealed the presence of some protein candidates, later confirmed by assessing EVs purified by ultrafiltration and size exclusion chromatography. These included human proteins (e.g. CD81) suggesting that the mechanism(s) underlying EV protein loading are conserved. In all, I developed an automated yeast genetic engineering and phenotyping research pipeline for high throughput screening of strategies to improve EV functionalities required for use as next-gen drug delivery vehicles.